Bispecific antibody against DLL3 and CD3 and use of bispecific antibody

Abstract

A bispecific antibody capable of specifically binding to DLL3 and CD3. The bispecific antibody can regulate the function of immune cells and can be used as a drug for treating malignant tumors. In addition, also provided are a polynucleotide encoding the bispecific antibody, a vector, a host cell, and a pharmaceutical composition comprising the bispecific antibody.

Description

Bispecific antibodies targeting DLL3 and CD3 and their applications

[0001] This disclosure claims priority to Chinese patent application No. 202410020775.9, filed with the Patent Office of China on January 5, 2024, entitled “Bispecific antibodies against DLL3 and CD3 and their applications”, and Chinese patent application No. 202411280343.8, filed with the Patent Office of China on September 12, 2024, entitled “Bispecific antibodies against DLL3 and CD3 and their applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of antibodies, and in particular, to bispecific antibodies against DLL3 and CD3. Background Art

[0003] Neuroendocrine lung cancer accounts for 20% of all lung cancers, of which small cell lung cancer accounts for about 14%. Most patients also have hematogenous metastases, and only about 1 / 3 of limited-stage patients have lesions confined to the chest. The current standard first-line treatment for patients with extensive-stage small cell lung cancer includes radiotherapy, carboplatin + etoposide + atezolizumab, carboplatin + etoposide + durvalumab, and cisplatin + etoposide + durvalumab. Untreated patients are very sensitive to chemotherapy and radiotherapy. However, due to a small number of residual cancer cells and insensitivity to tumor stem cell treatment, most patients will relapse in a short period of time, and the 5-year survival rate is less than 5%. For patients with relapse and refractory disease, there is currently a lack of effective treatment options. The recommended second-line treatment for patients who relapse within 6 months includes topotecan and rubicin, while for patients who relapse more than 6 months later, it is recommended to maintain the original treatment.

[0004] Bispecific antibodies (BsAb) are a type of antibody molecule that can simultaneously bind to two different antigens or two different epitopes on the same antigen. These two unique antigen-binding sites can advantageously achieve the binding of two targets and exert the synergistic effect of the two monoclonal antibodies. Bispecific antibodies can act as a bridge between target cells and functional molecules, producing a directed effector function. In preclinical research and clinical treatment, they may have more advantages than the mixed use of two monoclonal antibodies, and have broad application prospects in the fields of tumor immunotherapy and autoimmune diseases. Killing tumor cells through bispecific antibody-mediated cytotoxicity is a hot topic in current immunotherapy application research. It mainly uses the ability of bispecific antibodies to simultaneously bind to effector cells and tumor-associated antigens, directly triggering immune cells to specifically kill tumor cells.

[0005] CD3 is a specific surface molecule present on all T lymphocytes that recruits effector T cells with cytotoxic properties. DLL3 is largely absent in normal tissues but is highly expressed in small cell lung cancer samples, distributed across the cell membrane and matrix, and correlates with disease grade. Therefore, DLL3 is considered an effective target for small cell lung cancer or neuroendocrine cancer. Therefore, targeting CD3 and DLL3 may enable specific killing of DLL3-positive tumors. Summary of the Invention

[0006] In order to solve the above technical problems, this application provides the following technical solutions:

[0007] In a first aspect, the present disclosure provides a multispecific binding molecule, characterized in that the multispecific binding molecule comprises:

[0008] (A) Antigen-binding portion that specifically binds to DLL3;

[0009] (B) an antigen-binding portion that specifically binds CD3;

[0010] wherein the antigen-binding portion that specifically binds to DLL3 comprises a first antigen-binding portion and a second antigen-binding portion that bind to different epitopes of DLL3;

[0011] The antigen binding portion that specifically binds to CD3 is a Fab that binds to CD3.

[0012] In some embodiments, the antigen binding moieties are each independently selected from an antibody, an antibody fragment, F(ab')2, Fab', Fab, Fv, scFv or a nanobody (VHH).

[0013] In some embodiments, the multispecific binding molecule comprises three polypeptide chains: a first heavy chain, a second heavy chain, and a light chain, wherein:

[0014] (1) The first heavy chain and the second heavy chain are different and have the following structures:

[0015] (a) First heavy chain VL1 DLL3 -L1-VH1 DLL3 -L1-VH1 CD3 -CH1-Fc1 and second heavy chain VL2 DLL3 -L1-VH2 DLL3 -L2-Fc2;

[0016] (b) First heavy chain VH1 CD3 -CH1-Fc1 and second heavy chain VL3 DLL3 -L1-VH3 DLL3 -L1-VHH1DLL3 -L2-Fc2;

[0017] (c) First heavy chain VL1 DLL3 -L1-VH1 DLL3 -L1-VH1 CD3 -CH1-Fc1 and second heavy chain VHH1 DLL3 -L2-Fc2;

[0018] (d) First heavy chain VHH2 DLL3’ -L1-VH2 CD3 -CH1-Fc1 and second heavy chain VHH3 DLL3 -L2-Fc2; and,

[0019] (2) The light chain comprises the following structure: VL1 CD3 -CL, or VL2 CD3 -CL;

[0020] Among them, VH1 DLL3 、VH2 DLL3 and VH3 DLL3 The variable regions of the heavy chains with different specificities for binding to DLL3, VL1 DLL3 、VL2 DLL3 and VL3 DLL3 Different light chain variable regions specifically binding to DLL3, which respectively form different scFvs specifically binding to DLL3; VHH1 DLL3 、VHH2 DLL3 and VHH3 DLL3 Different nanobodies that specifically bind to DLL3; VH1 CD3 and VH2 CD3 VL1 is a heavy chain variable region that binds to CD3 with different specificities. CD3 and VL2 CD3 are different light chain variable regions that specifically bind to CD3, which respectively form different antigen-binding portions that specifically bind to CD3; Fc1 and Fc2 are the Fc regions of any antibody, and L1 and L2 are the same or different linkers, respectively.

[0021] In some embodiments, the Fc1 and Fc2 of the multispecific binding molecule are different, Fc1 is a knob-Fc, and Fc2 is a hole-Fc; preferably, the knob-Fc comprises a T366W mutation, and / or the hole-Fc comprises a T366S, L368A and / or Y407V mutation; preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4.

[0022] In some embodiments, the Fc1 and Fc2 of the multispecific binding molecule are different and have amino acid mutations that alter effector function; preferably, the mutations that alter effector function comprise L234A, L235A and / or G237A mutations; preferably, Fc1 and Fc2 are Fc regions of IgG1 or IgG4.

[0023] In some embodiments, the scFv that specifically binds to DLL3 comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conservative amino acid substitutions:

[0024] The heavy chain CDR1 sequence is shown in any one of SEQ ID NOs. 29, 32, 35, 68, 71, 74, 107, 110, and 113;

[0025] The heavy chain CDR2 sequence is shown in any one of SEQ ID NOs. 30, 33, 36, 69, 72, 75, 108, 111, and 114;

[0026] The heavy chain CDR3 sequence is shown in any one of SEQ ID NOs. 31, 34, 37, 70, 73, 76, 109, 112, and 115;

[0027] The light chain CDR1 sequence is shown in any one of SEQ ID NOs. 38, 41, 44, 77, 80, 83, 116, 119, and 122;

[0028] The light chain CDR2 sequence is shown in any one of SEQ ID NOs. 39, 42, 45, 78, 81, 84, 117, 120, and 123;

[0029] The light chain CDR3 sequence is shown in any one of SEQ ID NOs. 40, 43, 46, 79, 82, 85, 118, 121, and 124;

[0030] The Nanobodies that specifically bind to DLL3 comprise heavy chain CDRs selected from the following, or comprise heavy chain CDRs with 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably with conservative amino acid substitutions:

[0031] The heavy chain CDR1 sequence is shown in any one of SEQ ID NOs. 47, 50, 53, 86, 89, 92, 125, 128, and 131;

[0032] The heavy chain CDR2 sequence is shown in any one of SEQ ID NOs. 48, 51, 54, 87, 90, 93, 126, 129, and 132;

[0033] The heavy chain CDR3 sequence is shown in any one of SEQ ID NO. 49, 52, 55, 88, 91, 94, 127, 130, and 133.

[0034] In some embodiments, the heavy chain variable region (VH DLL3 ) comprises an amino acid sequence as shown in SEQ ID NO. 16, 18, 20, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of SEQ ID NO. 16, 18, 20; and / or the light chain variable region (VL DLL3 ) comprising the amino acid sequence as shown in SEQ ID NO. 17, 19, or 21, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of SEQ ID NO. 17, 19, or 21;

[0035] The Nanobody that specifically binds to DLL3 comprises an amino acid sequence as shown in SEQ ID NO. 22, 23, or 24, or an amino acid sequence that is 90%, 95%, 96%, 97%, 98%, 99% or higher identical to any one of SEQ ID NO. 22, 23, or 24.

