Bispecific antibodies targeting CD79b and CD3 and uses thereof

Abstract

The present invention relates to the field of biomedicine, and in particular, to a bispecific antibody comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to a second antigen, wherein the first antigen is CD79b and the second antigen is not CD79b. The present invention also relates to nucleic acid molecules, vectors and host cells encoding the bispecific antibody, derivatives of the bispecific antibody, and their use in treating diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to a bispecific antibody comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to a second antigen, wherein the first antigen is CD79b and the second antigen is not CD79b. The present invention also relates to nucleic acid molecules, vectors and host cells encoding the bispecific antibody, derivatives of the bispecific antibody, and their use in treating diseases. Technical Background

[0002] CD79 is a heterodimeric molecule composed of CD79a and CD79b, which is almost exclusively expressed on B cells and B cell tumors. As the signaling component of the B cell antigen receptor (BCR), CD79, together with the cell surface immunoglobulin (sIg) for antigen recognition, constitutes the BCR complex and plays a key role in B cell maturation and activation. Both CD79a and CD79b contain a single extracellular Ig domain, a transmembrane domain, and an intracellular signal transduction domain, which initiate BCR signaling after antigen binding, ultimately leading to B cell activation, antigen presentation, cytokine production, and cell proliferation and differentiation. During B cell ontogeny, the expression of CD79a and CD79b precedes immunoglobulin (Ig) heavy chain gene rearrangement and CD20 expression, and disappears later than CD20 in the late (plasma cell) stage of B cell differentiation. Therefore, antibodies targeting CD79a and CD79b can be used to differentiate B-cell neoplasms from T-cell neoplasms or myeloid neoplasms, or L&H lymphocyte-predominant Hodgkin lymphoma from classical Hodgkin lymphoma. In addition, anti-CD79a and anti-CD79b antibodies are effective markers for the diagnosis of precursor B-acute lymphoblastic leukemia (pre-B-ALL) because these tumors are negative for other B-cell markers such as CD20 and CD45RA.

[0003] After antigen binding to CD79, it is rapidly induced to be internalized and delivered to the major histocompatibility complex class II (MHCII) compartment (i.e., the lysosomal compartment, for class II antigen presentation by B cells). Because the drug is directly delivered to the target cell into the lysosomal compartment, the cytotoxic activity is enhanced, and it allows the use of more stable linkers that are cleaved in the MHCII compartment. This unique intracellular trafficking makes CD79 a potential target for targeted delivery of cytotoxic agents. In addition, CD79 is also considered a therapeutic target for antibodies because it is physiologically specifically expressed in mature B cells and the vast majority of B-cell non-Hodgkin's lymphomas (B-NHLs), including diffuse large B-cell lymphoma (DLBCL) (90-100%), acute B-lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy cell leukemia (HCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL).

[0004] The CD3 receptor complex is a protein complex consisting of four chains. In mammals, the complex contains one CD3y (γ) chain, one CD35 (δ) chain, and two CD3e (ε) chains. These chains bind to the T cell receptor (TCR) and the so-called ζ (ζ) chain to form the T cell receptor CD3 complex and generate activation signals in T lymphocytes. The CD3y (γ), CD35 (δ), and CD3e (ε) chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif, called the immunoreceptor tyrosine-based activation motif, or ITAM, which is essential for the signaling ability of the TCR.

[0005] In recent years, bispecific antibodies (bsAbs) that specifically recognize tumor-associated antigens and the T cell antigen CD3 have shown effectiveness in treating cancer. By bringing tumor cells and T cells together, they can trigger the activation and proliferation of T cells, thereby releasing cytotoxic molecules such as granzymes and perforins, inducing tumor cell lysis. Compared with monospecific antibodies, bispecific antibodies specific for T cell receptors have higher therapeutic potential. Summary of the invention

[0006] After in-depth research, the inventors have constructed a bispecific antibody that can simultaneously target CD79b and CD3, which, on the basis of treating diseases related to CD79b (such as B-cell lymphoma), further mediates T cells to kill tumor cells. The following invention is provided.

[0007] Bispecific Antibodies

[0008] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to a second antigen, wherein the first antigen is CD79b and the second antigen is not CD79b;

[0009] The first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0010] The VH comprises a CDR1 as shown in SEQ ID NO: 3, a CDR2 as shown in SEQ ID NO: 4, and a CDR3 as shown in SEQ ID NO: 5; and

[0011] The VL comprises CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:7, and CDR3 as shown in SEQ ID NO:8;

[0012] Preferably, the CDRs are defined by the Kabat numbering system.

[0013] In certain embodiments, the first antigen binding domain comprises a VH as shown in SEQ ID NO: 9 or a variant thereof, and a VL as shown in SEQ ID NO: 10 or a variant thereof;

[0014] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

[0015] In certain embodiments, the first antigen binding domain comprises the VH shown in SEQ ID NO:9, and the VL shown in SEQ ID NO:10.

[0016] In certain embodiments, the second antigen is selected from CD3, CD19, CD20, CD32B, CD137, CTLA-4, or BCMA.

[0017] In certain embodiments, the second antigen is CD3.

[0018] In certain embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0019] The VH comprises a CDR1 as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 14, and a CDR3 as shown in SEQ ID NO: 15; and the VL comprises a CDR1 as shown in SEQ ID NO: 16, a CDR2 as shown in SEQ ID NO: 17, and a CDR3 as shown in SEQ ID NO: 18;

[0020] The VH comprises CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:23, and CDR3 as shown in SEQ ID NO:24; and the VL comprises CDR1 as shown in SEQ ID NO:25, CDR2 as shown in SEQ ID NO:26, and CDR3 as shown in SEQ ID NO:27; or

[0021] The VH comprises a CDR1 as shown in SEQ ID NO:51, a CDR2 as shown in SEQ ID NO:52, and a CDR3 as shown in SEQ ID NO:53; and the VL comprises a CDR1 as shown in SEQ ID NO:54, a CDR2 as shown in SEQ ID NO:55, and a CDR3 as shown in SEQ ID NO:56;

[0022] Preferably, the CDRs are defined by the Kabat numbering system.

