CN118027210B - Bispecific antibodies targeting CD79b and CD3 and uses thereof - Google Patents

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

Bispecific antibodies targeting CD79b and CD3 and uses thereof

Technical Field

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

Technical Field

CD79 is a heterodimeric molecule consisting of CD79a and CD79B, expressed almost exclusively on B cells and B cell tumors. CD79, as a signaling component of the B cell antigen receptor (BCR), together with cell surface immunoglobulins (ig) for antigen recognition, constitute a BCR complex, playing 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 signaling domain that initiate BCR signaling upon antigen binding, ultimately leading to B cell activation, antigen presentation, cytokine production, and cell proliferation and differentiation. During B cell individuality, CD79a and CD79B are expressed prior to immunoglobulin (Ig) heavy chain gene rearrangement and CD20 expression and disappear later than CD20 in the late (plasma cell) stage of B cell differentiation. Therefore, antibodies targeting CD79a and CD79B can be used to differentially diagnose B cell tumors from T cell tumors or myeloid tumors, or L & H lymphomas as the primary and classical hodgkin lymphomas. In addition, anti-CD 79a and anti-CD 79B antibodies are effective markers for diagnosis of precursor B-acute lymphoblastic leukemia (pre-B-ALL) because these tumors are negative for other B cell markers, such as CD20 and CD45 RA.

Antigen binding to CD79 is rapidly induced to be endocytosed and delivered to the major histocompatibility complex class II (mhc II) compartment (i.e. lysosomal compartment for class II antigen presentation by B cells). Because drug delivery directly to the target cells into the lysosomal compartment enhances cytotoxic activity, and it allows the use of more stable linkers that cleave in the MHCII compartment, this unique intracellular transport 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 in the vast majority of B cell non-Hodgkin's lymphomas (B-NHLs), including Diffuse Large B Cell Lymphomas (DLBCL) (90-100%), acute B lymphoblastic leukemia (B-ALL), chronic Lymphoblastic Leukemia (CLL), B cell pre-lymphoblastic leukemia (PLL), splenic lymphomas with villous lymphocytes (SLVL), hairy Cell Leukemia (HCL), follicular Lymphomas (FL) and Mantle Cell Lymphomas (MCL).

The CD3 receptor complex is a protein complex, consisting of four chains. In mammals, the complex comprises 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, forming the T cell receptor CD3 complex and generating activation signals in T lymphocytes. The CD3y (γ), CD35 (δ) and CD3e (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily comprising single extracellular immunoglobulin domains. The intracellular tail of the CD3 molecule contains a single conserved motif, called the immunoreceptor tyrosine activation motif, abbreviated ITAM, which is critical for the signaling capacity of the TCR.

In recent years, bispecific antibodies (bsAbs) that specifically recognize tumor-associated antigens and the T cell antigen CD3 have shown efficacy in treating cancer. By combining tumor cells with T cells, they trigger activation and proliferation of T cells, thereby releasing cytotoxic molecules such as granzyme and perforin, and inducing tumor cell lysis. Bispecific antibodies specific for T cell receptors have a higher therapeutic potential than monospecific antibodies.

Disclosure of Invention

The present inventors have conducted intensive studies to construct bispecific antibodies capable of simultaneously targeting CD79B and CD3, which further mediate T cell killing of tumor cells upon treatment of CD 79B-related diseases (e.g., B cell lymphomas). The following invention is thereby provided.

Bispecific antibodies

In one aspect, the invention provides a bispecific antibody comprising a first antigen binding domain that specifically binds a first antigen that is CD79b and a second antigen binding domain that specifically binds a second antigen that 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 a CDR1 shown as SEQ ID NO. 6, a CDR2 shown as SEQ ID NO. 7 and a CDR3 shown as SEQ ID NO. 8;

preferably, the CDRs are defined by the Kabat numbering system.

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;

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 was derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1,2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to it, preferably the substitution is a conservative substitution.

In certain embodiments, the first antigen binding domain comprises a VH as set forth in SEQ ID NO. 9, and a VL as set forth in SEQ ID NO. 10.

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

In certain embodiments, the second antigen is CD3.

In certain embodiments, 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 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;

preferably, the CDRs are defined by the Kabat numbering system.

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;

The second antigen binding domain comprises a VH as shown in SEQ ID NO. 28 or a variant thereof, a VL as shown in SEQ ID NO. 29 or a variant thereof, or

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;

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 was derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1,2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to it, preferably the substitution is a conservative substitution.

In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO. 19, and a VL as set forth in SEQ ID NO. 20.

In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO. 28, and a VL as set forth in SEQ ID NO. 29.

In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO:57, and a VL as set forth in SEQ ID NO: 58.

In certain embodiments, the first antigen binding domain and the second antigen binding domain are selected from the group consisting of Fab, fab ', (Fab') ₂, fv, disulfide-linked Fv, scFv, and single domain antibodies (sdabs);

alternatively, the first antigen binding domain or the second antigen binding domain is of rabbit, murine, fully human or chimeric origin.