[0036] In some embodiments, the antigen binding portion that specifically binds to CD3 comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the following sequences; preferably having conservative amino acid substitutions:

[0037] The heavy chain CDR1 sequence is shown in any one of SEQ ID NOs. 56, 59, 95, 98, 134, and 137;

[0038] The heavy chain CDR2 sequence is shown in any one of SEQ ID NOs. 57, 60, 96, 99, 135, and 138;

[0039] The heavy chain CDR3 sequence is shown in any one of SEQ ID NOs. 58, 61, 97, 100, 136, and 139;

[0040] The light chain CDR1 sequence is shown in any one of SEQ ID NOs. 62, 65, 101, 104, 140, and 143;

[0041] The light chain CDR2 sequence is shown in any one of SEQ ID NOs. 63, 66, 102, 105, 141, and 144;

[0042] The light chain CDR3 sequence is shown in any one of SEQ ID NO. 64, 67, 103, 106, 142, and 145.

[0043] In some embodiments, the variable heavy chain region (VH) that specifically binds to CD3 CD3 ) comprises an amino acid sequence as shown in SEQ ID NO. 25, 27 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO. 25, 27; and / or the light chain variable region (VL) that specifically binds to CD3 CD3 ) comprises the amino acid sequence shown in SEQ ID NO. 26, 28, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of SEQ ID NO. 26, 28.

[0044] In some embodiments, the first heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, or 7, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity to any one of SEQ ID NO. 1, 4, or 7;

[0045] The second heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 3, 5, 6, or 9, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity to any one of SEQ ID NO. 3, 5, 6, or 9;

[0046] The light chain comprises an amino acid sequence as shown in SEQ ID NO. 2, 8 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of SEQ ID NO. 2, 8.

[0047] In some embodiments, the antigen binding portion is:

[0048] (1) Chimeric antibodies or fragments thereof;

[0049] (2) a humanized antibody or a fragment thereof; or

[0050] (3) Fully human antibodies or fragments thereof.

[0051] In some embodiments, the multispecific binding molecule is trivalent, tetravalent, pentavalent, or hexavalent; preferably, the multispecific binding molecule is trivalent.

[0052] In some embodiments, the multispecific binding molecule is further conjugated to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic drug or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

[0053] In a second aspect, the present disclosure provides an isolated nucleic acid fragment encoding the multispecific binding molecule of the first aspect.

[0054] In a third aspect, the present disclosure provides a vector comprising the nucleic acid fragment described in the second aspect.

[0055] In a fourth aspect, the present disclosure provides a host cell comprising the vector described in the third aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0056] In a fifth aspect, the present disclosure provides a method for preparing the multispecific binding molecule of the first aspect, comprising culturing the cell of the fourth aspect, and isolating the multispecific binding molecule expressed by the cell.

[0057] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the multispecific binding molecule, nucleic acid fragment, and vector described in the first aspect; the multispecific binding molecule prepared by the method described in the fifth aspect; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0058] In some embodiments, the pharmaceutical composition is packaged together with the anti-tumor agent, or packaged separately (eg, the anti-tumor agent is combined with the pharmaceutical composition prior to administration).

[0059] In a seventh aspect, the present disclosure provides the multispecific binding molecule of the first aspect, the nucleic acid fragment of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the multispecific binding molecule prepared by the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect, for use in preparing and / or preventing a medicament for cancer;

[0060] Preferably, the cancer is selected from lung cancer, neuroendocrine cancer, melanoma, glioblastoma multiforme, small cell bladder cancer, pancreatic cancer and other solid tumors expressing DLL3.

[0061] In some embodiments, the drug is used in combination with another therapeutic agent or with surgery; wherein the additional therapeutic agent or the surgery is selected from radiation therapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, surgical intervention, immunostimulatory antibodies, immunomodulatory drugs, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.

[0062] In an eighth aspect, the present disclosure provides a method for treating and / or preventing cancer, comprising administering to a subject a therapeutically effective amount of the multispecific binding molecule of the first aspect, the nucleic acid fragment of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the multispecific binding molecule prepared by the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect;

[0063] Preferably, the cancer is selected from lung cancer, neuroendocrine cancer, melanoma, glioblastoma multiforme, small cell bladder cancer, pancreatic cancer and other solid tumors expressing DLL3.

[0064] In some embodiments, the method further comprises administering an additional therapeutic agent or surgical treatment to the patient in need thereof; wherein the additional therapeutic agent or the surgery is selected from radiation therapy, chemotherapy, an oncolytic drug, a cytotoxic agent, a cytokine, surgery, an immunostimulatory antibody, an immunomodulatory drug, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, or a cellular immunotherapy.

[0065] Definitions and Explanations of Terms

[0066] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0067] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0068] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0069] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the term."

[0070] The term "Delta-Like Ligand 3 (DLL3)" herein refers to a single-pass transmembrane protein attached to the cell surface and a member of the Notch ligand family. The human DLL3 gene is located on chromosome 19q13, and its open reading frame is approximately 1800 base pairs long. The human DLL3 protein is composed of 619 amino acids, and its complete structure includes a DSL domain, an intracellular domain, and six epidermal growth factor-like domains. The DSL gene sequence at the N-terminus of the extracellular domain is highly conserved within the ligand family and is a functional domain required for binding to the Notch receptor. The DLL3 intracellular domain is relatively short, and its function remains unclear. Studies have found that DLL3 is highly expressed in SCLC and other neuroendocrine tumors, but is rarely expressed in normal tissues. Activation of DLL3 can exert either pro-oncogenic or anti-oncogenic effects. DLL3 is widely expressed in human cancers, including small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumors, endometriomas, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

[0071] The term "CD3" (cluster of differentiation 3) herein refers to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). In mammals, the CD3 molecule is a six-chain multiprotein complex, including a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain, wherein the CD3ζ chain is the intracellular tail of the CD3 molecule, and all of the CD3γ, CD3δ, and CD3ε chains contain an extracellular domain (ECD) expressed on the surface of T cells. Exemplary sequences of human CD3 include human CD3ε protein (NCBI Ref Seq No. NP_000724 or NCBI: AAH49847.1), human CD3δ protein (NCBI Ref Seq No. NP_000723), and human CD3γ protein (NCBI Ref Seq No. NP_000064). Exemplary sequences of non-human CD3 include Macaca fascicularis (monkey) CD3 epsilon protein (NCBI Ref Seq No. NP_001270544), Macaca fascicularis (monkey) CD3 delta protein (NCBI Ref Seq No. NP_001274617), Macaca fascicularis (monkey) CD3 gamma protein (NCBI Ref Seq No. NP_001270839); mouse CD3 epsilon protein (NCBI Ref Seq No. NP_031674), mouse CD3 delta protein (NCBI Ref Seq No. NP_038515), mouse CD3 gamma protein (NCBI Ref Seq No. AAA37400); Rattus norvegicus (rat) CD3 epsilon protein (NCBI Ref Seq No. NP_001101610), Rattus norvegicus (rat) CD3 delta protein (NCBI Ref Seq No. No.NP_037301), Rattus norvegicus (rat) CD3γ protein (NCBI Ref Seq No.NP_001071114).

[0072] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an affinity of 1×10 -7 M or lower, about 1×10 -8M or lower, about 1×10 -9 M or lower, about 1×10 -10 M or less, 1×10 -11 M or lower or 1×10 -12 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see Example 5 herein for methods for obtaining KD values.

[0073] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0074] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, etc. (the above contents are incorporated herein in their entirety).

[0075] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0076] The term "antibody" herein includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0077] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0078] As used herein, the term "nanoantibody" refers to a naturally occurring heavy chain antibody lacking a light chain that exists in camels. Cloning its variable region can yield a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0079] The terms "nanobody" and "single-domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy-chain variable region by cloning the variable region of a heavy-chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy-chain antibody naturally lacking the light chain and heavy-chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy-chain variable region.

[0080] For further description of “heavy chain antibodies” and “nanobodies”, see: Hamers-Casterman et al., Nature. 1993; 363; 446-8; the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO 03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and other prior art mentioned in these applications. (The above contents are incorporated herein in their entirety).

[0081] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0082] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0083] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0084] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0085] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0086] The term "Fd" herein refers to an antibody composed of a VH and CH1 domain. The term "Fv" herein refers to an antibody fragment composed of a single-arm VL and VH domain. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0087] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO. 148) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 3 (SEQ ID NO. 149), i.e., L1 as described herein, may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may exist between the VH and VL of an scFv, forming a disulfide-linked Fv (dsFv). (The above contents are incorporated herein in their entirety).

[0088] The terms "connector", "linker" and "Linker" are used interchangeably herein and refer to a sequence that connects two proteins or protein domains. In some embodiments of the present disclosure, the connector can be an amino acid or multiple amino acids, a non-functional amino acid sequence without secondary or higher structure. In some exemplary embodiments, the length of the linker can be about 5 to 100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 amino acids in length. Exemplarily, the linker can be a flexible linker or a rigid linker, such as repeated GGGGS, repeated KLAAA (SEQ ID NO.150), GGG (SEQ ID NO.151, as described herein L2).

[0089] The term "diabody" herein refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0090] The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or a tracer; the term "conjugated antibody" herein refers to an antibody that is conjugated to a therapeutic agent or a tracer.

[0091] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0092] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted onto human framework sequences.