[0023] In certain embodiments, the second antigen binding domain comprises a VH as shown in SEQ ID NO: 19 or a variant thereof, and a VL as shown in SEQ ID NO: 20 or a variant thereof;

[0024] The second antigen binding domain comprises a VH as shown in SEQ ID NO: 28 or a variant thereof, and a VL as shown in SEQ ID NO: 29 or a variant thereof; or

[0025] The second antigen binding domain comprises a VH as shown in SEQ ID NO: 57 or a variant thereof, and a VL as shown in SEQ ID NO: 58 or a variant thereof;

[0026] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

[0027] In certain embodiments, the second antigen binding domain comprises the VH shown in SEQ ID NO:19, and the VL shown in SEQ ID NO:20.

[0028] In certain embodiments, the second antigen binding domain comprises the VH shown in SEQ ID NO:28 and the VL shown in SEQ ID NO:29.

[0029] In certain embodiments, the second antigen binding domain comprises the VH shown in SEQ ID NO:57 and the VL shown in SEQ ID NO:58.

[0030] In certain embodiments, the first antigen binding domain and the second antigen binding domain are selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv and single domain antibody (sdAb);

[0031] Optionally, the first antigen binding domain or the second antigen binding domain is rabbit-derived, murine-derived, fully human or chimeric.

[0032] In certain embodiments, the first antigen binding domain is a Fab.

[0033] In certain embodiments, the second antigen binding domain is a scFv, and the VH of the second antigen binding domain is linked directly or through a peptide linker to the N-terminus or C-terminus of the VL of the second antigen binding domain.

[0034] In certain embodiments, the peptide linker is (GmS)n, where m and n are independently integers not less than 0, such as 1, 2, 3 or 4, respectively.

[0035] In certain embodiments, the peptide linker is GS.

[0036] In certain embodiments, the bispecific antibody further comprises an immunoglobulin Fc region.

[0037] In certain embodiments, the immunoglobulin Fc fragment is connected directly or via a peptide linker to the N-terminus and / or C-terminus of the first and second antigen-binding domains.

[0038] In certain embodiments, the immunoglobulin Fc fragment is the Fc fragment of human IgG (eg, IgG1, IgG2, IgG3, or IgG4).

[0039] In certain embodiments, the peptide linker is (GmS)n, where m and n are independently integers not less than 0, such as 1, 2, 3 or 4, respectively.

[0040] In certain embodiments, the peptide linker is GS.

[0041] In certain embodiments, the immunoglobulin Fc region comprises a knob or hole mutation.

[0042] In certain embodiments, the immunoglobulin Fc segment to which the first antigen-binding domain is connected comprises a knob mutation, and the immunoglobulin Fc segment to which the second antigen-binding domain is connected comprises a hole mutation.

[0043] In certain embodiments, the immunoglobulin Fc segment to which the first antigen-binding domain is connected comprises a hole mutation, and the immunoglobulin Fc segment to which the second antigen-binding domain is connected comprises a knob mutation.

[0044] In certain embodiments, the bispecific antibody is a knob-into-hole bispecific antibody comprising:

[0045] (1) a peptide chain IA, which comprises, from the N-terminus to the C-terminus, the VL and light chain constant region (CL) of the first antigen-binding domain; in certain embodiments, the CL is a human immunoglobulin κ or λ light chain;

[0046] (2) a peptide chain IB, which comprises, from the N-terminus to the C-terminus, the VH and heavy chain constant regions (CH) of the first antigen-binding domain; in certain embodiments, the CH is a human immunoglobulin IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0047] (3) A peptide chain IC, which comprises, from N-terminus to C-terminus, the VL of the second antigen-binding domain, a peptide linker, the VH of the second antigen-binding domain, a peptide linker and an Fc segment; preferably, the peptide linker is (GmS)n, m and n are independently integers not less than 0, for example, independently 1, 2, 3 or 4; in certain embodiments, the Fc segment is the Fc segment of human IgG (e.g., IgG1, IgG2, IgG3 or IgG4).

[0048] In certain embodiments, the CH3 domain of the Fc segment of the peptide chain IB comprises a hole mutation, and the CH3 domain of the Fc segment of the peptide chain IC comprises a knob mutation to promote heterodimerization.

[0049] In certain embodiments, the CH3 domain of the Fc segment of the peptide chain IB comprises mutations T366S, L368A, Y407V, and the CH3 domain of the Fc segment of the peptide chain IC comprises mutation T366W.

[0050] In certain embodiments, the CH3 domain of the Fc segment of the peptide chain IB comprises mutations H435R and Y436F to remove the protein A binding site.

[0051] In certain embodiments, the CH1 domain of the Fc segment of the peptide chain IB comprises the mutation N159S to eliminate the deamination reaction.

[0052] In certain embodiments, the CH2 domain of the Fc segment of peptide chains IB and IC comprises the mutation N297G to reduce ADCC effector function.

[0053] In certain embodiments, the peptide chain IA comprises the sequence shown in SEQ ID NO:12, the peptide chain IB comprises the sequence shown in SEQ ID NO:11, and the peptide chain IC comprises the sequence shown in any one of SEQ ID NOs:21, 30 and 59.

[0054] Antibody preparation

[0055] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the antibody of the present invention is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions and the antibody of the present invention is expressed. The antigen-binding fragment of the present invention can be obtained by hydrolyzing the complete antibody molecule.

[0056] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the bispecific antibody of the present invention.

[0057] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like.

[0058] On the other hand, the invention provides a host cell comprising the nucleic acid molecule or vector separated as above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0059] In another aspect, a method for preparing the bispecific antibody of the present invention is provided, comprising the steps of:

[0060] The host cells as described above are cultured under conditions that allow protein expression, and the bispecific antibody is recovered from the cultured host cell culture.

[0061] Conjugate

[0062] In another aspect, the present invention also provides a conjugate comprising the bispecific antibody of the present invention and a coupling moiety.

