CN108659112B - Asymmetric bispecific antibody - Google Patents

Abstract

The present invention relates to a novel asymmetric bispecific antibody. The invention also relates to methods of making asymmetric bispecific antibodies and methods of using these antibodies to treat diseases.

Description

Asymmetric bispecific antibody

Technical Field

The present invention relates to an asymmetric bispecific antibody.

Background

Recently, many recombinant antibody types have been developed, for example, tetravalent bispecific antibodies obtained by fusing, for example, an IgG antibody type and a single chain domain. Some other new classes have also been developed in which the core structure of the antibody (IgA, IgD, IgE, IgG or IgM) is no longer preserved, such as diabodies, triabodies or tetrabodies, minibodies (minibodies) and some single-chain antibodies (scFv, Bis-scFv) capable of binding two or more antigens.

Bispecific antibodies are capable of binding 2 different targets simultaneously, and bispecific antibodies specific for certain cellular targets have been described to date.

Tumors are one of the major diseases currently causing human death. The conventional treatment means of tumors depend on operations, chemotherapy and radiotherapy, but most patients have drug resistance and relapse after chemotherapy. Although surgery in combination with chemotherapy and radiation therapy can prolong the survival of patients, many tumor patients eventually develop tumor metastasis and die. The monoclonal antibody has high drug specificity, small toxic and side effects, unique biological effect and more important position in the field of tumor targeted therapy. The FDA in the united states has approved the marketing of several dozen therapeutic antibody drugs. Among these drugs, antibody drugs represented by Rituxan, Avastin, Herceptin, etc. show good therapeutic effects in treating malignant tumors such as breast cancer, colon cancer, etc.

Currently accepted anti-tumor mechanisms for antibodies include inhibition of key signaling, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), among others. Among them, antibody-mediated cytotoxicity, ADCC, is one of the major mechanisms by which antibodies kill tumor cells. When the antibody binds to an antigen on the surface of a tumor cell, the Fc portion of the antibody binds to Fc receptors on the surface of immune effector cells, which in turn trigger killing of target cells by effector cells (e.g., NK cells). Many monoclonal antibody drugs exert an antitumor effect mainly through ADCC.

ADCC effects of common IgG antibodies tend to be limited, IgG Fc has a limited ability to recruit effector cells, and the presence of inhibitory IgG Fc receptors can attenuate recruitment of effector cells. Bispecific antibodies (BsAbs) are an effective way to enhance ADCC, and have been studied and applied in tumor immunotherapy. For example, Blinatumomab approved by FDA in the U.S. is a monoclonal antibody against CD19 and CD3, and has the structural characteristic that an anti-CD19 single-chain antibody (scFv) and an anti-CD 3scFv are directly connected by a connecting sequence, so that the technology platform is called BiTE (bipolar T cell engage). However, bispecific antibodies prepared by Blinatumomab or BiTE technology have very short half-life in vivo, and influence the effect of the drug.

In order to obtain bispecific antibodies with good pharmaceutical and pharmacokinetic properties, researchers have designed many different bispecific antibodies, most of which have various deficiencies, mainly characterized by: 1) difficult to mass produce, 2) too short half-life, 3) bivalent binding to immune effector cells, resulting in mutual killing of immune effector cells. Therefore, although bispecific antibodies are a hot spot for antibody drug research, few varieties of bispecific antibodies approved for clinical use are expected to be technically innovative.

Disclosure of Invention

The inventor designs an asymmetric bispecific antibody with a brand-new structure, which can show better anti-tumor effect and obviously improve the defects of the existing bispecific antibody.

In particular, it is an object of the present invention to construct bispecific antibodies capable of binding to immune effector cells by a monovalent binding, which bind to tumor cell surface proteins on the one hand and to immune effector cells by a monovalent binding, i.e. one bispecific antibody molecule binds to only one immune effector cell, thereby avoiding killing of the effector cells against each other. At the same time, it must be ensured that such bispecific antibodies have a good half-life in vivo.