[0093] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0094] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). (The above contents are incorporated herein in their entirety). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0095] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0096] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0097] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). (The above contents are incorporated herein in their entirety).

[0098] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0099] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0100] The term "Fc" herein refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody and is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447. Typically, it comprises the CH3 and CH2 domains of the antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. The "CH2 region" of a human IgG Fc region generally extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 region. The CH2 domain herein can be a native sequence CH2 region or a variant CH2 region. A "CH3 region" comprises the residues at the C-terminus of the CH2 region in the Fc region (i.e., from amino acid residue approximately at position 341 to amino acid residue approximately at position 447 of IgG). The CH3 region herein can be a native sequence CH3 region or a variant CH3 region. Fc variants generated from such variant CH3 regions can form space-filling effects, electrostatic guidance, hydrogen bonding, hydrophobic interactions, and other interactions between Fc variants, contributing to the formation of stable heterodimeric proteins.

[0101] Mutational design techniques for Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" approach proposed by Cater et al.; the electrostatic steering approach used by Amgen researchers to form Fc-containing heterodimers (US20100286374 A1); the SEEDbodies approach proposed by Jonathan H. Davis et al. to form heterodimers via IgG / Ig chain exchange; bispecific molecules formed using Genmab's DuoBody platform technology; Xencor researchers' integrated structural calculations and Fc amino acid mutations to create heterodimeric proteins with different modes of action; and Suzhou Alphamab's charge network-based Fc engineering approach (CN201110459100.7) to generate heterodimeric proteins. Other genetic engineering approaches based on Fc amino acid changes or functional modifications to achieve heterodimeric functional proteins are also being explored. The knob / hole structure on the Fc variant fragment described in this application refers to two Fc fragments that are each mutated, and after the mutations, they can be combined in a "knob-into-hole" form. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site mutations on the Fc region so that the resulting first Fc variant and second Fc variant can be combined in a "knob-into-hole" form to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the scope of those skilled in the art. Preferably, the Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4 is used, and more preferably the Fc region of human antibodies IgG1. Randomly select one of the first Fc variant or the second Fc variant to form a knob mutation (knob chain) and the other to form a hole mutation (hole chain). In some specific embodiments, the CH3 region of the knob chain contains a T366W mutation, and the hole chain contains T366S, L368A, and / or Y407V mutations, thereby generating an Fc variant. (The above contents are incorporated herein in their entirety.) In some embodiments, the first Fc variant may be Fc1 (SEQ ID NO. 152), and the second Fc variant may be Fc2 (SEQ ID NO. 153).

[0102] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0103] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0104] 1) Alanine (A), serine (S), threonine (T);

[0105] 2) Aspartic acid (D), glutamic acid (E);

[0106] 3) Asparagine (N), glutamine (Q);

[0107] 4) Arginine (R), Lysine (K), Histidine (H);

[0108] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0109] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0110] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0111] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0112] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0113] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0114] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid (SEQ ID NO. 1) molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0115] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for direct expression of antibodies of the present disclosure in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into the subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823B1). (the foregoing is incorporated herein in its entirety). "Isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0116] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0117] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0118] The term "pharmaceutical composition" herein refers to a formulation that is in a form that allows for the effective biological activity of the active ingredient contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. The "pharmaceutical composition" may further comprise an additional therapeutic agent, such as an anti-tumor agent; more preferably, the anti-tumor agent may be a PD-1 axis binding antagonist, a small molecule anti-tumor agent, or a cell therapy agent, wherein the cell therapy agent may be CAR-T, CAR-NK, etc.

[0119] The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, etc. and combinations thereof, which are known to those skilled in the art. Except in the case of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0120] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals being treated for a disease or condition.

[0121] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject of treatment, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0122] The term "immunostimulatory antibodies" herein refers to antibodies that promote anti-tumor immunity by directly modulating immune function, i.e., by blocking otherwise inhibitory targets or enhancing immunostimulatory proteins. These include: 1) antagonistic antibodies that target inhibitory immune checkpoints and agonistic antibodies that enhance immunostimulatory proteins.

[0123] The term "immunomodulatory drug" herein may refer to, for example, thymosin alpha 1. Principle: Thymosin alpha 1 (Tα1) is a naturally occurring thymosin peptide that acts as an endogenous regulator of the innate and adaptive immune systems. It is used worldwide to treat conditions associated with immune dysfunction, including viral infections such as hepatitis B and C, certain cancers, and for vaccine enhancement. In particular, recent advances in immunomodulatory research have indicated a beneficial effect of Tα1 treatment in septic patients.

[0124] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0125] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0126] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figures 1A-1D are diagrams showing the construction structures of bispecific antibodies Bis01, Bis02, Bis03, and Bis04, respectively.

[0128] FIG2A shows the ELISA assay for the binding activity of Bis01, Bis02, and Bis03 to human DLL3 protein.

[0129] FIG2B shows the ELISA assay for the binding activity of Bis04 to human DLL3 protein.

[0130] FIG2C shows the binding activity of Bis01, Bis02 and Bis03 to monkey DLL3 protein detected by ELISA.

[0131] FIG2D shows the binding activity of Bis04 to monkey DLL3 protein detected by ELISA.

[0132] FIG3A shows the ELISA assay for the binding activity of Bis01, Bis02 and Bis03 to human CD3e-His.

[0133] FIG3B is an ELISA test showing the binding activity of Bis04 to human CD3e-His.

[0134] FIG3C shows the binding activity of Bis01, Bis02 and Bis03 to monkey CD3e-His detected by ELISA.

[0135] FIG3D shows the binding activity of Bis04 to monkey CD3e-His detected by ELISA.

[0136] FIG4A shows the expression levels of DLL3 on the surface of tumor cell lines SHP77, H2171 and A549 detected by FACS.

[0137] FIG4B is a FACS analysis of the expression levels of DLL3 on the surface of CHOK1 in human and monkey DLL3-overexpressing cells.

[0138] 5A-5B are FACS assays showing the binding reactions of Bis01, Bis02, Bis03 and Bis04 to the human endogenous tumor cell line SHP77.

[0139] 5C-5D are FACS assays showing the binding reactions of Bis01, Bis02, Bis03, and Bis04 to the human endogenous tumor cell line NCI-H2171.

[0140] 6A-6B are FACS assays showing the binding reactions of Bis01, Bis02, Bis03 and Bis04 to human negative tumor cells A549.

[0141] 7A-7B show the binding reactions of Bis01, Bis02, Bis03 and Bis04 to human DLL3-overexpressing cells CHOK1-hDLL3 detected by FACS.

[0142] 7C-7D show the binding reactions of Bis01, Bis02, Bis03 and Bis04 to monkey DLL3-overexpressing cells CHOK1-cynoDLL3 detected by FACS.

[0143] 8A-8B show the binding reactions of Bis01, Bis02, Bis03 and Bis04 to human Jurkat cells detected by FACS.

[0144] 9A-9B show the binding activities of BisO1, BisO2, BisO3 and BisO4 to human PBMCs detected by FACS.

[0145] 9C-9D show the binding activities of Bis01, Bis02, Bis03 and Bis04 to monkey PBMCs detected by FACS.

[0146] 10A-10B are luciferase reporter gene assays to detect the activation of Bis01, Bis02, Bis03, and Bis04 after co-incubation of H2171 and Jurkat-luc.

[0147] 10C-10D are luciferase reporter gene assays to detect the activation of Bis01, Bis02, Bis03, and Bis04 after co-incubation of A549 cells with Jurkat-luc.

[0148] 11A-11C show the evaluation of the killing activity of Bis01, Bis02, Bis03 and Bis04 against the tumor cell line SHP77.

[0149] 12A-12C show the evaluation of the killing activity of Bis01, Bis02, Bis03 and Bis04 against the tumor cell line H2171.

[0150] FIG13 is an evaluation of the killing activity of Bis01, Bis02 and Bis03 against the negative tumor cell line A549.

[0151] 14A-14C show that bispecific antibodies Bis01, Bis02, Bis03, and Bis04 stimulate PBMC to secrete IFNγ in the presence of H2171 cells.

[0152] 15A-15C show that bispecific antibodies Bis01, Bis02, Bis03, and Bis04 stimulate PBMC to secrete TNFα in the presence of H2171 cells.

[0153] Figures 16A-16C show that bispecific antibodies Bis01, Bis02, Bis03, and Bis04 stimulate PBMC to secrete IL-6 in the presence of H2171 cells.

[0154] FIG17A is a PBMC reconstitution model used to evaluate the inhibitory ability of bispecific antibodies Bis01, Bis02, and Bis03 against SHP77 tumors.

[0155] FIG17B shows the changes in mouse body weight during administration of bispecific antibodies Bis01, Bis02, and Bis03.

[0156] FIG18A is a PBMC reconstitution model used to evaluate the inhibitory ability of the bispecific antibody Bis04 on SHP77 tumors.

[0157] FIG18B shows the changes in mouse body weight during administration of the bispecific antibody Bis04.

[0158] FIG19A is a PBMC reconstitution model used to evaluate the inhibitory ability of bispecific antibodies Bis01, Bis02, and Bis03 against NCI-H2171 tumors.