[0063] In certain embodiments, the bispecific antibodies of the invention are conjugated to the coupling moiety, optionally via a linker.

[0064] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select a suitable protein tag (e.g., a purification tag, a detection tag, or a tracer tag) according to the desired purpose. In certain exemplary embodiments, the C-terminus of the bispecific antibody of the present invention is connected to a purification tag.

[0065] In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material) or biotin. The detectable label of the present invention can be any material that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads (e.g., ), detection heat markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above markers. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels as described above can be connected to the bispecific antibodies of the present invention through linkers of different lengths to reduce potential steric hindrance.

[0066] In certain embodiments, the conjugated moiety is selected from a therapeutic agent, such as an anti-tumor drug.

[0067] In certain embodiments, the conjugation moiety is selected from additional biologically active polypeptides.

[0068] Pharmaceutical composition

[0069] In another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present invention, and one or more pharmaceutically acceptable excipients.

[0070] In certain embodiments, the pharmaceutical composition may further comprise an additional anti-tumor drug.

[0071] In certain embodiments, in the pharmaceutical composition, the bispecific antibody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present invention and the additional anti-tumor drug can be provided as separate components or as mixed components. Therefore, the bispecific antibody, isolated nucleic acid molecule, vector, host cell, or conjugate of the present invention and the additional anti-tumor drug can be administered simultaneously, separately or sequentially.

[0072] In certain embodiments, the one or more pharmaceutically acceptable excipients may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0073] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, or conjugates of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0074] Therapeutic applications

[0075] On the other hand, the present invention provides a method for preventing and / or treating a disease associated with CD79b and / or CD3 in a subject, comprising administering the bispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition of the present invention to a subject in need thereof. The present invention also relates to the use of the bispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition for preparing a drug for preventing and / or treating a disease associated with CD79b and / or CD3 in a subject.

[0076] In certain embodiments, the CD79b-associated disease is characterized by elevated CD79b expression and / or excessive CD79b activity.

[0077] In certain embodiments, the CD3-related disease is characterized by elevated CD3 expression and / or excessive CD3 activity.

[0078] In certain embodiments, the disease associated with CD79b is a B-cell lymphoma-related disease, such as diffuse large B-cell lymphoma (DLBCL), acute B-lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy cell leukemia (HCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL).

[0079] In certain embodiments, the CD3-associated disease is an inflammatory disease or an autoimmune disease.

[0080] In certain embodiments, the subject is a mammal, such as a human.

[0081] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition is used alone or in combination with another anti-tumor drug.

[0082] The bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, conjugates or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0083] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention in an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0084] The bispecific antibodies of the present invention, isolated nucleic acid molecules, vectors, host cells, conjugates or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician will understand that the route of administration and / or mode will change according to the intended purpose. In certain embodiments, the bispecific antibodies of the present invention, isolated nucleic acid molecules, vectors, host cells or conjugates or pharmaceutical compositions of the present invention are administered by intravenous injection or push injection.

[0085] Detection Application

[0086] In another aspect, the present invention provides a method for detecting the presence or content of CD79b and / or CD3 in a sample, which comprises using the bispecific antibody or conjugate of the present invention.

[0087] In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0088] In certain embodiments, the conjugate used in the methods comprises a bispecific antibody of the invention and a detectable label.

[0089] In certain embodiments, the bispecific antibodies used in the methods are detectably labeled.

[0090] In certain embodiments, the bispecific antibody used in the method does not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) with a detectable label to detect the bispecific antibody of the invention.

[0091] In certain embodiments, the method comprises the following steps:

[0092] (1) contacting the sample with the bispecific antibody or conjugate of the present invention;

[0093] (2) detecting the formation of a complex between the bispecific antibody or conjugate and the antigen or detecting the amount of the complex.

[0094] The formation of the complex indicates the presence of the antigen or a cell expressing the antigen;

[0095] Wherein, the antigen is selected from CD79b or CD3.

[0096] The methods may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0097] In certain embodiments, the method is used to diagnose whether a subject suffers from a disease related to CD79b and / or CD3. In such embodiments, the method may also include a step of comparing the amount of the CD79b and / or CD3 in a sample from the subject with a reference value. The reference value may be the level of CD79b and / or CD3 in a sample from a subject (e.g., a healthy control) known not to have a disease related to CD79b and / or CD3 (also referred to as a "negative reference value"). For example, if the amount of CD79b and / or CD3 in a sample from the subject increases relative to a negative reference value, it indicates that the subject suffers from a disease related to CD79b and / or CD3.

[0098] In certain embodiments, the disease associated with CD79b is characterized by elevated CD79b expression and / or excessive CD79b activity. In certain embodiments, the disease associated with CD79b is a B-cell lymphoma-related disease, such as diffuse large B-cell lymphoma (DLBCL), acute B-lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy cell leukemia (HCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL).

[0099] In certain embodiments, the CD3-related disease is characterized by elevated CD3 expression and / or excessive CD3 activity. In certain embodiments, the CD3-related disease is an inflammatory disease or an autoimmune disease.

[0100] In certain embodiments, the sample can be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue or fine needle aspirate tissue), histological preparations, etc.

[0101] In certain embodiments, the CD79b is human CD79b.

[0102] In certain embodiments, the CD3 is human CD3.

[0103] On the other hand, provided is the use of the bispecific antibody or conjugate of the present invention in the preparation of a detection reagent for detecting the presence or level of CD79b and / or CD3 in a sample or for diagnosing whether a subject suffers from a disease associated with CD79b and / or CD3.

[0104] In certain embodiments, the conjugate used to prepare the detection reagent comprises a bispecific antibody of the invention and a detectable label.

[0105] In certain embodiments, the bispecific antibody used to prepare the detection reagent is detectably labeled.

[0106] In certain embodiments, the bispecific antibody used to prepare the detection reagent does not carry a detectable label. In such embodiments, the detection reagent may further comprise other reagents (such as a second antibody) capable of detecting the bispecific antibody of the invention.