The T cell immune response is an important link in the human immune system, so that the construction of bispecific antibody to T cell and tumor cell surface antigen can activate the killing of static T cell to target tumor, thereby achieving the purpose of treating malignant tumor, and the T cell surface antigen CD3 can be used as target. In addition, other immune effector cell surface markers can be used as bispecific antibody targets. Studies have shown that the IgA receptor Fc α RI (CD89) is expressed on neutrophils, monocytes, macrophages, eosinophils and Dendritic Cells (DCs) and mediates a variety of immune effector processes that occur between IgA and myeloid cells such as neutrophils, monocytes and macrophages. The number of neutrophils is the largest number of leukocytes, and the number of neutrophils in the blood of an adult human accounts for about 55-70% of the total number of leukocytes. Neutrophils can mediate tumor killing reactions more efficiently. Therefore we are also interested in bispecific antibodies with CD89 cells as effector cells.

The tumor surface antigens CD19 and CD20 are both non-glycosylated transmembrane proteins and have high conservation. CD19, CD20 are expressed in most B lymphocytes, but not in plasma cells, lymphoid stem cells and other tissues, and over 95% of B cell lymphomas have CD19, CD20 expression without significant endocytosis and shedding, which make CD19, CD20 ideal target antigens for treating B cell lymphomas.

Through constructing the bispecific antibody, the marrow effector cells expressing markers such as CD3 and the like can be recruited while targeting tumor cells, thereby achieving the purpose of killing the tumor cells more effectively.

In addition to tumor immunotherapy, bispecific antibodies can also be used for other diseases, for example by targeting viral antigens and CD3, can direct T cell killing of viruses, and can be used to treat viral infections or diseases caused by viruses. According to the nature of the target, the bispecific antibody can have different functions, thereby achieving the purpose of treating different diseases.

Although researchers have designed a wide variety of bispecific antibodies, they still do not meet the needs of drug development. The main content of the present invention is to design a new type of bispecific antibody, which has Asymmetric structure and thus called Asymmetric bispecific antibodies (AsBs Ab), which have longer half-life in vivo and can be better expressed from cells, and more importantly, the bispecific antibody can be monovalent combined with immune effector cells, i.e. one antibody molecule only binds to an epitope on one immune effector cell, and can prevent the immune effector cells from being coupled and killed with each other.

The present invention provides an isolated asymmetric bispecific antibody comprising:

a first chain comprising, in order from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL);

a second chain comprising, in order from N-terminus to C-terminus, a heavy chain variable region, a heavy chain constant region, and a single chain antibody (scFv) region, wherein VL and CL of the first chain and the second chain heavy chains VH and CH1 together comprise an antigen binding fragment (Fab) for a first antigen or epitope; the heavy chain constant region of the second chain comprises the CH1 and Fc regions of an antibody heavy chain; the scFv region of the second chain comprises, in order from N-terminus to C-terminus, a heavy chain variable region and a light chain variable region for a second antigen, or comprises, in order from N-terminus to C-terminus, a light chain variable region and a heavy chain variable region for a second antigen or epitope; and

a third chain comprising a third chain heavy chain Fc region, wherein the third chain heavy chain Fc region comprises an antibody hinge region, CH2, and CH 3.

In particular, the second chain comprises a hinge region located between the CH1 and CH2 domains, and the third chain comprises, in order from N-terminus to C-terminus, a third chain hinge region and a heavy chain Fc region.

In particular, the third chain comprises a hinge region, a CH2 domain, and a CH3 domain.

In particular, the end of the light chain constant region of the first chain is linked to the end of the heavy chain constant region of the second chain by a disulfide bridge.

In particular, the hinge region of the second chain has a disulfide bridge with the hinge region of the third chain, as is present in natural antibodies.

In particular, the light chain constant region of the first chain has a disulfide bond linkage with the heavy chain constant region of the second chain, and/or wherein there are 0, 1 or 2 disulfide bridges between the CH3 domain of the second chain and the CH3 domain of the third chain, preferably wherein the disulfide bridges are introduced by introducing a cysteine at the respective position of the second heavy chain variable region of the second chain and the light chain variable region of the second chain and/or the CH3 domain of the second chain and the CH3 domain of the third chain.