[0159] FIG19B shows the changes in mouse body weight during administration of bispecific antibodies Bis01, Bis02, and Bis03.

[0160] FIG20A is a PBMC reconstitution model evaluating the inhibitory ability of the bispecific antibody Bis04 on NCI-H2171 tumors.

[0161] FIG20B shows the changes in mouse body weight during administration of the bispecific antibody Bis04.

[0162] FIG21A is a PBMC reconstitution model used to evaluate the inhibitory ability of bispecific antibodies Bis01 and Bis04 against QGP-1 tumors.

[0163] FIG21B shows the changes in mouse body weight during administration of bispecific antibodies Bis01 and Bis04. DETAILED DESCRIPTION

[0164] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0165] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0166] Example 1. Design and Construction of Bispecific Antibodies

[0167] A bispecific antibody molecule targeting DLL3×CD3 was constructed using humanized anti-DLL3 and anti-CD3 antibody sequences. The bispecific antibody comprises three chains, the specific structures of which are shown in Figures 1A-1D. To reduce homologous mispairing, the antibody molecule adopts an asymmetric structure, with KIH mutations introduced into the Fc regions of the two heavy chains. Furthermore, to reduce the antibody's ADCC and CDC functions, and to avoid damage to T cells and potential toxic side effects, mutations L234A, L235A, and G237A were introduced into the heavy chain constant region. The constructed bispecific antibodies were named Bis01, Bis02, Bis03, and Bis04, respectively. The amino acid sequences of each chain are shown in Table 1 below, and the structural composition of each bispecific antibody is shown in Table 2. The sequence of the control antibody AMG757 is derived from the published patent CN108271376B, and the sequence of the control antibody BI764532 is derived from the published patent CN105143266B. The specific sequences are shown in Table 1.

[0168] Table 1 Bispecific antibody amino acid sequence list

[0169] Table 2 Structural composition of bispecific antibodies

[0170] Table 3 Variable region sequences of bispecific antibodies

[0171] Table 4 CDR analysis results of bispecific antibodies

[0172] Example 2. Design and expression of DLL3 antigen

[0173] The amino acid sequence encoding a truncated extracellular domain of human DLL3 protein (UniProt: Q9NYJ7) was cloned into a His-tagged pTT5 vector (Ubao Bio, VT2202). Plasmids were prepared using a plasmid extraction kit and transiently expressed in Expi 293F cells (Gibco, A14527). Cellular components were removed by centrifugation, and the culture supernatant was obtained. Proteins from the cell culture supernatant were purified using a Ni affinity chromatography column (purchased from GE Healthcare). After equilibration with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4), the clarified culture supernatant was loaded onto the Ni affinity column at a flow rate of 5 mL / min. After loading, the column was washed with equilibration buffer (3-5 times the bed volume of the Ni affinity column). A gradient elution was performed using 0-500 mM imidazole, and elution was monitored using a nucleic acid protein detector (A280 UV absorbance peak). The eluted protein was collected and dialyzed into PBS phosphate buffer at 4°C using a dialysis card (purchased from Thermo Scientific). Sterile filtration was performed using a 0.22 μm filter (purchased from Millipore) and stored aseptically to obtain the disclosed antigen and detection protein. The preparation method for the truncated extracellular region of cynomolgus macaque DLL3 protein is similar to that for human recombinant protein. The cynomolgus macaque DLL3 sequence is obtained from Uniprot No. A0A2K5WSR4. The specific sequence information of the recombinant protein is shown below:

[0174] Human DLL3-his (His-tagged human DLL3 protein extracellular domain fusion protein) SEQ ID NO. 146

[0175] Cyno DLL3-his (His-tagged monkey DLL3 protein extracellular domain fusion protein) SEQ ID NO. 147

[0176] Example 3. Expression and purification of bispecific antibodies

[0177] 3.1 Transfection of double antibody plasmid

[0178] The light and heavy chain nucleotide sequences encoding AMG757, BI764532, Bis01, Bis02, Bis03, and Bis04 were cloned into the pTT5 vector. Plasmids and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (Thermofisher, Catalog No. A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day of transfection, cell supernatants were collected.

[0179] 3.2 Expression and purification of bispecific antibodies

[0180] 3.2.1 Purification method of AMG757 and BI764532 control bispecific antibodies

[0181] After collecting the culture supernatant, the protein was purified using AKTA Pure Protein A affinity and molecular sieve purification. The resulting antibody was quantitatively and qualitatively analyzed by UV spectrophotometry, SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification method is as follows: Initial purification was performed using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The Protein A column was first equilibrated with 3–5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and the clarified culture supernatant was then loaded at a flow rate of 8 mL / min. After loading, the column was washed with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3–5 column volumes. Protein bound to the Protein A column was eluted with an eluent (20 mM citrate buffer, pH 3.5), and protein elution was monitored by the A280 UV absorbance peak. The eluted protein was collected, neutralized to pH 5-6 by adding 1M Tris-HCl, pH 8.0, and dialyzed into molecular sieve buffer (10mM Hac, 150mM NaCl, pH 5.5). Next, the protein was purified using molecular sieves (purchased from Boglund) and the target sample was collected. After concentration, the sample was dialyzed into 559 buffer (10mM Hac, 9% sucrose, pH 5.5), sterile filtered using a 0.22μm filter, and stored aseptically to obtain the purified antibody.

[0182] 3.2.2 Bis01 purification method

[0183] After collecting the culture supernatant, the protein was purified using AKTA Pure Protein A affinity chromatography and Capto MMC ImpRes composite medium. The resulting antibody was quantitatively and qualitatively analyzed by UV spectrophotometry, SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification method is as follows.

[0184] 1. Initial purification was performed using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The Protein A column was equilibrated with 3-5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading, the column was eluted with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3-5 column volumes. Protein bound to the Protein A column was eluted with an eluent (50 mM sodium acetate buffer, pH 3.5), and protein elution was monitored by the A280 UV absorbance peak. The eluted protein was collected and neutralized to pH 5-6 by adding 1 M Tris-HCl, pH 8.0.

[0185] 2. Purification was performed using Capto MMC ImpRes composite packing (purchased from Cytiva). The column was first equilibrated with 3–5 column volumes of equilibration buffer (Buffer A: 10 mM PB buffer, pH 6.0), and the pre-purified protein solution was loaded at a flow rate of 5 mL / min. After loading, the protein solution was washed with Buffer A for 3–5 column volumes, followed by a salt gradient elution: 20%–60% of Buffer B (Buffer A + 1 M NaCl, pH 6.0). Fractions were collected by monitoring the A280 UV absorbance peak, and the target fraction was identified by SDS-PAGE, SEC-HPLC, and CE-HPLC. Proteins meeting the required purity were pooled, concentrated, and dialyzed into 559 buffer (10 mM HaC, 9% sucrose, pH 5.5). The purified bispecific antibody was sterile filtered through a 0.22 μm filter and stored aseptically.

[0186] 3.2.3 Purification of BisO2 and BisO3

[0187] After collecting the culture supernatant, the protein was purified using AKTA Pure using Protein A affinity chromatography and Capto SPImpRes cationic medium. The resulting antibody was quantitatively and qualitatively analyzed by UV spectrophotometry, SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification method is as follows.

[0188] 1. Perform initial purification using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The purification method is similar to the Protein A column initial purification step in 3.2.2.

[0189] 2. Purify using Capto SPImpRes cationic column (purchased from Cytiva). Equilibrate with 3-5 column volumes of equilibration buffer (Buffer A: 50 mM sodium acetate, pH 5.5). Then, load the pre-purified protein solution at a flow rate of 5 mL / min. After loading, elute with 3-5 column volumes of Buffer A, followed by a salt gradient elution: 0%-40% Buffer B (Buffer A + 500 mM NaCl, pH 5.5). Fractions were collected by monitoring the A280 UV absorbance peak and identified by SDS-PAGE, SEC-HPLC, and CE-HPLC. Finally, proteins meeting the required purity were pooled, concentrated, and dialyzed into 559 buffer (10 mM HAc, 9% sucrose, pH 5.5). Sterile filtration was performed through a 0.22 μm filter and stored aseptically to obtain the purified bispecific antibody.

[0190] 3.2.4 Purification of Bis04

[0191] After collecting the culture supernatant, the protein was purified using AKTA Pure Protein A affinity, HiTrap KappaSelect affinity, and Capto SPImpRes cationic medium. The resulting antibody was quantitatively and qualitatively analyzed by UV spectrophotometry, SDS-PAGE, SEC-HPLC, and CE-SDS. Specific purification methods are as follows.

[0192] 1. Perform initial purification using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The purification method is similar to the Protein A column initial purification step in 3.2.2.

[0193] 2. Purify with HiTrap KappaSelect affinity (purchased from Cytiva). First, equilibrate with 3 to 5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and then load the pre-purified protein solution at a flow rate of 5 mL / min. After loading, elute with PBS for 3 to 5 column volumes, followed by elution with 10 mM phosphate buffer (pH 7.4) for 10 column volumes. Elute the protein bound to the KappaSelect column with eluent (50 mM glycine, pH 3.2), and monitor the protein elution by the A280 UV absorption peak. Collect the eluted protein and neutralize it to pH 5-6 by adding 1 M pH 8.0 Tris-HCl. After concentration, dialyze and exchange the buffer into 550 buffer (10 mM Hac, pH 5.5).