[0107] Definition of terms

[0108] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0109] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0110] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0111] As used herein, the term "CD79b" refers to a single-stranded membrane protein that constitutes CD79, comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain interacts with the extracellular domain of the Igα chain to form the external structure of the B cell receptor complex. The intracellular domain participates in the signal transduction pathway and regulates the biological functions of B cells. The sequence of CD79b is well known to those skilled in the art (see, for example, NCBIProtein database accession number: AAH32651).

[0112] As used herein, the term "CD3" refers to a protein complex consisting of four chains. In mammals, the complex contains a CD3y (γ) chain, a CD35 (δ) chain, and two CD3e (ε) chains. These chains bind to the T cell receptor (TCR) and the so-called ζ (ζ) chain to form a T cell receptor CD3 complex and generate an activation signal in T lymphocytes. The sequence of CD3 is well known to those skilled in the art (see, for example, NCBIProtein database accession number: NP_000724).

[0113] As used herein, the term "antibody" refers to a molecule that specifically binds to an antigenic determinant in its broadest sense and may include various antibody structures as long as they exhibit the desired antigen binding activity. Typically, an antibody may be an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains may be classified as κ (kappa) and λ (lambda) light chains. Heavy chains may be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0114] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). A bispecific antibody comprises two antigen binding domains that have binding specificity to different antigens (or epitopes), thereby being able to bind to two different binding sites and / or target molecules.

[0115] As used herein, the term "Fab fragment" refers to two identical antigen-binding fragments that make up an intact antibody, each Fab fragment comprising a heavy chain variable domain, a light chain variable domain, a light chain constant domain, and the first constant domain (CH1) of the heavy chain.

[0116] As used herein, the term "single-chain variable fragment (scFv)" is a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, connected to a short linker peptide of 10 to about 25 amino acids. VH is connected to the N-terminus or C-terminus of VL through a short peptide. ScFv does not contain a constant region, but retains the specificity of the original antibody.

[0117] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0118] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0119] As used herein, the term "knob-into-hole" is a development technology for a bispecific antibody that involves introducing a "knob" (protrusion) into a first polypeptide and introducing a corresponding "hole" (hole) into a second polypeptide, so that the "knob" can be positioned in the "hole" to promote the formation of heterodimers and hinder the formation of homodimers. The "knob" is constructed by replacing a small amino acid side chain from the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory "hole" of the same or similar size as the "knob" is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). "Knobs" and "holes" can be prepared by changing nucleic acids encoding polypeptides, such as by site-specific mutagenesis or by peptide synthesis.

[0120] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0121] The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, surface plasmon resonance (SPR) can be used to measure the dissociation constant in Biacore. In addition, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

[0122] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0123] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, etc. Host cells may include a single cell or a cell population.

[0124] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using 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.

[0125] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0126] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0127] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.

[0128] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease associated with CD79b and / or CD3) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0129] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a disease associated with CD79b. In certain embodiments, the subject (e.g., a human) suffers from a disease associated with CD3.

[0130] In the present invention, unless otherwise specified, the "first" (e.g., the first antigen or the first antigen-binding domain) and the "second" (e.g., the second antigen or the second antigen-binding domain) are mainly for reference distinction and do not have a typical order meaning.

[0131] Advantageous Effects of the Invention

[0132] The present invention provides a bispecific antibody capable of simultaneously targeting CD79b and CD3, which, on the basis of treating diseases associated with CD79b (such as B-cell lymphoma), further mediates T cells to kill tumor cells, and has a higher therapeutic potential compared with a monospecific antibody.

[0133] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 : Schematic diagram of antibody structure.

[0135] Figure 2 : Binding of different CD79b / CD3 bispecific antibodies to CD79b.

[0136] Figure 3 : Binding of different CD79b / CD3 bispecific antibodies to CD3.

[0137] Figure 4 : Binding of different CD79b / CD3 bispecific antibodies to Jurkat cells.

[0138] Figure 5 : Activation of T cells by different CD79b / CD3 bispecific antibodies.

[0139] Figure 6 :Different CD79b / CD3 bispecific antibodies target cancer cells and kill them.

[0140] Figure 7 :Different CD79b / CD3 bispecific antibodies affect the changes of granzyme B during CD8 T cell activation and execution of killing function.

[0141] Figure 8 :Different CD79b / CD3 bispecific antibodies affect Ki67 changes during CD8 T cell activation and cytotoxicity.

[0142] Fig. 9 :The release of cytokine TNFa induced by different CD79b / CD3 bispecific antibodies.

[0143] Fig.10 : Release of cytokine IFNr induced by different CD79b / CD3 bispecific antibodies.

[0144] Sequence information

[0145] Table 1: The information of the sequences involved in the present invention is described in the table below:

[0146]

[0147]

[0148]

[0149]

[0150] DETAILED DESCRIPTION

[0151] The invention is now described in the following non-limiting examples.

[0152] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0153] Example 1: Molecular Construction

[0154] Build as Figure 1 Bispecific antibody structures shown

[0155] To express chimeric rabbit / human IgG1 antibodies, human IgG1 heavy chain constant region (CH1-3) (SEQ ID NO: 1) and human light chain kappa constant region (CL-κ) (SEQ ID NO: 2) were synthesized and cloned into pcDNA3.4 (GeneScript). pcDNA3.4 containing CH1-3 of human IgG1 (pcDNA3.4-huIgG1-Hc) was further digested with EcoRI / NheI to clone the VH sequence. The VL sequence of the vector pcDNA3.4 (pcDNA3.4-huKappa-Lc) expressing human CL-κ was obtained by digestion with EcoRI / BsiWI. The VH (SEQ ID NOs: 9, 37 and 47) and VL sequences (SEQ ID NOs: 10, 38 and 48) of the two selected rabbit anti-CD79b antibodies (23D8 and 44G2 clones) and the reference antibody Polatuzumab (hereinafter referred to as Pola) were synthesized from Integrated DNA Technologies, with overlapping sequences designed at both the 5' and 3' ends, which were annealed and assembled with the corresponding ends of the resulting vector (NEB HiFi DNA Assembly).