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

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

In certain embodiments, the peptide linker is (GmS) n, m, n are independently integers not less than 0, e.g., independently 1,2, 3, or 4.

In certain embodiments, the peptide linker is GS.

In certain embodiments, the bispecific antibody further comprises an immunoglobulin Fc segment.

In certain embodiments, the immunoglobulin Fc segment is linked directly or through a peptide linker to the N-terminus and/or C-terminus of the first antigen binding domain and the second antigen binding domain.

In certain embodiments, the immunoglobulin Fc fragment is an Fc fragment of a human IgG (e.g., igG1, igG2, igG3, or IgG 4).

In certain embodiments, the peptide linker is (GmS) n, m, n are independently integers not less than 0, e.g., independently 1,2, 3, or 4.

In certain embodiments, the peptide linker is GS.

In certain embodiments, the immunoglobulin Fc fragment comprises a knob or hole mutation.

In certain embodiments, the first antigen binding domain linked immunoglobulin Fc fragment comprises a knob mutation and the second antigen binding domain linked immunoglobulin Fc fragment comprises a hole mutation.

In certain embodiments, the first antigen binding domain linked immunoglobulin Fc fragment comprises a hole mutation and the second antigen binding domain linked immunoglobulin Fc fragment comprises a knob mutation.

In certain embodiments, the bispecific antibody is a knob-into-hole form of bispecific antibody comprising:

(1) Peptide chain I-A comprising, in order from the N-terminus to the C-terminus, a VL and a light chain constant region (CL) of the first antigen-binding domain, in certain embodiments, the CL is a human immunoglobulin kappa or lambda light chain;

(2) Peptide chain I-B comprising, in order from N-terminus to C-terminus, a VH and a heavy chain constant region (CH) of the first antigen binding domain, in certain embodiments, the CH is a human immunoglobulin IgG, such as IgG1, igG2, igG3, or IgG4;

(3) A peptide chain I-C comprising, in order from the N-terminus to the C-terminus, a VL of the second antigen binding domain, a peptide linker, a VH of the second antigen binding domain, a peptide linker and an Fc segment, preferably the peptide linker is (GmS) N, m, N are independently integers not less than 0, such as independently 1,2,3 or 4, in certain embodiments the Fc segment is that of a human IgG (e.g.IgG 1, igG2, igG3 or IgG 4).

In certain embodiments, the CH3 domain of the Fc segment of the peptide chain I-B comprises a hole mutation and the CH3 domain of the Fc segment of the peptide chain I-C comprises a knob mutation to promote heterodimerization.

In certain embodiments, the CH3 domain of the Fc fragment of the peptide chain I-B comprises the mutation T366S, L368A, Y V and the CH3 domain of the Fc fragment of the peptide chain I-C comprises the mutation T366W.

In certain embodiments, the CH3 domain of the Fc segment of peptide chain I-B comprises a mutation H435R, Y436F to remove a Protein a (Protein a) binding site.

In certain embodiments, the CH1 domain of the Fc segment of the peptide chain I-B comprises the mutation N159S to eliminate deamination.

In certain embodiments, the CH2 domain of the Fc segment of peptide chains I-B and I-C comprises a mutation N297G to reduce ADCC effector function.

In certain embodiments, the peptide chain I-A comprises the sequence set forth in SEQ ID NO. 12, the peptide chain I-B comprises the sequence set forth in SEQ ID NO. 11, and the peptide chain I-C comprises the sequence set forth in any one of SEQ ID NOs:21, 30 and 59.

Preparation of antibodies

The antibodies of the invention may be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, a DNA molecule encoding an antibody of the invention is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention. The antigen binding fragments of the invention may be obtained by hydrolysis of the intact antibody molecule.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a bispecific antibody of the invention.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells, such as E.coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In another aspect, there is provided a method of preparing a bispecific antibody of the present invention comprising the steps of:

Culturing a host cell as described above under conditions that allow expression of the protein, and recovering the bispecific antibody from the cultured host cell culture.

Conjugate(s)

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

In certain embodiments, the bispecific antibodies of the invention are conjugated to the coupling moiety, optionally through a linker.

In certain embodiments, the coupling moiety is selected from the group consisting of a protein tag (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 it is known to those skilled in the art how to select an appropriate protein tag (e.g., a purification tag, a detection tag, or a tracer tag) depending on the intended purpose. In certain exemplary embodiments, the bispecific antibodies of the invention have a purification tag attached to the C-terminus.

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 substance (e.g., a chemiluminescent substance), or biotin. The detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-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 substances (e.g., chemiluminescent substances such as acridine esters), magnetic beads (e.g.,) Detection of thermal labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, a detectable label as described above may be attached to a bispecific antibody of the invention by linkers (linker) of different lengths to reduce potential steric hindrance.