[0194] 3. Purify with Capto SPImpRes cationic ion (purchased from Cytiva). Purify the dialyzed protein according to the Capto SPImpRes cationic ion purification steps in 3.2.3 to obtain the purified bispecific antibody.

[0195] 3.3 Purity testing of bispecific antibodies

[0196] 3.3.1 SEC-HPLC analysis

[0197] The SEC-HPLC method was used to analyze the test samples, characterize the molecular size homogeneity of the bispecific antibodies, and determine the purity of the bispecific antibodies. The HPLC used in this method was Agilent 1260, the chromatographic column was TSKgel G3000SWXL from Tosoh Bioscience, the mobile phase was 200mM phosphate buffer, pH 7.0 / isopropanol (v / v 9:1) (Batch No.: 20220616101), the detection temperature was 25°C, the flow rate was 0.5mL / min, the detection wavelength was 280nm, the target protein was diluted 10 times with DI water, the sample load was 50μg, and the analysis time was 40 minutes. For the SEC-HPLC data, the chromatogram was analyzed by manual integration, and the protein purity was calculated according to the area normalization method. The main peak was considered to be a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragment. The purity information of the obtained bispecific antibodies is shown in the following table.

[0198] Table 5 Purity of bispecific antibodies detected by SEC-HPLC

[0199] 3.3.2 CE-SDS analysis

[0200] The non-reducing CE-SDS method was used to analyze the test samples, determine the purity of the bispecific antibody, and characterize the size homogeneity of the test samples. The capillary electrophoresis instrument used in this method was an AB Sciex PA 800Plus, the detector was a PDA, the detection wavelength was 220 nm, the capillary detection effective length was 20 cm, the protein separation voltage was 15.0 kV, and the NR-CE SDS detection time was 40 min. The test sample amount was 100 μg. For the NR-CE-SDS method, SDS Sample Buffer (20 mM PB, 5 mM citric acid, 1% SDS, pH 6.5) was added to the protein sample and dissolved to 94 μL. 5 μL of 100 mmol / L NEM and 1 μL of 10 KD marker were added, and the sample was incubated at 70°C for 10 min. The NR-CE-SDS test sample was used. The chromatograms of the NR-CE SDS data were analyzed by manual integration, and the protein purity was calculated by area normalization. The purity information of the obtained bispecific antibodies is shown in the following table.

[0201] Table 6 Purity of bispecific antibodies detected by NR-CE-SDS

[0202] Example 4. Binding activity determination of bispecific antibodies

[0203] 4.1 ELISA detection of binding of bispecific antibodies to human and monkey DLL3 proteins

[0204] Human DLL3-his protein was diluted with PBS to a final concentration of 2 μg / mL, and then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking solution [PBS + 2% (w / w) BSA] at room temperature for 2 hours. The blocking solution was discarded, and 10-fold serial dilutions of bispecific antibody starting at 100 nM, as well as 50 μl of positive and negative control antibodies were added to each well. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Merck, catalog number: AP113P) was added, incubated at 37°C for 1 hour, and then washed 5 times with PBST. TMB substrate 50 μl was added to each well, incubated at room temperature for 10 minutes, and then stop solution (1.0 M HCl) 50 μl was added to each well. The OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). 450nmFigures 2A and 2B show the binding activity of the bispecific antibodies to human DLL3. The negative control, anti-FITC×CD3, showed no binding to human DLL3, while the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all effectively bound to human DLL3-his, with binding abilities comparable to those of the positive control molecule, AMG757.

[0205] Monkey DLL3-his protein was assayed and analyzed by ELISA according to the method described in 4.1 above. The results are shown in Figures 2C and 2D , indicating that the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all showed strong binding activity to monkey DLL3 protein.

[0206] 4.2 ELISA detection of binding of bispecific antibodies to human and monkey CD3e proteins

[0207] Human CD3e-His protein (Sino Biological, CAT#10977-H08H) was diluted in PBS to a final concentration of 2 μg / mL. 50 μl was then added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking buffer (PBS + 2% (w / w) BSA) for 2 hours at room temperature. The blocking buffer was discarded, and 50 μl of bispecific antibody starting at 100 nM and serially diluted 10-fold, as well as positive and negative control antibodies, were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-conjugated secondary antibody (Merck, Cat. No. AP113P) was added and incubated at 37°C for 1 hour. The plate was then washed five times with PBST. 50 μl of TMB substrate was added to each well and incubated at room temperature for 10 minutes. Then, 50 μl of stop buffer (1.0 M HCl) was added to each well. OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) 450nm Figures 3A and 3B show the binding activity of bispecific antibodies to human CD3e protein. The results show that the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all effectively bound to the human CD3e-his protein. Bis01, Bis02, and Bis03 had slightly weaker binding to human CD3e protein than AMG757 (Figure 3A), while Bis04 had comparable binding to the positive control antibody AMG757 (Figure 3B).

[0208] ELISA assays and data analysis were performed using monkey CD3e-his protein (ACRO, CAT#CDE-C5226) according to the method described in 4.2 above. As shown in Figures 3C and 3D , the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all showed binding activity to the monkey CD3e-his protein. The binding abilities of Bis01, Bis02, and Bis03 to monkey CD3e protein were slightly weaker than those of AMG757 (Figure 3C). The binding ability of Bis04 to monkey CD3e protein was comparable to that of the positive control antibody AMG757 (Figure 3D).

[0209] 4.3 Flow cytometry (FACS) detection of the binding of bispecific antibodies to human DLL3

[0210] SHP77 is a human small cell lung cancer cell line that highly expresses DLL3 protein on its cell surface. The DLL3 expression level detected by FACS is shown in Figure 4A. SHP77 cells were cultured in a T-175 culture flask until the logarithmic growth phase. The culture medium was aspirated and the cells were washed twice with PBS buffer. The cells were trypsinized and then digested with complete culture medium. The cells were pipetted to a single cell suspension. After cell counting, the cells were centrifuged and the cell pellet was resuspended in FACS buffer (PBS + 2% fetal bovine serum) to a concentration of 2×10 6 For every milliliter of cells, 100 μl was added to each well of a 96-well FACS reaction plate, centrifuged, the supernatant was discarded, 50 μl of the antibody sample to be tested (100 nM as the starting concentration, 5-fold serial dilution) was added to each well, mixed with the cells, and incubated at 4°C for 1 hour. Washed three times by centrifugation with PBS buffer, 50 μl of Alexa Fluor® was added to each well. Incubate with 647 AffiniPure Goat Anti-Human IgG, Fcγ fragment-specific secondary antibody (purchased from Jackson, Cat. No. 109-605-098) at 4°C for 1 hour. Wash cells three times with PBS buffer by centrifugation, resuspend in 100 μl of PBS, and analyze using FACS (FACS Canto™, purchased from BD Biosciences). Data were analyzed using FlowJo software to obtain the mean fluorescence intensity (MFI). GraphPad Prism 8 software was then used for data fitting and EC50 calculation. As shown in Figure 5A, the bispecific antibodies Bis01, Bis02, and Bis03 all specifically bound to SHP77 cells, with superior binding to the control antibodies AMG757 and BI764532. Bis04 also exhibited strong binding activity to SHP77 tumor cells, surpassing the control antibody AMG757 (Figure 5B).

[0211] The same method was used to test the binding of bispecific antibodies to NCI-H2171 small cell lung cancer cells, which have low DLL3 expression (DLL3 expression levels are shown in Figure 4A). Figure 5C shows that bispecific antibodies Bis01, Bis02, and Bis03 all specifically bind to NCI-H2171 cells, with stronger binding than AMG757. Figure 5D shows that Bis04 binds to NCI-H2171 cells more strongly than the control AMG757.

[0212] 4.4 Flow cytometry (FACS) detection of the binding of bispecific antibodies to negative cells A549

[0213] Tumor A549 cells (Nanjing Kebai, CBP60084, DLL3 expression levels are shown in Figure 4A) were expanded in T-175 culture flasks to the logarithmic growth phase. The culture medium was removed, and the cells were washed twice with PBS buffer. The cells were trypsinized, then digested with complete culture medium, and the cells were pipetted to a single-cell suspension. The binding of the bispecific antibodies to negative tumor cells was detected and analyzed using the method described in Section 4.3 above. As shown in Figures 6A and 6B, the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 showed no binding activity to negative A549 cells, indicating that the bispecific antibodies disclosed herein do not bind nonspecifically.

[0214] 4.5 Flow cytometry (FACS) detection of the binding of bispecific antibodies to DLL3-overexpressing cells

[0215] The nucleotide sequence encoding the human DLL3 fragment was cloned into the pcDNA5 vector, and plasmids were prepared to construct overexpression cell lines. Monoclonal cell lines with high fluorescence intensity were selected for subsequent testing. The constructed overexpression cell line was named CHOK1-hDLL3. FACS expression levels are shown in Figure 4B.