[0156] To generate the CD79b / CD3 bispecific antibody, the scFv (single-chain fragment variable VL-GS-VH) of the CD3 antibody was linked to a shortened Fc (CH2-3) knob, while the CD79b VH was linked to the Fc hole. Fragments of the scFv and Fc knobs derived from the CD3 binding portion of Mosunetuzumab (hereafter MsnCD3), 38E4, and TRX4 (otelixizumab) were synthesized and assembled (NEB HiFi DNA Assembly) into pcDNA3.4-huIgG1-Hc or pcDNA3.4-huKappa-Lc carrying the above-mentioned required fragments. The partial composition and sequence of the CD79b antibody are shown in Table 2 below, the partial composition and sequence of the CD3 antibody are shown in Table 3 below, and the specific composition and sequence of the CD79b / CD3 bispecific antibody are shown in Table 4 below.

[0157] Table 2: Partial composition and sequence of CD79b antibodies

[0158]

[0159] Table 3: CD3 antibody partial composition and sequence

[0160] CD3 Antibody VH (SEQ ID NO: VL (SEQ ID NO: msnCD3 57 58 TRX4 19 20 38E4 28 29

[0161] Table 4: Composition and sequence of CD79b / CD3 bispecific antibodies

[0162]

[0163] The assembled plasmid was transformed into competent Escherichia coli ( 5-alpha), and clones with the correct sequence were cloned according to the sequencing results (Elim Biopharm), and further cultured with LB containing carbenicillin (100 μg / ml) for plasmid purification (QIAGEN Plasmid Plus Kits). The plasmid was eluted with nuclease-free water (Sigma) and stored at -80°C.

[0164] Example 2: Transfection and purification of antibodies

[0165] The paired VH and VL sequences were expressed in pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc cells by transfection into CHO cells (ExpiCHO TMExpression System, Gibco. ExpiCHO cells were cultured using ExpiCHO expression medium and maintained at 0.3 to 6 × 10 cells / mL at 37 °C, 125 rpm, 5% CO2, and 80% humidity according to the manufacturer’s recommendations. 6 In a 125ml culture flask, 3×10 6 Prepare 25 ml of fresh ExpiCHO cells (6 × 10 6 / ml and viability>95%). 1 ml serum-free medium (OptiPRO TM SFM, Gibco) and 80 μl of transfection reagent (ExpiFectamine TM 1ml OptiPRO from CHOReagent, Gibco) TM Mix well by pipetting SFM. Then add the transfection mixture to 25 ml of ExpiCHO cells and incubate at 37°C. The next day, add 150 μl of ExpiFectamine TM CHO Enhancer, 6ml ExpiCHO TM After feeding and 1X penicillin-streptomycin (Gibco), the transfected culture was transferred to a 32°C incubator. The cell density and viability of the transfected culture were monitored, and the IgG1 antibody titer in the culture medium was determined using BLI technology and Protein A biosensor (GatorBio). After about 5 days, the culture medium containing the secreted IgG1 antibody was collected (2000g, 10 minutes) and filtered (Thermo Scientific TM Nalgene TM Rapid-Flow TM Sterile disposable filter) and filled with Protein A resin (TOYOPEARL AF-rProtein A Hc-650F) in a gravity flow column (Bio-Rad). IgG1 antibody was eluted with 3.5 ml of glycine-HCl (100 mM, pH 2.7), immediately neutralized with 1 M Tris-HCl (pH 8.5), and eluted with ThermoScientific TM Slide-A-Lyzer TM G2 dialysis cassettes (20K MWCO) were dialyzed against 1x PBS buffer (Ph 7.2) and stored at 4°C. TM One / OneC Micro-Volume UV-Vis Spectrophotometer (Thermo ScientificTM ) The concentration of the purified IgG1 antibody was determined, and the quality of the IgG1 antibody was checked by SDS-PAGE gel under denaturing and non-denaturing conditions. Five CD79b / CD3 bispecific antibodies, 23D8 / TRX4, 23D8 / 38E4, 23D8 / msnCD3, 44G2 / 38E4 and Pola / msnCD3, were obtained and used in the following examples.

[0166] Example 3: CD79b / CD3 bispecific antibody binds to CD79b

[0167] The binding affinity of CD79b / CD3 bispecific antibody to human CD79b recombinant protein was tested by ELISA. 1 μg / ml (in PBS buffer) human CD79b recombinant protein was coated on ELISA plates overnight. The ELISA plates were washed and blocked with blocking buffer (PBS + 1% BSA), and then incubated with serially diluted CD79b / CD3 bispecific antibody primary antibodies. Anti-CD79b antibody binding was quantified using anti-human IgG HRP secondary antibody (Biolegend catalog number 410902) and HRP substrate. The ELISA binding affinity of anti-CD79b antibody to human CD79b recombinant protein is shown in Figure 2 The results showed that 23D8 / TRX4, 23D8 / 38E4, 23D8 / msnCD3, 44G2 / 38E4 and Pola / msnCD3 could bind to CD79b.

[0168] Example 4: CD79b / CD3 bispecific antibody binds to CD3

[0169] The binding affinity of CD79b / CD3 bispecific antibody to human CD3 recombinant protein was tested by ELISA. 1 μg / ml (in PBS buffer) human CD3 recombinant protein was coated on ELISA plates overnight. The ELISA plates were washed and blocked with blocking buffer (PBS + 1% BSA), and then incubated with serially diluted CD79b / CD3 bispecific antibody primary antibodies. Anti-CD3 antibody binding was quantified using anti-human IgG HRP secondary antibody (Biolegend catalog number 410902) and HRP substrate. The ELISA binding affinity of anti-CD79b antibody to human CD3 recombinant protein is shown in Figure 3 The results showed that the CD79b / CD3 bispecific antibodies 23D8 / TRX4, 23D8 / 38E4, 23D8 / msnCD3, 44G2 / 38E4 and Pola / msnCD3 could bind to CD3.