In certain embodiments, the coupling moiety is selected from therapeutic agents, such as anti-tumor agents.

In certain embodiments, the coupling moiety is selected from the group consisting of additional biologically active polypeptides.

Pharmaceutical composition

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

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

In certain embodiments, the bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, or conjugates of the invention and the additional anti-tumor agent in the pharmaceutical composition may be provided as separate components or as a mixed component. Thus, the bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, or conjugates of the invention and the additional anti-tumor agent may be administered simultaneously, separately or sequentially.

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 the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a bispecific antibody, isolated nucleic acid molecule, vector, host cell, or conjugate of the invention. "prophylactically effective amount" means an amount sufficient to prevent, arrest, or delay the onset of a disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest 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 general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

Therapeutic application

In another aspect, the invention provides a method of preventing and/or treating a CD79 b-related and/or CD 3-related disease in a subject comprising administering to a subject in need thereof a bispecific antibody, an isolated nucleic acid molecule, a vector, a host cell, a conjugate or a pharmaceutical composition of the invention. The invention also relates to the use of said bispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a CD79 b-related and/or CD 3-related disease in a subject.

In certain embodiments, the CD79 b-associated disease is characterized by increased expression of CD79b and/or excessive CD79b activity.

In certain embodiments, the CD3 associated disease is characterized by increased CD3 expression and/or excessive CD3 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-lymphoblastic leukemia (B-ALL), chronic Lymphoblastic Leukemia (CLL), B-cell pre-lymphoblastic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy Cell Leukemia (HCL), follicular Lymphoma (FL), and Mantle Cell Lymphoma (MCL).

In certain embodiments, the CD 3-associated disease is an inflammatory disease or an autoimmune disease.

In certain embodiments, the subject is a mammal, e.g., a human.

In certain embodiments, the bispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is used alone or in combination with an additional anti-tumor agent.

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

One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the antibodies or antigen-binding fragments thereof of the present invention in the necessary amount in an appropriate solvent, and optionally with the addition of other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonizing agents, preservatives, diluents, or any combination thereof), followed by filtered sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, conjugates of the invention, or pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, intrainguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. For many therapeutic uses, however, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the bispecific antibodies, isolated nucleic acid molecules, vectors, host cells, or conjugates of the invention or the pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

Detection application

In another aspect, the invention provides a method of detecting the presence or amount of CD79b and/or CD3 in a sample comprising the use of a bispecific antibody or conjugate of the invention.

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

In certain embodiments, the conjugates used in the methods comprise a bispecific antibody of the invention and a detectable label.

In certain embodiments, the bispecific antibodies used in the methods bear a detectable label.

In certain embodiments, the bispecific antibodies used in the methods do not bear a detectable label. Thus, the method may further comprise detecting the bispecific antibody of the invention using other reagents (e.g. secondary antibodies) bearing a detectable label.

In certain embodiments, the method comprises the steps of:

(1) Contacting the sample with a bispecific antibody or conjugate of the invention;

(2) Detecting the formation of a complex between the bispecific antibody or conjugate and an antigen or detecting the amount of said complex.

The formation of the complex indicates the presence of an antigen or cells expressing an antigen;

wherein the antigen is selected from CD79b or CD3.

The method may be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

In certain embodiments, the methods are used to diagnose whether a subject has a disease associated with CD79b and/or with CD3. In such embodiments, the method may further comprise the step of comparing the amount of CD79b and/or CD3 in the sample from the subject to 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 associated with CD79b and/or with 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 is increased relative to a negative reference value, the subject is indicated to have a CD79 b-related and/or CD 3-related disease.

In certain embodiments, the CD79 b-associated disease is characterized by increased expression of CD79b 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-lymphoblastic leukemia (B-ALL), chronic Lymphoblastic Leukemia (CLL), B-cell pre-lymphoblastic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy Cell Leukemia (HCL), follicular Lymphoma (FL), and Mantle Cell Lymphoma (MCL).

In certain embodiments, the CD3 associated disease is characterized by increased CD3 expression and/or excessive CD3 activity. In certain embodiments, the CD 3-associated disease is an inflammatory disease or an autoimmune disease.

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

In certain embodiments, the CD79b is human CD79b.

In certain embodiments, the CD3 is human CD3.

In another aspect, there is provided the use of a bispecific antibody or conjugate of the 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 has a CD79 b-related and/or CD 3-related disease.

In certain embodiments, the conjugates used to prepare the detection reagents comprise a bispecific antibody of the invention and a detectable label.

In certain embodiments, the bispecific antibodies used to prepare the detection reagents are provided with a detectable label.

In certain embodiments, the bispecific antibodies used to prepare the detection reagents are free of a detectable label. In such embodiments, the detection reagent may further comprise other reagents (e.g., a secondary antibody) capable of detecting the bispecific antibodies of the invention.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of 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) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "CD79b" refers to a single-ply membrane protein comprising 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. Intracellular domains are involved in signaling pathways that regulate the biological function of B cells. The sequence of CD79b is well known to those skilled in the art (see, e.g., NCBIProtein database accession number: AAH 32651).