[0216] CHOK1-hDLL3 cells were subjected to FACS analysis and data analysis according to the method described in 4.4 above. Antibody samples were diluted 5-fold starting at 100 nM. As shown in Figures 7A and 7B, the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 effectively bound to CHOK1-hDLL3 cells, with binding abilities significantly stronger than the control AMG757.

[0217] Recombinant cells overexpressing monkey DLL3 (CHOK1-cynoDLL3, Figure 4B) were constructed using the same method as above, and FACS analysis and data analysis were performed using the same detection methods as above. As shown in Figures 7C and 7D, the bispecific antibodies Bis01, Bis02, Bis03, and Bis04 effectively bound to CHOK1-cynoDLL3 cells, with binding abilities significantly stronger than the control AMG757.

[0218] 4.6 Flow cytometry (FACS) detection of the binding of bispecific antibodies to Jurkat cells

[0219] To test the binding ability of bispecific antibodies to cell surface CD3, we used FACS to perform binding assays on Jurkat cells (highly expressing CD3, Nanjing Kebai, CBP60520). Jurkat cells were cultured and harvested. Antibody samples were diluted 5-fold starting at 100 nM and subjected to FACS analysis and data analysis according to the method described in 4.3 above. The test results, shown in Figures 8A and 8B, show that bispecific antibodies Bis01, Bis02, Bis03, and Bis04 effectively bind to Jurkat cells, though with weaker binding than AMG757. This weak binding to CD3 may help mitigate drug side effects.

[0220] 4.7 Flow cytometry (FACS) assay for binding of bispecific antibodies to human and monkey PBMCs

[0221] Peripheral blood mononuclear cells (PBMCs) contain naturally unactivated CD3+ T lymphocytes. We used PBMCs to represent CD3+ T cells to evaluate the binding ability of bispecific antibodies to natural T lymphocytes. After overnight culture, human PBMCs (SailyBio, CAT#XFB-HP010B) and monkey PBMCs (Hekai Bio, CAT#5208) were collected and Fc Block (BD, 564220) was added. The antibody samples to be tested were diluted 5-fold with a starting concentration of 100nM and FACS detection and data analysis were performed according to the method in 4.3 above. The results are shown in Figures 9A-9B. The bispecific antibodies Bis01, Bis02, Bis03 and Bis04 can effectively bind to human PBMC cells, but the binding ability is weaker than AMG757. The weaker CD3 binding ability may help to reduce the cytokine storm of the drug. The same detection method was used to analyze the binding activity of bispecific antibodies to monkey PBMCs. The results are shown in Figures 9C-9D. The bispecific antibodies Bis01, Bis02, Bis03 and Bis04 had weaker binding abilities to monkey PBMC cells than AMG757.

[0222] 4.8 Luciferase reporter gene assay to detect the effect of bispecific antibodies on T cell activity

[0223] NCI-H2171 (DLL3 positive cells) and Jurkat-luc cells (using conventional methods in the art, based on Jurkat cells, overexpressing the luciferase gene) were cultured and collected, and resuspended to 5×10 4 / 25μL. The antibody sample to be tested was diluted 4-fold with a starting concentration of 30nM. At the same time, 25μL of NCI-H2171 and 25μL of Jurkat-luc cells were mixed with 50μL of antibody diluent and added to a 96-well white plate with a transparent bottom microplate (corning, CAT#3610). After incubation at 37°C for 5 hours, 50μL of Nano-Glo Luciferase reagent (Promega, CAT#N1130) was added. After incubation at room temperature at 450rpm for 10 minutes, the results were read using an Envision microplate reader (Perkin Elmer, Envision2105). As shown in Figure 10A, the bispecific antibodies Bis01, Bis02, and Bis03 can all effectively activate Jurkat cells. The same method was used to detect the activation of Bis04 after co-incubation. The results are shown in Figure 10B. The antibodies can also activate Jurkat cells, and the activation performance is significantly better than the positive reference AMG757. The same method was used to examine activation of A549 cells (DLL3-negative) after co-incubation with Jurkat-luc. The results, shown in Figures 10C-10D, show that none of the antibodies activated Jurkat cells. This suggests that T cell activation by the DLL3 / CD3 bispecific antibody is DLL3-specific.

[0224] Example 5. Affinity detection of bispecific antibodies

[0225] The binding strength of antibodies to antigens was determined using the anti-human antibody capture method using a BIAcore 8K instrument. First, anti-human IgG antibodies were immobilized on a CM5 chip (Cytiva; 29-1496-03) using the amino coupling method according to the instructions of the Human Antibody Capture Kit (Cytiva; 29-2346-00). HBS-EP+ pH 7.4 (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cytiva; BR-1006-69) was used as the mobile phase. After mixing NHS and EDC, the chip was activated for approximately 600 seconds. Anti-human IgG antibodies were diluted to 15 μg / mL in 10 mM sodium acetate, pH 5.0, and injected for 420 seconds. Finally, the remaining active sites were blocked with ethanolamine. Next, a multi-cycle kinetic method was used to determine the affinity of the antibody and antigen. In each cycle, the test antibody was first captured, followed by the injection of a single concentration of the antigen protein. The binding and dissociation processes between the antibody and antigen protein were recorded, and the chip was regenerated with 3M MgCl2. The mobile phase was HBS-EP (pH 7.4), with a flow rate of 30 μL / min, a regeneration time of 30 seconds, and a detection temperature of 25°C. Finally, the data were analyzed according to a 1:1 binding model, and the antibody-antigen binding kinetic parameters, including the association rate constant ka, the dissociation rate constant kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax, were fitted. The association rates (ka), dissociation rates (Kd), and binding affinities (KD) of the bispecific antibodies Bis01, Bis02, Bis03, Bis04, and AMG757 with the DLL3 protein were measured. The results are shown in Table 7.

[0226] Table 7 Affinity of bispecific antibodies to DLL3 protein detected by SPR (Biacore)

[0227] Example 6: Bispecific Antibody-Mediated Tumor Cell Killing in Vitro

[0228] The bispecific antibody-mediated PBMC killing of tumor cells was performed by quantitatively measuring the number of target tumor cells. Target tumor cells were labeled by stably overexpressing luciferase (using conventional methods in the art to overexpress the luciferase gene in target tumor cells). The number of cells was proportional to the luciferase expression level. The Bright-Glo Luciferase Assay System was used to measure the luciferase content in the cells and quantify the number of tumor cells. Target tumor cells included DLL3-positive tumor cell lines (SHP77 and NCI-H2171; DLL3 expression levels are shown in Figure 4A) and a DLL3-negative control cell line, A549.

[0229] 1.0E4 tumor cells per well and 1.0E5 PBMCs (Allcells, FPB004F-C) per well were added to a 96-well plate in RPMI Medium 1640 supplemented with 10% FBS. Control and test antibodies were diluted to the target concentration in RPMI Medium 1640 and added to each well of the 96-well plate containing tumor cells and PBMCs, respectively. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. The final antibody concentration in the drug incubation system started at 100 nM, and the control and test antibodies were serially diluted using appropriate dilutions. Finally, the number of tumor cells in each well was quantified using the Bright-Glo Luciferase Assay System (Promega, E2620) to quantify luciferase in viable tumor cells, reflecting antibody-mediated tumor cell killing by PBMCs. Tumor cell killing rate = (100 * (wells without antibody - wells treated with antibody) / (wells without antibody - wells treated with PBMCs only))%. The results of cell killing on SHP77 cells are shown in Figures 11A-11C. Bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all showed significant cytotoxicity against DLL3-positive SHP77 tumor cells, and outperformed the positive reference AMG757. The results of cell killing on NCI-H2171 cells are shown in Figures 12A-12C. Bispecific antibodies Bis01, Bis02, Bis03, and Bis04 all showed significant cytotoxicity against DLL3-positive H2171 tumor cells, and outperformed the positive reference AMG757. None of the bispecific antibodies showed cytotoxicity against DLL3-negative A549 cells (Figure 13). Specific cytotoxicity data for each tumor cell type are shown in Table 8 below.

[0230] Table 8 PBMC-mediated in vitro tumor cell killing EC50 (pM)

[0231] Example 7: Bispecific Antibody-Mediated Cytokine Secretion

[0232] T cells are activated under the mediation of bispecific antibodies and release cytokines while killing target cells. The level of cytokine release reflects the activity of the bispecific antibody, which is generally positively correlated with the activation level of T cells, as well as the in vitro killing activity and in vivo tumor suppression activity. The cell supernatant in Example 6 was collected, centrifuged at 3000rpm for 10 minutes, and frozen in a -80 degree refrigerator for future use. The secretion levels of IFNγ (BD, 555142), TNFα (BD, 555212) and IL-6 (BD, 555220) were detected by ELISA. The operating steps are detailed in the instructions in the kit. The results of IFNγ secretion are shown in Figures 14A-14C. The bispecific antibodies Bis01, Bis02, Bis03 and Bis04 can effectively induce PBMC to secrete IFNγ when PBMC and DLL3-positive tumor cells coexist, and the overall secretion level is better than that of the positive control AMG757. The secretion levels of TNFα and IL6 are shown in Figures 15A-16C. The bispecific antibodies Bis01, Bis02, Bis03 and Bis04 can effectively induce PBMC to secrete TNFα and IL-6 when PBMC and DLL3-positive tumor cells coexist. The overall secretion level is comparable to that of the positive control AMG757.