[0170] Example 5: Binding of CD79b / CD3 bispecific antibody to Jurkat cells

[0171] In order to compare the binding capacity of different CD3 binding parts in CD79b / CD3 bispecific antibodies, Jurkat cells were used as target cells. Jurkat cells were washed twice with FACS buffer (1xPBS+2% BSA+2mM EDTA). 5x10 4 Target cells were seeded into each well of a V-bottom 96-well plate. Then, 50 μl of serially diluted CD79b / CD3 bispecific antibodies 23D8 / 38E4, 23D8 / TRX4, 23D8 / msnCD3, Mosunetuzumab, and negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) were added to each well. The mixture was incubated on ice for 30 minutes; then, the cells were washed twice with FACS buffer (centrifuged at 1200 rpm for 5 minutes). The cells were resuspended in 100 μl FACS buffer containing PE-conjugated goat anti-human IgG Fc (1:1000) (Invitrogen) and incubated on ice in the dark for 30 minutes. After removing the staining buffer and washing twice with FACS buffer, the cells were resuspended in 100 μl FACS buffer and analyzed with a Cytek cytometer. The results are shown in Tables 5 and Figure 4 As shown, the bispecific antibody 23D8 / TRX4 exhibited superior binding ability to other bispecific antibodies.

[0172] Table 5: Binding of CD79b / CD3 bispecific antibodies to Jurkat cells

[0173] Antibody IC50 23D8 / 38E4 9.393 23D8 / TRX4 6.624 23D8 / msnCD3 11.35 Mosunetuzumab 2.483

[0174] Example 6: Activation of T cells by CD79b / CD3 bispecific antibody

[0175] In order to detect the ability of different CD79b / CD3 bispecific antibodies to induce CD8 T cell activation, a co-culture system of human CD8 T cells and BJAB target cells was established. CD8 T cells were isolated and enriched from human PBMCs using the StemCell kit. The frozen CD8 T cells were thawed and washed once with 10 ml of fresh medium. 15 μl of CD79b / CD3 bispecific antibodies 23D8 / TRX4, pola / msnCD3, 23D8 / 38E4, 44G2 / 38E4, Mosunetuzumab and negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) serially diluted in PBS were added to each well of a U-bottom 96-well plate. 1x105 CD8 T cells and 2x10 4 BJAB target cells (5:1 ratio) were added to 135 μl complete medium (RPMI1640 + 10% FBS). The mixture was incubated at 37°C + 5% CO2 for 48 hours. The cells were centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in FACS buffer, stained and analyzed with a Cytek cytometer. The staining group included live / dead dyes syntox blue, PEcy7CD8, AF700 CD4, APC CD25 and APCcy7 CD69, and CD8+CD25+CD69+ cells stained by L / D were circled as active CD8 T cells. The results are shown in Figure 5 As shown, 23D8 / TRX4 exhibited the most potent ability to activate CD8 T cells.

[0176] Example 7: CD79b / CD3 bispecific antibody against T cell-mediated tumor cell killing

[0177] In order to detect the efficacy of different CD79b / CD3 bispecific antibodies in killing target tumor cells, a co-culture system of human CD8 T cells and BJAB target cells was established. CD8 T cells were isolated and enriched from human PBMCs using the StemCell kit. BJAB cells were stained with CFSE (Invitrogen). 15 μl of CD79b / CD3 bispecific antibodies 23D8 / TRX4, pola / msnCD3, 23D8 / 38E4, 44G2 / 38E4, Mosunetuzumab and negative control Human IgGIsotype Control (Invitrogen Catalog #02-7102) serially diluted in PBS were added to each well of a U-bottom 96-well plate. 1x10 5 CD8 T cells and 2x10 4 BJAB target cells (5:1 ratio) were in 135 μl complete medium (RPMI1640 + 10% FBS). The mixture was incubated at 37°C + 5% CO2 for 48 hours. The cells were centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in FACS buffer, stained and analyzed with a Cytek cytometer. The staining group included the live / dead dye syntoxblue, BV421 CD19. L / D CFSE+ cells were circled as viable tumor cells. The killing efficacy was calculated as follows: Killing = (control tumor cell number - control tumor cell number) / control tumor cell number. The results are shown in Figure 6 As shown, 23D8 / TRX4 exhibited the most potent killing efficacy among the candidates tested.

[0178] Example 8: Cytokine Release by CD79b / CD3 Bispecific Antibody

[0179] In order to understand the changes of granzyme B and Ki67 during CD8 T cell activation and killing function, a co-culture system of human CD8 T cells and BJAB target cells was established. CD8 T cells were isolated and enriched from human PBMCs using the StemCell kit. BJAB cells were stained with CFSE (Invitrogen). 15 μl of CD79b / CD3 bispecific antibodies 23D8 / TRX4, pola / msnCD3, 23D8 / 38E4, 44G2 / 38E4, Mosunetuzumab and negative control HumanIgG Isotype Control (Invitrogen Catalog #02-7102) serially diluted in PBS were added to each well of a U-bottom 96-well plate. 1x10 5 CD8 T cells and 2x10 4 BJAB target cells (5:1 ratio) were in 135 μl complete medium (RPMI1640 + 10% FBS). The mixture was incubated at 37°C + 5% CO2 for 48 hours. The cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was collected for ELISA determination of cytokine release. The cell pellet was resuspended in FACS buffer and stained with surface markers and fixable L / D dye Zombie Aqua (Invitrogen). The staining group included PEcy7 CD8, APC CD25, APCcy7CD69. After surface marker and Zoombie aqua staining, the cells were washed twice with FACS buffer and intraocular staining was performed with TONBO's kit (TNB-0607-kit). 200 μl Fix / perm buffer was added to each well, and the plate was incubated at 40°C for 1 hour. The cells were centrifuged and the supernatant was discarded. The cells were washed once with Perm buffer. 50 μl of intracellular staining mixture (PE Granzyme B and PCPcy5.5 Ki67) was added to each well and the plate was placed at room temperature for 30 minutes. The cells were rinsed once with Perm buffer. After washing once more with FACS buffer, the cells were resuspended in 120 μl FACS buffer and analyzed in a Cytek flow cytometer. Target cells were gated by dye-CD8+ Granzyme B+ and dye-CD8+ Ki67+ cells. Figure 7 , Figure 8 The results showed that 23D8 / TRX4 significantly increased the levels of granzyme B and Ki67 in CD8 T cells compared with other candidates.