As used herein, the term "CD3" refers to a protein complex consisting of four chains. In mammals, the complex comprises 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, forming the T cell receptor CD3 complex and generating activation signals in T lymphocytes. The sequence of CD3 is well known to those skilled in the art (see, e.g., NCBIProtein database accession number NP-000724).

As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds an epitope and may include a variety of antibody structures so 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 one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can 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 linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 (CH 1, CH2 and CH 3). 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 domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The allocation of amino acids in each region or domain can be as defined by Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens (or epitopes). Bispecific antibodies comprise two antigen binding domains with binding specificities for different antigens (or epitopes) so as to be able to bind to two different binding sites and/or target molecules.

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 constant domain, and a first constant domain of a heavy chain (CH 1).

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, linked to a short linker peptide of 10 to about 25 amino acids. VH is linked to the N-or C-terminus of VL via a short peptide. scFv do not contain constant regions, but retain the specificity of the original antibody.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as may be defined in 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)、Chothia (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, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see, for example, LEFRANC ET al. Dev. Comparat. Immunol.27:55-77,2003).

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

As used herein, the term "knob-into-hole" is a development technique for bispecific antibodies that involves the introduction of a "knob" (protuberance) in a first polypeptide and the introduction of a corresponding "hole" in a second polypeptide, such that the "knob" can be positioned in the "hole" in order to promote the formation of heterodimers and hinder the formation of homodimers. "knob" is constructed by substituting a small amino acid side chain from a first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating "hole" of the same or similar size as "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). "knob" and "hole" can be prepared by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

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 against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a particular 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 antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see MALMQVIST M, nature,1993, 361:186-187). The kdis/kon ratio 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, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby 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 (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may 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 origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells. Host cells may include single cells or cell populations.

As used herein, the term "identity" is used to refer to the match 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 two DNA molecules is occupied by adenine, or a position in each of 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 matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.48:443-453, which may 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 integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1,2,3, 4,5 or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. 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 the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids 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).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., ,Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),, incorporated by reference herein. 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 indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable adjuvant" refers to an adjuvant that is pharmacologically and/or physiologically compatible with the subject and 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 modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that retard absorption, preservatives, e.g., pH modifiers including, but not limited to, phosphate buffers, surfactants including, but not limited to, cationic, anionic or nonionic surfactants, e.g., tween-80, ionic strength enhancers including, but not limited to, sodium chloride, preservatives including, but not limited to, various antibacterial and antifungal agents, e.g., parabens, t-butanol, phenol, sorbic acid, and the like.

As used herein, the term "preventing" refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease associated with CD79b and/or CD 3) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has a disease associated with CD79 b. In certain embodiments, the subject (e.g., human) has a disease associated with CD 3.

In the present invention, the "first" (e.g., first antigen or first antigen binding domain) and "second" (e.g., second antigen or second antigen binding domain) are intended primarily to refer to distinction and do not have the meaning in the order that they are typically used, unless otherwise specified.

Advantageous effects of the invention

The invention provides bispecific antibodies capable of simultaneously targeting CD79B and CD3, which further mediate T cell killing of tumor cells on the basis of treatment of CD 79B-related diseases (e.g., B cell lymphomas), with higher therapeutic potential than monospecific antibodies.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram of the structure of an antibody.

FIG. 2 binding of different CD79b/CD3 bispecific antibodies to CD79 b.

FIG. 3 binding of different CD79b/CD3 bispecific antibodies to CD 3.

FIG. 4 binding of different CD79b/CD3 bispecific antibodies to Jurkat cells.

FIG. 5 activation of T cells by different CD79b/CD3 bispecific antibodies.

FIG. 6 different CD79b/CD3 bispecific antibodies target cancer cell killing.

FIG. 7 granzyme B changes in CD 8T cells when activated and performing killing functions with different CD79B/CD3 bispecific antibodies.

FIG. 8 Ki67 changes in different CD79b/CD3 bispecific antibodies on CD 8T cells when activating and performing killing functions.

FIG. 9 shows cytokine TNFa release induced by different CD79b/CD3 bispecific antibodies.

FIG. 10 shows cytokine IFNr release induced by different CD79b/CD3 bispecific antibodies.

Sequence information

Table 1 information on sequences according to the invention is described in the following table:

Detailed Description

The invention will now be described in the following non-limiting examples.