[0233] Example 8: PBMC-reconstructed mouse efficacy model - SHP77

[0234] The anti-tumor efficacy of bispecific antibodies in vivo was evaluated using a human PBMC-reconstructed NPG mouse model (NPG: female 5-6 weeks, Beijing Weitongda Biotechnology Co., Ltd.). SHP77 cells were cultured to the logarithmic growth phase, collected by centrifugation, and plated at 10×10 6 Inoculate mice subcutaneously, and resuscitate human PBMC cells three days in advance and culture at a rate of 5×10 6 Each mouse was intravenously inoculated. The mice were then raised normally. When the tumor volume of the tumor-bearing mice reached 100 mm 3 Around 14:00, mice were randomly divided into groups of 8 per group, and the reconstruction rate was measured at the same time. The day of grouping was defined as day 0 of the experiment. Subsequently, intravenous administration was performed twice a week for a total of 4 times. Tumors were observed and measured twice a week, and tumor volume was calculated according to the following formula: Tumor volume (TV) = 1 / 2 (height * width) 2 ). Width is the smaller of the two measurements and height is defined as the larger of the two measurements. Throughout the dosing cycle, mouse body weights were recorded twice weekly, and changes in mouse body weight were calculated. The results are shown in Figure 17A. One week after dosing, the bispecific antibodies Bis01, Bis02, and Bis03 were all able to effectively inhibit tumor growth, and the tumor inhibition rate in the low-dose group was superior to that of the control antibody AMG757. There was no significant change in mouse body weight during the dosing cycle (Figure 17B).

[0235] Table 9 Effects of the test substances on tumor volume in SHP77 cell transplanted human PBMC mice * Calculate the dose and administer according to equimolar concentration.

[0236] The same model was used to verify the effect of Bis04 on the growth of mouse tumor tissue. The results are shown in Figure 18A. One week after administration, the bispecific antibody was able to effectively inhibit tumor production. The tumor inhibition rates of the high-dose and low-dose groups were comparable to those of the control antibody AMG757. There was no downward trend in the weight of the mice during the entire dosing period (Figure 18B).

[0237] Table 10 Effects of Bis04 on tumor volume in SHP77 cell-transplanted human PBMC mice * Calculate the dosage and administer the drug according to the equal mass concentration.

[0238] Example 9: PBMC-reconstructed mouse efficacy model - H2171

[0239] The anti-tumor efficacy of bispecific antibodies in vivo was evaluated using a human PBMC-reconstructed NPG mouse model (NPG: female 5-6 weeks, Beijing Weitongda Biotechnology Co., Ltd.). NCI-H2171 cells were cultured to the logarithmic growth phase, collected by centrifugation, and plated at 10×10 6 Inoculate mice subcutaneously, and resuscitate human PBMC cells three days in advance and culture at a rate of 5×10 6 After that, the mice were raised normally. When the tumor volume of the tumor-bearing mice reached 100-200mm 3 Around 14:00, mice were randomly divided into groups of 7 per group, and the reconstruction rate was tested simultaneously. The day of grouping was defined as day 0 of the experiment. Subsequently, intravenous administration was performed twice a week for a total of 4 times. Tumors were observed and measured twice a week, and tumor volume was calculated according to the following formula: Tumor volume (TV) = 1 / 2 (height * width) 2 ). The width is the smaller of the two measurements and the height is defined as the larger of the measurements. During the entire dosing cycle, the weight of the mice was recorded twice a week, and the change in mouse weight was calculated. The results are shown in Figure 19A. One week after dosing, the bispecific antibodies Bis01, Bis02, and Bis03 were all able to effectively inhibit tumor growth, and the tumor inhibition rates of the high-dose and low-dose groups were better than those of the control antibody AMG757. There was no significant change in the weight of the mice during the dosing cycle (Figure 19B).

[0240] Table 11 Effects of the test substances on tumor volume in mice transplanted with human PBMCs using NCI-H2171 cells *Bis01-Bis03 molecules were dosed and administered at equimolar concentrations.

[0241] The same model was used to verify the effect of Bis04 on the growth of mouse tumor tissue. The results are shown in Figure 20A. One week after administration, the bispecific antibody was able to effectively inhibit tumor production. The tumor inhibition rates of the high-dose and medium-dose groups were better than those of the control antibody AMG757. There was no downward trend in the weight of the mice during the entire administration period (Figure 20B).

[0242] Table 12 Effects of Bis04 on tumor volume in mice transplanted with human PBMCs using NCI-H2171 cells *Bis04 molecules are dosed and administered according to the same mass concentration.

[0243] Example 10: PBMC-reconstructed mouse pharmacodynamic model QGP-1

[0244] The anti-tumor efficacy of bispecific antibodies in vivo was evaluated using a human PBMC-reconstructed NPG mouse model (NPG: female 5-6 weeks, Beijing Weitongda Biotechnology Co., Ltd.). The day of drug treatment was designated as day 0 of the experiment. On day -11 of the experiment, human pancreatic cancer cells QGP1 (derived from the JCRB cell bank in Japan) were harvested and inoculated, cultured to the desired number using 1640 complete medium (RPMI-1640 medium + 1% P / S + 10% FBS) and in the logarithmic growth phase (confluence of approximately 80%). The cell density was adjusted to 200×10 6 Before inoculation, the cell suspension was mixed with Matrigel in equal proportions and 100 μL of the cell mixture was inoculated subcutaneously in the right axilla of each mouse. On day -10 of the experiment, the required number of human peripheral blood mononuclear cells (PBMC, P122090210C, purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.) was revived using 1640 medium containing 2% FBS (RPMI-1640 medium + 2% FBS) and the cell density was adjusted to 25×10 using serum-free medium. 6 200 μL of the cell suspension was inoculated into the tail vein of each mouse to establish a PBMC humanized model. After inoculation of human pancreatic cancer cells QGP1, tumor growth was monitored. When the average tumor volume was 150 mm 3 When the hCD45+ / total CD45+ ratio in peripheral blood was higher than 0.5%, mice were randomly divided into groups based on tumor volume and hCD45+ / total CD45+ cell ratio, with 7 mice per group. The corresponding drug was administered via the tail vein (Day 0) according to the protocol, twice a week for a total of 4 doses. Tumor volume and mouse body weight changes were also measured.

[0245] The results, shown in Table 13 and Figure 21, show that on Day 17 after dosing, all candidate molecule-administered groups demonstrated excellent efficacy and dose-response. The 0.2 mg / kg Bis01 group exhibited superior tumor inhibition, with a tumor inhibition rate of 107.26% (p<0.0001), and five mice experienced complete remission, a number of complete responses (CRs) exceeding that of the AMG757 equivalent. The 0.2 mg / kg Bis04 group also demonstrated superior efficacy compared to the positive reference (AMG757 equivalent), with two mice experiencing complete remission, a number of complete responses (CRs) exceeding that of the AMG757 equivalent. There were no significant changes in mouse body weight during the dosing period, indicating that the tumor-bearing mice tolerated the test drug well at this experimental dose.

[0246] Table 13 Effects of the test substances on tumor volume in QGP1 cell-transplanted human PBMC mice

Claims

1. A multispecific binding molecule, characterized in that, The multispecific binding molecule comprises: (A) an antigen-binding portion that specifically binds to DLL3; (B) an antigen-binding portion that specifically binds to CD3; wherein the antigen-binding portion that specifically binds to DLL3 comprises a first antigen-binding portion and a second antigen-binding portion that bind to different epitopes of DLL3; the antigen-binding portion that specifically binds to CD3 is a Fab that binds to CD3.

2. The multispecific binding molecule according to claim 1, wherein The antigen-binding portions are each independently selected from an antibody, an antibody fragment, F(ab’)2, Fab’, Fab, Fv, scFv, or a nanobody (VHH).