[0180] In order to detect the cytokine release induced by the CD79b / CD3 bispecific antibody, the supernatant of the 48-hour co-culture system of CD8 T cells and BJAB tumor cells was collected for ELISA cytokine determination (IFNg (invitrogen KitCat#88-7066) and TNFa (88-346-88)). The high binding ELISA plate was coated with the collected antibodies overnight. The next day, after washing 3 times with washing buffer (PBST+0.05% Tween 20), 100 μl of blocking buffer (1% BSAPBST) was added to each well, and the plate was placed at room temperature for one hour. After washing 3 times with washing buffer (PBST), 50 μl of sample (supernatant diluted 5 times) / well (50 μl 1% FBS PBS in the blank well) was added to the designated well. The plate was sealed with a plastic sealing film and incubated at room temperature for 2 hours. After incubation, the plate was washed 3 times with washing buffer. Then, 100 μl of detection antibodies 23D8 / TRX4, pola / msnCD3, 23D8 / 38E4, 44G2 / 38E4, Mosunetuzumab and negative control Human IgGIsotype Control (Invitrogen Catalog #02-7102) were added to each well. The plate was sealed with a plastic sealing film and incubated for another hour at room temperature. After washing 3 times with wash buffer, 100 μl of SP-avidin HRP was added to each well, and the plate was incubated for another 30 minutes at room temperature. After washing 6 times with wash buffer, 100 μl of TMB solution was added to each well, and the plate was incubated at room temperature for 15 minutes. Then, 100 μl of stop solution was added to each well. The OD at 450 nm was read in a spectrometer. Fig. 9 , Fig.10 The results showed that 23D8xTRX4 induced the most potent TNFa and IFNr production among the candidates tested.

[0181] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to a first antigen and a second antigen-binding domain that specifically binds to a second antigen, wherein the first antigen is CD79b and the second antigen is not CD79b; The first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises a CDR1 as shown in SEQ ID NO: 3, a CDR2 as shown in SEQ ID NO: 4, and a CDR3 as shown in SEQ ID NO: 5; and The VL comprises CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO:

8.

2. The bispecific antibody according to claim 1, wherein The first antigen binding domain comprises VH as shown in SEQ ID NO: 9, and VL as shown in SEQ ID NO:

10.

3. The bispecific antibody according to claim 1, wherein The second antigen is selected from CD3, CD19, CD20, CD32B, CD137, CTLA-4 or BCMA.

4. The bispecific antibody according to claim 3, wherein The second antigen is CD3.

5. The bispecific antibody of claim 4, wherein the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises a CDR1 as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 14, and a CDR3 as shown in SEQ ID NO: 15; and the VL comprises a CDR1 as shown in SEQ ID NO: 16, a CDR2 as shown in SEQ ID NO: 17, and a CDR3 as shown in SEQ ID NO: 18; The VH comprises a CDR1 as shown in SEQ ID NO: 22, a CDR2 as shown in SEQ ID NO: 23, and a CDR3 as shown in SEQ ID NO: 24; and the VL comprises a CDR1 as shown in SEQ ID NO: 25, a CDR2 as shown in SEQ ID NO: 26, and a CDR3 as shown in SEQ ID NO: 27; or The VH comprises CDR1 as shown in SEQ ID NO: 51, CDR2 as shown in SEQ ID NO: 52, and CDR3 as shown in SEQ ID NO: 53; and the VL comprises CDR1 as shown in SEQ ID NO: 54, CDR2 as shown in SEQ ID NO: 55, and CDR3 as shown in SEQ ID NO:

56.

6. The bispecific antibody according to claim 5, wherein The second antigen binding domain comprises VH as shown in SEQ ID NO: 19, and VL as shown in SEQ ID NO: 20; The second antigen binding domain comprises VH as shown in SEQ ID NO: 28, and VL as shown in SEQ ID NO: 29; or The second antigen binding domain comprises VH as shown in SEQ ID NO: 57 and VL as shown in SEQ ID NO:

58.

7. The bispecific antibody according to claim 6, wherein The second antigen binding domain comprises VH as shown in SEQ ID NO: 19, and VL as shown in SEQ ID NO:

20.

8. The bispecific antibody according to claim 6, wherein The second antigen binding domain comprises VH as shown in SEQ ID NO: 28 and VL as shown in SEQ ID NO:

29.

9. The bispecific antibody according to claim 6, wherein The second antigen binding domain comprises VH as shown in SEQ ID NO: 57, and VL as shown in SEQ ID NO:

58.

10. The bispecific antibody according to any one of claims 1 to 9, wherein: The first antigen binding domain and the second antigen binding domain are independently selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv and scFv.

11. The bispecific antibody according to any one of claims 1, 3, and 4, wherein: The first antigen-binding domain or the second antigen-binding domain is rabbit-derived, mouse-derived, fully human or chimeric.

12. The bispecific antibody according to claim 11, wherein The first antigen binding domain is Fab.

13. The bispecific antibody according to claim 11, wherein The second antigen-binding domain is a scFv, and the VH of the second antigen-binding domain is connected directly or through a peptide linker to the N-terminus or C-terminus of the VL of the second antigen-binding domain.

14. The bispecific antibody according to claim 13, wherein The peptide linker is (GmS)n, where m and n are independently integers not less than 0.

15. The bispecific antibody according to claim 14, wherein The m and n are independently 1, 2, 3 or 4.

16. The bispecific antibody according to any one of claims 1 to 9, wherein: The bispecific antibody further comprises an immunoglobulin Fc segment.