Those skilled in the art will appreciate that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are all conventional methods unless otherwise specified. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 molecular construction

Construction of bispecific antibody structures as shown in FIG. 1

To express chimeric rabbit/human IgG1 antibodies, human IgG1 heavy chain constant region (CH 1-3) (SEQ ID NO: 1) and human light chain kappa constant region (CL-kappa) (SEQ ID NO: 2) were synthesized and cloned into pcDNA3.4 (GENESCRIPT), respectively. The pcDNA3.4 (pcDNA3.4-huIgG 1-Hc) containing CH1-3 of human IgG1 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-kappa was obtained by digestion with EcoRI/BsiWI. Two selected rabbit anti-CD 79b antibodies (23D 8 and 44G2 clones) were synthesized from INTEGRATED DNA Technologies and VH (SEQ ID NOs:9, 37 and 47) and VL sequences (SEQ ID NOs:10, 38 and 48) of reference antibody Polatuzumab (hereinafter Pola) designed with overlapping sequences at both the 5 'and 3' ends, which were annealed and assembled with the corresponding ends of the resulting vector (NEBHiFi DNA Assembly)。

To generate the CD79b/CD3 bispecific antibody, the scFv of the CD3 antibody (single chain fragment variable VL-GS-VH) was linked to a shortened Fc (CH 2-3) pestle, while the CD79b VH was linked to the Fc mortar. Fragments of scFv derived from CD3 binding portion Mosunetuzumab (hereinafter MsnCD 3), 38E4 and TRX4 (oteliximab) and Fc pestle were synthesized and assembled (NEBHiFi DNA Assembly) into pcDNA3.4-huIgG1-Hc or pcDNA3.4-huKappa-Lc carrying the desired fragment described above. The composition and sequence of the antibody portion of CD79b is shown in Table 2 below, and the composition and sequence of the antibody portion of CD3 is shown in Table 3 below, and specific compositions and sequences of the CD79b/CD3 bispecific antibodies are shown in Table 4 below.

TABLE 2 antibody partial composition and sequence of CD79b

TABLE 3 antibody partial composition and sequence of CD3

CD3 antibodies	VH(SEQ ID NO:)	VL(SEQ ID NO:)
			msnCD3	57	58
TRX4	19	20
			38E4	28	29

TABLE 4 CD79b/CD3 bispecific antibody compositions and sequences

Transforming the assembled plasmid into competent colibacillus5-Alpha) and clones with the correct sequence were cloned according to the sequencing result (Elim Biopharm) and further cultured with LB containing carbenicillin (100. Mu.g/ml) for plasmid purification (QIAGEN PLASMID Plus kit). Plasmids were eluted with nuclease-free water (Sigma) and stored at-80 ℃.

Example 2 transfection and purification of antibodies

Antibodies were expressed by transfection of pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc containing paired VH and VL sequences into CHO cells (ExpiCHO ^TM Expression System, gibco). ExpiCHO cells were cultured using ExpiCHO expression medium and maintained between 0.3 and 6X 10 ⁶/ml at 37℃at 125rpm, 5% CO ₂ and 80% humidity as per manufacturer's instructions. 25ml of fresh ExpiCHO cells (6X 10 ⁶/ml and viability > 95%) were prepared in 125ml flasks at 3X 10 ⁶/ml of 1 day of inoculation culture. 1ml of serum-free medium (OptiPRO ^TM SFM, gibco) containing pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc (12. Mu.g per plasmid) was pipetted into 1ml OptiPRO ^TM SFM containing 80. Mu.l of transfection Reagent (ExpiFectamine ^TM CHO Reagent, gibco). the transfection mixture was then added to 25ml ExpiCHO cells and cultured at 37 ℃. The following day, after addition of 150 μ l ExpiFectamine ^TMCHO Enhancer、6ml ExpiCHO^TM Feed and 1X penicillin-streptomycin (Gibco), the transfected cultures were transferred to a 32 ℃ incubator. Cell density and viability of the transfected cultures were monitored and IgG1 antibody titer in the medium was determined using BLI technology and Protein a biosensor (GatorBio). After about 5 days, medium containing secreted IgG1 antibodies (2000 g,10 min) was collected, filtered (Thermo Scientific ^TMNalgene^TMRapid-Flow^TM sterile disposable filter) and gravity flow column (Bio-Rad) was packed with Protein A resin (TOYOPEARL AF-rProtein A Hc-650F). The IgG1 antibody was eluted with 3.5ml glycine-HCl (100 mM, pH 2.7), immediately neutralized with 1M Tris-HCl (pH 8.5), dialyzed against buffer (Ph 7.2) in 1xPBS with Thermo Scientific ^TMSlide-A-Lyzer^TM G2 dialysis cartridge (20K MWCO) and stored at 4 ℃. The concentration of purified IgG1 antibodies was determined using a NanoDrop ^TM One/OneC micro ultraviolet-visible spectrophotometer (Thermo Scientific ^TM) and the quality of the IgG1 antibodies was checked by SDS-PAGE gel under denaturing and non-denaturing conditions. Five CD79b/CD3 bispecific antibodies were obtained for 23D8/TRX4, 23D8/38E4, 23D8/msnCD3, 44G2/38E4 and Pola/msnCD for the following examples.