To make such bispecific antibodies, we designed a three vector antibody expression system. In this system, the bispecific antibody is composed of three polypeptide chains. The first polypeptide chain is an antibody light chain having intact antibody variable and constant region sequences; the second polypeptide chain is a fusion protein chain of a complete antibody heavy chain and another single-chain antibody (scFv), and from N-terminus to C-terminus are the heavy chain variable region, the heavy chain constant region, and the SCFV of another antibody, respectively; the third polypeptide chain contains only the heavy chain hinge region and the Fc fragment. During the expression of the three polypeptide chains in the cell, the first polypeptide chain (the light chain of the first antibody) is paired with the N-terminal of the second polypeptide chain (the heavy chain of the first antibody) to form an antibody Fab structure, which can bind an epitope; the C-terminus of the second polypeptide chain comprises an scFv of an antibody which binds to another epitope; the third polypeptide chain forms a disulfide bond with the second polypeptide chain at the hinge region, forming an Fc dimer. Thus, the bispecific antibody molecule is a three-chain molecule with an asymmetric structure, and the three-chain antibody is combined with one antigen through a Fab fragment at the N terminal and then combined with another antigen through a scFv at the C terminal, thereby achieving the purpose of the bispecific antibody.

The bispecific antibody designed by the invention is characterized by the asymmetric structure of the Fc of the antibody. The bispecific antibody comprises two protein chains containing Fc, wherein one protein chain containing Fc is longer and comprises the following components from the N end: (1) the heavy chain variable region and heavy chain of the first antibody CH1, (2) the Fc region (hinge region, CH2, CH3), (3) the scFv of another antibody at the C-terminus, e.g., in one embodiment, the molecular weight of the protein chain is about 77 kD; another Fc-containing protein chain is an Fc-containing hinge region, e.g., in one embodiment the protein chain has a molecular weight of 29 kD. In order to realize the heterologous pairing of long-chain and short-chain Fc, concave-convex pairing structures are designed in the two Fc sequences, the formation of homodimer Fc is reduced through the amino acid mutation of an Fc region, the heterologous pairing of long-chain and short-chain Fc is increased, and the purposes of monovalent and bispecific functions are achieved. Due to the large difference in molecular weight between the two Fc fragment-containing polypeptides, even small amounts of Fc that form homodimers can be removed by subsequent purification.

The basic structure of the asymmetric bispecific antibody of the present invention can be seen in FIG. 1. This structure can be used for expression of bispecific antibodies for a variety of different purposes. Wherein, two antigen binding regions in the bispecific antibody structure, namely Fab and scFv are combined with different antigens, and Fab can be combined with tumor antigen, surface antigen of immune effector cell, or other antigen epitope; the scFv can bind to a tumor antigen, to a surface antigen of an immune effector cell, or to other epitopes. The structure of the antibody of the invention is particularly suitable for use in tumor/cancer therapy, such as B cell-associated tumors and the like. When Fab and scFv of the bispecific antibody are combined with a tumor antigen and an antigen on the surface of an immune effector cell respectively, the immune effector cell can be coupled with the tumor cell, so that the tumor cell is killed, and the immunotherapy effect is achieved.

Tumor antigens useful in the present invention include, but are not limited to, AFP, BCMA, CEA, Claudin, CA19-9, CA125, DR5, EMP2, GPA33, EGFR, Folate, HER2, HER3, FGFR1, c-MET, PDGFR, VEGFR, CD16, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD47, CD123, CD133, CD137, CD138, PSMA, TAG72, Tim-3, Trop-2, P-cadherin, gp100, PD-L1, and EpCAM.

There are also many effector cell surface antigens that can be used as bispecific antibodies described herein, including but not limited to: fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIB (CD32), Fc γ RIIIA (CD16a), Fc γ RIIIB (CD16b), Fc α RI (CD89), CD3, and PD 1. T lymphocytes are commonly used, with CD3 as the optional cell surface marker, but other T cell surface markers may also be used. In addition to T cells, many other cells also have an immune effector function, such as neutrophils, monocytes, etc., which can be bound by different cell markers. In particular, CD89 is a surface marker expressed on neutrophils and monocytes, and in bispecific antibodies of the invention, one example is the killing of CD19 positive cells, including CD19 positive tumor cells, by effector cells by targeting CD89 on the one hand and CD19 on the other hand.