3. The multispecific binding molecule according to claim 1 or 2, characterized in that, The multispecific binding molecule comprises three polypeptide chains: a first heavy chain, a second heavy chain, and a light chain, wherein: (1) The first heavy chain and the second heavy chain are different and have the structures shown below: (a) The first heavy chain VL1 DLL3 -L1-VH1 DLL3 -L1-VH1 CD3 -CH1-Fc1 and the second heavy chain VL2 DLL3 -L1-VH2 DLL3 -L2-Fc2; (b) The first heavy chain VH1 CD3 -CH1-Fc1 and the second heavy chain VL3 DLL3 -L1-VH3 DLL3 -L1-VHH1 DLL3 -L2-Fc2; (c) First heavy chain VL1 DLL3 -L1-VH1 DLL3 -L1-VH1 CD3 -CH1-Fc1 and second heavy chain VHH1 DLL3 -L2-Fc2; (d) The first heavy chain VHH2 DLL3 -L1-VH2 CD3 -CH1-Fc1 and the second heavy chain VHH3 DLL3 -L2-Fc2; and, (2) The light chain contains the following structure: VL1 CD3 -CL, or VL2 CD3 -CL; Among them, VH1 DLL3 、VH2 DLL3 and VH3 DLL3 are different heavy chain variable regions specifically binding to DLL3, VL1 DLL3 、VL2 DLL3 and VL3 DLL3 are different light chain variable regions specifically binding to DLL3, which respectively form different single-chain variable fragments (scFvs) specifically binding to DLL3; VHH1 DLL3 、VHH2 DLL3 and VHH3 DLL3 are different nanobodies specifically binding to DLL3; VH1 CD3 and VH2 CD3 are different heavy chain variable regions specifically binding to CD3, VL1 CD3 and VL2 CD3 are different light chain variable regions specifically binding to CD3, which respectively form different antigen-binding portions specifically binding to CD3; Fc1 and Fc2 are the Fc regions of any antibody, and L1 and L2 are respectively the same or different linkers.

4. The multispecific binding molecule according to any one of claims 1 - 3, characterized in that, The Fc1 and Fc2 of the multispecific binding molecule are different, Fc1 is a knob-Fc, and Fc2 is a hole-Fc; preferably, the knob-Fc comprises a T366W mutation, and / or the hole-Fc comprises T366S, L368A, and / or Y407V mutations; preferably, Fc1 and Fc2 are Fc regions of IgG1 or IgG4.

5. The multispecific binding molecule according to any one of claims 1-3, characterized in that, The Fc1 and Fc2 of the multispecific binding molecule are different and have amino acid mutations that alter effector functions; preferably, the mutations that alter effector functions comprise L234A, L235A, and / or G237A mutations; preferably, Fc1 and Fc2 are Fc regions of IgG1 or IgG4.

6. The multispecific binding molecule according to any one of claims 1-5, characterized in that, The scFv that specifically binds to DLL3 comprises heavy chain CDRs and / or light chain CDRs selected from the following, or comprises heavy chain CDRs and / or light chain CDRs having 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to the following sequences; preferably having conservative amino acid substitutions: The heavy chain CDR1 sequence is as shown in any one of SEQ ID NO.29, 32, 35, 68, 71, 74, 107, 110, 113; The heavy chain CDR2 sequence is as shown in any one of SEQ ID NO.30, 33, 36, 69, 72, 75, 108, 111, 114; The heavy chain CDR3 sequence is as shown in any one of SEQ ID NO.31, 34, 37, 70, 73, 76, 109, 112, 115; The light chain CDR1 sequence is as shown in any one of SEQ ID NO.38, 41, 44, 77, 80, 83, 116, 119, 122; The light chain CDR2 sequence is as shown in any one of SEQ ID NO.39, 42, 45, 78, 81, 84, 117, 120, 123; The light chain CDR3 sequence is as shown in any one of SEQ ID NO.40, 43, 46, 79, 82, 85, 118, 121, 124; The nanobody specifically binding to DLL3 comprises heavy-chain CDRs selected from the following, or heavy-chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared with the following sequences; preferably having conservative amino acid substitutions: The heavy-chain CDR1 sequence is as shown in any one of SEQ ID NO.47, 50, 53, 86, 89, 92, 125, 128, 131; The heavy-chain CDR2 sequence is as shown in any one of SEQ ID NO.48, 51, 54, 87, 90, 93, 126, 129, 132; The heavy-chain CDR3 sequence is as shown in any one of SEQ ID NO.49, 52, 55, 88, 91, 94, 127, 130, 133.

7. The multispecific binding molecule according to claim 6, wherein The heavy-chain variable region specifically binding to DLL3 comprises the amino acid sequence as shown in SEQ ID NO.16, 18, 20 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared with any one of SEQ ID NO.16, 18, 20; and / or the light-chain variable region specifically binding to DLL3 comprises the amino acid sequence as shown in SEQ ID NO.17, 19, 21 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared with any one of SEQ ID NO.17, 19, 21; The nanobody specifically binding to DLL3 comprises the amino acid sequence as shown in SEQ ID NO.22, 23, 24 or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared with any one of SEQ ID NO.22, 23, 24.

8. The multispecific binding molecule according to any one of claims 1-5, characterized in that, The antigen-binding portion specifically binding to CD3 comprises heavy-chain CDRs and / or light-chain CDRs selected from the following, or heavy-chain CDRs and / or light-chain CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared with the following sequences; preferably having conservative amino acid substitutions: The heavy-chain CDR1 sequence is as shown in any one of SEQ ID NO.56, 59, 95, 98, 134, 137; The heavy-chain CDR2 sequence is as shown in any one of SEQ ID NO.57, 60, 96, 99, 135, 138; The heavy-chain CDR3 sequence is as shown in any one of SEQ ID NO.58, 61, 97, 100, 136, 139; The light-chain CDR1 sequence is as shown in any one of SEQ ID NO.62, 65, 101, 104, 140, 143; The light-chain CDR2 sequence is as shown in any one of SEQ ID NO.63, 66, 102, 105, 141, 144; The light-chain CDR3 sequence is as shown in any one of SEQ ID NO.64, 67, 103, 106, 142, 145.

9. The multispecific binding molecule according to claim 8, characterized in that, The heavy chain variable region specifically binding to CD3 comprises the amino acid sequences shown in SEQ ID NO. 25 and 27, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to any one of SEQ ID NO. 25 and 27; and / or the light chain variable region specifically binding to CD3 comprises the amino acid sequences shown in SEQ ID NO. 26 and 28, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to any one of SEQ ID NO. 26 and 28.

10. The multispecific binding molecule according to any one of claims 3-9, characterized in that, The first heavy chain comprises the amino acid sequences shown in SEQ ID NO. 1, 4, and 7, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to any one of SEQ ID NO. 1, 4, and 7; The second heavy chain comprises the amino acid sequences shown in SEQ ID NO. 3, 5, 6, and 9, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to any one of SEQ ID NO. 3, 5, 6, and 9; The light chain comprises the amino acid sequences shown in SEQ ID NO. 2 and 8, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared to any one of SEQ ID NO. 2 and 8.

10. The multispecific binding molecule according to any one of claims 1-9, wherein the antigen-binding portion is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or a fragment thereof.

11. The multispecific binding molecule according to any one of claims 1 to 10, characterized in that, The multispecific binding molecule is trivalent, tetravalent, pentavalent or hexavalent; preferably, the multispecific binding molecule is trivalent.

12. The multispecific binding molecule according to any one of claims 1 to 11, characterized in that, The multispecific binding molecule is further conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from drugs, toxins, radioisotopes, chemotherapeutic drugs or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.

13. An isolated nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the multispecific binding molecule according to any one of claims 1-12.

14. A vector, characterized in that, The vector comprises the nucleic acid fragment according to claim 13.

15. A host cell, characterized in that, The host cell comprises the vector according to claim 14; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (E. coli), a fungus (yeast), an insect cell or a mammalian cell (CHO cell line or 293T cell line).

16. A method for preparing a multispecific binding molecule according to any one of claims 1-12, characterized in that, The method comprises culturing the cell according to claim 15, and isolating the multispecific binding molecule expressed by the cell.

17. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the multispecific binding molecule according to any one of claims 1-12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14; or the multispecific binding molecule obtained by the method according to claim 16; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is packaged together with or separately from the anti-tumor agent.

19. Use of the multispecific binding molecule according to any one of claims 1 to 12, the isolated nucleic acid fragment according to claim 13, the vector according to claim 14, the host cell according to claim 15, the multispecific binding molecule prepared by the method according to claim 16, or the pharmaceutical composition according to claim 17 or 18, in the preparation of and / or for the prophylaxis of a medicament for cancer; Preferably, the cancer is selected from lung cancer, neuroendocrine cancer, melanoma, glioblastoma multiforme, small cell bladder cancer, pancreatic cancer, and other solid tumors expressing DLL3.

20. The use according to claim 19, characterized in that, The medicament is used in combination with another therapeutic agent or in combination with surgery; wherein the another therapeutic agent or the surgery is selected from radiotherapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, surgery, immune-stimulating antibodies, immunomodulatory drugs, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cellular immunotherapy.

21. A method for treating and / or preventing cancer, characterized in that, The method comprises administering to a subject a therapeutically effective amount of the multispecific binding molecule according to any one of claims 1 to 12, the isolated nucleic acid fragment according to claim 13, the vector according to claim 14, the host cell according to claim 15, the multispecific binding molecule prepared by the method according to claim 16, or the pharmaceutical composition according to claim 17 or 18; Preferably, the cancer is selected from lung cancer, neuroendocrine cancer, melanoma, glioblastoma multiforme, small cell bladder cancer, pancreatic cancer, and other solid tumors expressing DLL3.

22. The method according to claim 21, characterized in that The method further comprises administering to a patient in need thereof another therapeutic agent or surgical treatment; wherein the another therapeutic agent or the surgery is selected from radiotherapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, surgery, immune-stimulating antibodies, immunomodulatory drugs, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cellular immunotherapy.

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