Example 3 binding of CD79b/CD3 bispecific antibody to CD79b

The binding affinity of the CD79b/CD3 bispecific antibodies to human CD79b recombinant proteins was tested by ELISA. 1 μg/ml of human CD79b recombinant protein (in PBS buffer) was coated on ELISA plates overnight. ELISA plates were washed and blocked with blocking buffer (PBS+1% BSA) and then incubated with serial dilutions of CD79b/CD3 bispecific antibody primary antibody. anti-CD 79b antibody binding was quantified using anti-human IgG HRP secondary antibody (Biolegend cat No. 410902) and HRP substrate. The ELISA binding affinity of the anti-CD 79b antibodies to human CD79b recombinant proteins is shown in figure 2. The results showed that 23D8/TRX4, 23D8/38E4, 23D8/msnCD3, 44G2/38E4 and Pola/msnCD3 all bound to CD79 b.

Example 4 binding of CD79b/CD3 bispecific antibody to CD3

The binding affinity of the CD79b/CD3 bispecific antibodies to human CD3 recombinant proteins was tested by ELISA. 1 μg/ml of human CD3 recombinant protein (in PBS buffer) was coated on ELISA plates overnight. ELISA plates were washed and blocked with blocking buffer (PBS+1% BSA) and then incubated with serial dilutions of CD79b/CD3 bispecific antibody primary antibody. anti-CD 3 antibody binding was quantified using anti-human IgG HRP secondary antibody (Biolegend cat No. 410902) and HRP substrate. The ELISA binding affinity of the anti-CD 79b antibodies to human CD3 recombinant proteins is shown in figure 3. The results show that CD79b/CD3 bispecific antibodies 23D8/TRX4, 23D8/38E4, 23D8/msnCD3, 44G2/38E4 and Pola/msnCD3 can all bind to CD 3.

Example 5 binding of CD79b/CD3 bispecific antibody to Jurkat cells

To compare the binding capacity of different CD3 binding moieties 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). Mu.l of 5X10 ⁴ target cells in FACS buffer were seeded into each well of a V-bottomed 96-well plate. Then, 50. Mu.l of serial dilutions of CD79b/CD3 bispecific antibody 23D8/38E4, 23D8/TRX4, 23D8/msnCD3, mosunetuzumab and negative control Human IgG Isotype Control (Invitrogen Catalog # 02-7102) were added to each well, respectively. The mixture was incubated on ice for 30 min, and then the cells were washed twice with FACS buffer (centrifuged at 1200rpm for 5 min). Cells were resuspended in 100 μl FACS buffer containing PE-conjugated goat anti-human IgG Fc (1:1000) (Invitrogen) and incubated for 30 min in ice protected from light. After removal of 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 table 5 and fig. 4, with bispecific antibody 23D8/TRX4 showing superior binding capacity to other bispecific antibodies.

TABLE 5 binding of CD79b/CD3 bispecific antibodies to Jurkat cells

Antibodies to	IC50
		23D8/38E4	9.393
23D8/TRX4	6.624
		23D8/msnCD3	11.35
Mosunetuzumab	2.483

Example 6 activation of T cells by CD79b/CD3 bispecific antibodies

To test the ability of different CD79b/CD3 bispecific antibodies to induce activation of CD 8T cells, a co-culture system of human CD 8T cells with BJAB target cells was established. CD 8T cells were isolated and enriched from human PBMCs using the stescell kit. Frozen CD 8T cells were thawed and washed once with 10ml fresh medium. Mu.l of CD79b/CD3 bispecific antibody 23D8/TRX4, pola/msnCD3, 23D8/38E4, 44G2/38E4, mosunetuzumab serially diluted in PBS and negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) were added to each well of the U-bottom 96-well plate. 1x10 ⁵ CD 8T cells and 2x10 ⁴ BJAB target cells (5:1 ratio) were added to 135. Mu.l of complete medium (RPMI 1640+10% FBS) per well, respectively. The mixture was incubated at 37 ℃ plus 5% co ₂ for 48 hours. The cells were centrifuged at 1500rpm 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, PEcy CD8, AF700 CD4, APC CD25 and APCcy CD69, and L/D stained cd8+cd25+cd69+ cells were circled as active CD 8T cells. The results are shown in FIG. 5, where 23D8/TRX4 demonstrated the most potent ability to activate CD 8T cells.