Furthermore, it is worth pointing out that the bispecific antibody of the present invention can also bind to two tumor antigens, to two target molecules separately, or to different epitopes of the same target molecule, to block tumor signals, or to kill tumor cells by other mechanisms. Furthermore, bispecific antibodies of the present invention may also be used in diseases other than tumor therapy, for example, by simultaneously binding two non-tumor associated targets and, depending on the mechanism of action, performing their biological function. These structural or functional bispecific antibodies are within the scope of the present technology.

The bispecific antibody vector with the three polypeptide chains can be constructed by gene cloning, can be plasmids, viruses, DNA fragments and the like, and can be realized by conventional molecular biology technology. The commonly used method is that three polypeptide chains can be amplified and synthesized by PCR and cloned into bacterial plasmid. A number of plasmids, such as pcDNA3.1 and the like, can be used for this purpose. The plasmid can be used for transfected cell expression after being determined by gene sequencing. A variety of cells can be used to express the bispecific antibodies of the present invention, such as mammalian cells, insect cells, yeast cells, bacterial cells, and the like. CHO cells (Chinese hamster approach cells) are commonly used in mammalian cells, and HEK293 cells, myeloma cells, and the like can also be used for expression. The bispecific antibodies of the present invention involve three polypeptide chains, which can be cloned into one, two, or three vectors for expression. Taking HEK293 cells as an example, DNA encoding three polypeptides can be cloned into three plasmids, respectively, purified, and transfected into cells using a co-transfection technique. The transfection method may be a DNA transfection kit. Transfection methods are divided into transient transfection and stable transfection. Transient transfection can obtain relatively small amounts of protein in a short time, stable transfection can obtain stable cell lines, and larger-scale protein expression can be achieved.

Drawings

FIG. 1 shows an exemplary structural diagram of an asymmetric bispecific antibody of the present invention, in which are both an asymmetric bispecific antibody of the present invention and a normal (normal) form antibody.

FIG. 2 shows the electrophoresis chart of the target gene detected by colony PCR.

FIG. 3 shows an SDS-PAGE electrophoresis of anti-CD 3-CD19 bispecific antibody proteins.

FIG. 4 shows the flow cytometry detection of the binding of anti-CD 89-anti-CD 20 bispecific antibodies to PMN, Raji cells.

FIG. 5 shows flow cytometry for the detection of binding of anti-CD 19-anti-CD 3 bispecific antibodies to Raji, Jurkat cells.

FIG. 6 shows the killing ability of anti-CD 89-anti-CD 20 bispecific antibodies against tumor cells.

FIG. 7 shows the T cell killing capacity mediated by anti-CD 19-anti-CD 3 bispecific antibody in vitro.

Figure 8 shows the serum half-life of the bispecific antibody in mice.

FIG. 9 shows in vivo therapeutic experiments for tumors with anti-CD 89-anti-CD 20 bispecific antibodies.

FIG. 10 shows in vivo therapeutic experiments for tumors with anti-CD 19-anti-CD 3 bispecific antibodies.

Detailed Description

The present invention provides an isolated asymmetric bispecific antibody comprising:

a first chain comprising, in order from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL);

a second chain comprising, in order from N-terminus to C-terminus, a heavy chain variable region, a heavy chain constant region, and a single chain antibody (scFv) sequence. Wherein the VL and CL of the first chain and the heavy chain VH and CH1 of the second chain together comprise an antigen binding fragment (Fab) of a first antibody; the heavy chain constant region of the second chain comprises the CH1 and Fc regions of an antibody heavy chain; the scFv region of the second chain comprises, in order from N-terminus to C-terminus, the heavy chain variable region and the light chain variable region of the second antibody, which are linked by a suitable linking sequence, or comprises, in order from N-terminus to C-terminus, the light chain variable region and the heavy chain variable region of the second antibody, which are linked by a suitable linking sequence; and

a third chain comprising a third chain heavy chain Fc region, wherein the third chain heavy chain Fc region comprises a third chain CH2 domain and a CH3 domain.