17. The bispecific antibody according to claim 16, wherein The immunoglobulin Fc segment is connected to the N-terminus and / or C-terminus of the first antigen-binding domain and the second antigen-binding domain directly or through a peptide linker.

18. The bispecific antibody according to claim 17, wherein The immunoglobulin Fc segment is the Fc segment of human IgG.

19. The bispecific antibody according to claim 18, wherein The IgG is IgG1, IgG2, IgG3 or IgG4.

20. The bispecific antibody according to claim 17, wherein The peptide linker is (GmS)n, where m and n are independently integers not less than 0.

21. The bispecific antibody according to claim 20, wherein The m and n are independently 1, 2, 3 or 4.

22. The bispecific antibody according to claim 17, wherein The immunoglobulin Fc segment comprises a knob or a hole mutation.

23. The bispecific antibody according to any one of claims 1 to 9, wherein the bispecific antibody is a knob-into-hole bispecific antibody, comprising: (1) a peptide chain IA, which comprises, from N-terminus to C-terminus, the VL and light chain constant region (CL) of the first antigen-binding domain; (2) peptide chain IB, which comprises, from N-terminus to C-terminus, the VH and heavy chain constant region (CH) of the first antigen-binding domain; (3) Peptide chain IC, which comprises, from N-terminus to C-terminus, the VL of the second antigen-binding domain, a peptide linker, the VH of the second antigen-binding domain, a peptide linker and an Fc segment.

24. The bispecific antibody according to claim 23, wherein The CL is a human immunoglobulin kappa or lambda light chain.

25. The bispecific antibody according to claim 23, wherein The CH is human immunoglobulin IgG.

26. The bispecific antibody according to claim 25, wherein The IgG is IgG1, IgG2, IgG3 or IgG4.

27. The bispecific antibody according to claim 23, wherein The peptide linker is (GmS)n, where m and n are independently integers not less than 0.

28. The bispecific antibody according to claim 27, wherein The m and n are independently 1, 2, 3 or 4.

29. The bispecific antibody according to claim 23, wherein The Fc fragment is the Fc fragment of human IgG.

30. The bispecific antibody according to claim 29, wherein The IgG is IgG1, IgG2, IgG3 or IgG4.

31. The bispecific antibody according to claim 23, wherein The CH3 domain of the Fc segment of the peptide chain IB comprises a hole mutation, and the CH3 domain of the Fc segment of the peptide chain IC comprises a knob mutation.

32. The bispecific antibody according to claim 23, wherein The peptide chain IA comprises the sequence shown in SEQ ID NO: 12, the peptide chain IB comprises the sequence shown in SEQ ID NO: 11, and the peptide chain IC comprises the sequence shown in any one of SEQ ID NOs: 21, 30 and 59.

33. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1-32.

34. A vector comprising the nucleic acid molecule of claim 33.

35. The vector of claim 34, wherein The vector is a cloning vector or an expression vector.

36. A host cell comprising the nucleic acid molecule of claim 33 or the vector of claim 34 or 35.

37. A method for preparing the bispecific antibody according to any one of claims 1 to 32, comprising the steps of: Cultivating the host cell under conditions that allow protein expression, and recovering the bispecific antibody from the cultured host cell culture, wherein The host cell comprises the nucleic acid molecule of claim 33, or comprises a vector, wherein the vector is an expression vector, and the vector comprises the nucleic acid molecule of claim 33.

38. A conjugate comprising the bispecific antibody of any one of claims 1 to 32 and a coupling moiety.

39. The conjugate of claim 38, wherein The conjugated moiety is selected from a protein tag; a detectable label; a therapeutic agent; or, another biologically active polypeptide.

40. The conjugate of claim 39, wherein The protein tag is a purification tag.

41. The conjugate of claim 39, wherein The detectable label is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

42. The conjugate of claim 41, wherein The enzyme is horseradish peroxidase.

43. The conjugate of claim 41, wherein The luminescent substance is a chemiluminescent substance.

44. The conjugate of claim 39, wherein The therapeutic agent is an anti-tumor drug.

45. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-32, the nucleic acid molecule of claim 33, the vector of claim 34 or 35, the host cell of claim 36, or the conjugate of any one of claims 38-44; and one or more pharmaceutically acceptable excipients.

46. ​​The pharmaceutical composition of claim 45, wherein The pharmaceutical composition also contains another anti-tumor drug.

47. Use of the bispecific antibody or pharmaceutical composition according to any one of claims 4 to 32 for preparing a medicament, wherein: The pharmaceutical composition comprises the bispecific antibody according to any one of claims 4 to 32, and one or more pharmaceutically acceptable excipients; The medicament is used for preventing and / or treating diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma in a subject.

48. The use according to claim 47, wherein The subject is a mammal.

49. The use of claim 48, wherein The mammal is a human.

50. The use of claim 47, wherein The bispecific antibody or pharmaceutical composition is used alone or in combination with another anti-tumor drug.

51. A method for detecting the presence or level of CD79b and / or CD3 in a sample for non-diagnostic purposes, comprising the step of using the bispecific antibody of any one of claims 1 to 32 or the conjugate of any one of claims 38 to 44.

52. The method of claim 51, wherein: The method is an immunological assay.

53. The method of claim 52, wherein: The immunological detection is selected from immunoblotting, enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

54. The method of claim 53, wherein: The enzyme immunoassay is ELISA.

55. Use of the bispecific antibody or conjugate according to any one of claims 4 to 32 in the preparation of a detection reagent for detecting the presence or level of CD79b and / or CD3 in a sample or for diagnosing whether a subject suffers from a disease associated with CD79b and / or CD3, wherein: The conjugate comprises the bispecific antibody of any one of claims 4 to 32 and a detectable marker, the disease associated with CD79b is characterized by elevated CD79b expression and / or excessive CD79b activity, and the disease associated with CD3 is characterized by elevated CD3 expression and / or excessive CD3 activity.

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