Example 7T cell mediated tumor cell killing by CD79b/CD3 bispecific antibody

To test the efficacy of different CD79b/CD3 bispecific antibodies to kill target tumor cells, a co-culture system of human CD 8T cells and BJAB target cells was established. CD 8T cells were isolated and enriched from human PBMCs using the stescell kit. BJAB cells were stained with CFSE (Invitrogen). Mu.l of CD79b/CD3 bispecific antibody 23D8/TRX4, pola/msnCD3, 23D8/38E4, 44G2/38E4, mosunetuzumab serially diluted in PBS and negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) were added to each well of the U-bottom 96-well plate. 1x10 ⁵ CD 8T cells and 2x10 ⁴ BJAB target cells (5:1 ratio) were added to 135. Mu.l of complete medium (RPMI 1640+10% FBS) per well, respectively. The mixture was incubated at 37 ℃ plus 5% co ₂ for 48 hours. The cells were centrifuged at 1500rpm for 5 minutes. The cell pellet was resuspended in FACS buffer, stained and analyzed with a Cytek cytometer. The staining group included live/dead dye syntox blue, BV421 CD19.L/D CFSE+ cells are circled as viable tumor cells. Killing efficacy is calculated as follows, killing= (control tumor cell number-control tumor cell number)/control tumor cell number. The results are shown in fig. 6, with 23D8/TRX4 exhibiting the most effective killing efficacy among the candidates tested.

Example 8 cytokine Release of CD79b/CD3 bispecific antibody

To understand the changes in granzyme B and Ki67 of CD 8T cells when activating and performing killing functions, a co-culture system of human CD 8T cells with BJAB target cells was established. CD 8T cells were isolated and enriched from human PBMCs using the stescell kit. BJAB cells were stained with CFSE (Invitrogen). Mu.l of CD79b/CD3 bispecific antibody 23D8/TRX4, pola/msnCD3, 23D8/38E4, 44G2/38E4, mosunetuzumab serially diluted in PBS and negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) were added to each well of the U-bottom 96-well plate. 1x10 ⁵ CD 8T cells and 2x10 ⁴ BJAB target cells (5:1 ratio) were added to 135. Mu.l of complete medium (RPMI 1640+10% FBS) per well, respectively. The mixture was incubated at 37 ℃ plus 5% co ₂ for 48 hours. The cells were centrifuged at 1500rpm for 5 minutes. Supernatants were collected for ELISA assays for cytokine release. The cell pellet was resuspended in FACS buffer and stained with surface markers and the fixable L/D dye Zombie Aqua (Invitrogen). The staining group included PEcy CD8, APC CD25, APCcy7 CD69. After surface labeling and Zoombie aqua staining, cells were washed twice with FACS buffer and stained intraocular with the kit of TONBO (TNB-0607-kit). Mu.l of Fix/perm buffer was added to each well and the plates incubated for 1 hour at 40 ℃. The cells were centrifuged and the supernatant discarded. Cells were washed once with Perm buffer. Mu.l of the intracellular staining mixture (PE Granzyme B and PCPcy5.5 Ki 67) was added to each well and the plates were left at room temperature for 30 minutes. Cells were washed once with Perm buffer. After one more wash 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. The results in FIGS. 7, 8 show that 23D8/TRX4 significantly increased the levels of granzyme B and Ki67 in CD 8T cells as compared to the other candidates.

To detect cytokine release induced by CD79b/CD3 bispecific antibodies, supernatants from the CD 8T cell and BJAB tumor cell co-culture system for 48 hours were collected for ELISA cytokine assays (IFNg (invitrogen Kit Cat # 88-7066) and TNFa (88-346-88)). High binding ELISA plates were coated overnight with the collected antibodies. The next day, after 3 washes with wash buffer (pbst+0.05% tween 20), 100 μl of blocking buffer (1% BSA PBST) was added to each well and the plate was left at room temperature for one hour. After 3 washes with wash buffer (PBST), 50. Mu.l of sample (supernatant diluted 5-fold) per well (50. Mu.l of 1% FBS PBS in blank wells) was added to the indicated wells. The plates were sealed with a plastic sealing film and incubated for 2 hours at room temperature. After incubation, the plates were washed 3 times with wash buffer. Then, 100. Mu.l of the detection antibodies 23D8/TRX4, pola/msnCD3, 23D8/38E4, 44G2/38E4, mosunetuzumab and the negative control Human IgG Isotype Control (Invitrogen Catalog #02-7102) were added to each well. The plates were sealed with a plastic sealing film and incubated for an additional hour at room temperature. After 3 washes with wash buffer, 100 μl SP-AVIDIN HRP was added to each well and the plate incubated for an additional 30 minutes at room temperature. After 6 washes with wash buffer, 100 μl of TMB solution was added to each well and the plate incubated for 15 minutes at room temperature. Then, 100. Mu.l of stop solution was added to each well. OD at 450nm was read in a spectrometer. The results of fig. 9, 10 demonstrate that 23D8xTRX4 induced the most efficient TNFa and IFNr production among the candidates tested.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims (55)

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds a first antigen that is CD79b and a second antigen-binding domain that specifically binds a second antigen that 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 a CDR1 as shown in SEQ ID NO. 6, a CDR2 as shown in SEQ ID NO. 7, and a CDR3 as shown in SEQ ID NO. 8.

2. The bispecific antibody of claim 1, wherein the first antigen-binding domain comprises a VH as set forth in SEQ ID No. 9, and a VL as set forth in SEQ ID No. 10.

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

4. The bispecific antibody of 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 of claim 5, wherein,

The second antigen binding domain comprises a VH as shown in SEQ ID No. 19, and a VL as shown in SEQ ID No. 20;

The second antigen binding domain comprises a VH as shown in SEQ ID NO. 28 and a VL as shown in SEQ ID NO. 29, or

The second antigen binding domain comprises a VH as shown in SEQ ID NO. 57 and a VL as shown in SEQ ID NO. 58.

7. The bispecific antibody of claim 6, wherein the second antigen-binding domain comprises a VH as set forth in SEQ ID No. 19, and a VL as set forth in SEQ ID No. 20.

8. The bispecific antibody of claim 6, wherein the second antigen-binding domain comprises a VH as set forth in SEQ ID No. 28, and a VL as set forth in SEQ ID No. 29.

9. The bispecific antibody of claim 6, wherein the second antigen-binding domain comprises a VH as set forth in SEQ ID No. 57, and a VL as set forth in SEQ ID No. 58.

10. The bispecific antibody of any one of claims 1-9, wherein the first and second antigen binding domains are independently selected from the group consisting of Fab, fab ', (Fab') ₂, fv, disulfide-linked Fv, and scFv.

11. The bispecific antibody of any one of claims 1, 3,4, wherein the first or second antigen binding domain is of rabbit, murine, fully human or chimeric origin.

12. The bispecific antibody of claim 11, wherein the first antigen binding domain is a Fab.

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

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

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

16. The bispecific antibody of any one of claims 1-9, wherein the bispecific antibody further comprises an immunoglobulin Fc segment.

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

18. The bispecific antibody of claim 17, wherein the immunoglobulin Fc fragment is an Fc fragment of a human IgG.

19. The bispecific antibody of claim 18, wherein the IgG is IgG1, igG2, igG3, or IgG4.

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

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

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

23. The bispecific antibody of any one of claims 1-9, which is a knob-into-hole form of bispecific antibody comprising:

(1) A peptide chain I-a comprising, in order from the N-terminus to the C-terminus, a VL and a light chain constant region (CL) of the first antigen binding domain;

(2) A peptide chain I-B comprising, in order from the N-terminus to the C-terminus, a VH and a heavy chain constant region (CH) of the first antigen binding domain;

(3) A peptide chain I-C comprising, in order from the N-terminus to the C-terminus, a VL of the second antigen binding domain, a peptide linker, a VH of the second antigen binding domain, a peptide linker, and an Fc segment.

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

25. The bispecific antibody of claim 23, wherein the CH is human immunoglobulin IgG.

26. The bispecific antibody of claim 25, wherein the IgG is IgG1, igG2, igG3, or IgG4.

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

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

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

30. The bispecific antibody of claim 29, wherein the IgG is IgG1, igG2, igG3, or IgG4.

31. The bispecific antibody of claim 23, wherein the CH3 domain of the Fc segment of peptide chain I-B comprises a hole mutation and the CH3 domain of the Fc segment of peptide chain I-C comprises a knob mutation.

32. The bispecific antibody of claim 23, wherein the peptide chain I-a comprises the sequence shown in SEQ ID No. 12, the peptide chain I-B comprises the sequence shown in SEQ ID No. 11, and the peptide chain I-C 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 of preparing the bispecific antibody of any one of claims 1-32, comprising the steps of:

Culturing a host cell under conditions that allow expression of the protein, 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 that 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 1-32 and a coupling moiety.

39. The conjugate of claim 38, wherein the coupling moiety is selected from a protein tag, a detectable label, a therapeutic agent, or an additional biologically active polypeptide.

40. The conjugate according to claim 39, wherein the protein tag is a purification tag.

41. The conjugate according to claim 39, wherein the detectable label is an enzyme, radionuclide, fluorescent dye, luminescent material, or biotin.

42. The conjugate according to claim 41, wherein the enzyme is horseradish peroxidase.

43. The conjugate according to claim 41, wherein the luminescent material is a chemiluminescent material.

44. The conjugate according to claim 39, wherein the therapeutic agent is an anti-neoplastic 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 further comprises an additional anti-tumor agent.

47. Use of the bispecific antibody or pharmaceutical composition of any one of claims 4-32 for the preparation of a medicament, wherein the pharmaceutical composition comprises the bispecific antibody of any one of claims 4-32, 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 of claim 47, wherein the subject is a mammal.

49. The method 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 an additional anti-tumor agent.

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 1-32 or the conjugate of any one of claims 38-44.

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

53. The method of claim 52, wherein the immunological assay is selected from the group consisting of 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 of any one of claims 4-32 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 has CD79 b-related and/or CD 3-related disease characterized by increased CD79b expression and/or excessive CD79b activity, wherein the conjugate comprises the bispecific antibody of any one of claims 4-32 and a detectable label.