In some specific embodiments, the interface at which the CH3 domain of the second strand and the CH3 domain of the third strand are in contact is modified to reduce homodimer formation, wherein the modification is:

a) replacing the amino acid residue in the CH3 domain of the second strand located within the above-mentioned interface with an amino acid residue having a larger side chain volume, thereby generating a bulge on the second strand side of the interface,

b) replacing an amino acid residue in the CH3 domain of the third strand located within the above-described interface with an amino acid residue having a smaller side chain volume, thereby creating a cavity on the third strand side of the interface,

wherein the protrusion is positioned in the cavity.

In some specific embodiments, the amino acid residue having a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the replacement of an amino acid residue in the CH3 domain of the second strand, which is located within the above-mentioned interface, with an amino acid residue having a larger side chain volume is a T366W mutation (corresponding to the position of amino acid 146 in SEQ ID NO: 5 of the present application).

In some specific embodiments, the amino acid residue having a smaller side chain volume is selected from the group consisting of: alanine (a), serine (S), threonine (T), and valine (V). Preferably, the replacement of amino acid residues in the CH3 domain of the second strand that are located within the above-described interface with amino acid residues having a smaller side chain volume is a mutation of T366S, L368A and Y407V (corresponding to positions 146, 148 and 187 of amino acids in SEQ ID NO: 4 of the present application).

In some specific embodiments, there are 1, 2 or 3 disulfide bridges between the hinge region of the second chain and the hinge region of the third chain.

If the Fc sequence used in the bispecific antibody has the property of binding native Fc to fcyr, it may result in NK equivalents binding to the bispecific antibody and induce ADCC effect, which may enhance the killing ability of the bispecific antibody against tumor cells by binding the bispecific antibody to the tumor target, but may also reduce the anti-tumor effect by killing effector cells by binding the bispecific antibody to the corresponding cell target, such as CD 3. In some specific embodiments, to prevent ADCC from affecting the efficacy of the bispecific antibody, we further modify the Fc region of the second chain and the heavy chain Fc region of the third chain to remove ADCC effector functions, e.g., mutation of Asn 297 to Ala.

In some specific embodiments, the light chain variable region of the first chain and the heavy chain variable region of the second chain are capable of specifically binding to a first antigen or epitope.

In some specific embodiments, the scFv region of the second chain is capable of specifically binding a second antigen or epitope.

In some specific embodiments, the first antigen or epitope is different from the second antigen or epitope. In other specific embodiments, the first antigen or epitope and the second antigen or epitope are the same.

In some specific embodiments, the first antigen or epitope is a tumor cell surface antigen or epitope and the second antigen or epitope is an immune effector cell surface antigen or epitope. In other specific embodiments, the first antigen or epitope is an immune effector cell surface antigen or epitope and the second antigen or epitope is a tumor cell surface antigen or epitope. In yet other specific embodiments, the first antigen or epitope is a tumor cell surface antigen or epitope and the second antigen or epitope is another tumor cell surface antigen or epitope.

In particular, the tumor cell surface antigen or epitope is selected from: AFP, BCMA, CA19-9, CA125, CEA, Claudin, DR5, EMP2, GPA33, EGFR, Folate, HER2, HER3, FGFR1, c-MET, PDGFR, VEGFR, CD16, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD47, CD123, CD133, CD137, CD138, PSMA, TAG72, Tim-3, Trop-2, P-dhcalin, gp100, PD-L1, and EpCAM.

In particular, the immune effector cell surface antigen or epitope may be selected from: fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIB (CD32), Fc γ RIIIA (CD16a), Fc γ RIIIB (CD16b), Fc α RI (CD89), CD3, and PD 1.

In some specific embodiments, each of the portions of the antibodies of the invention (including, but not limited to, the VL domain and the CL domain of the first chain, the heavy chain VH domain, the heavy chain constant domain (including the CH1 domain, the hinge domain, the CH2 domain, the CH3 domain), and the scFv domain of the second chain, the heavy chain Fc domain of the third chain, including the hinge domain, the CH2 domain, the CH3 domain, each of which may be independently derived from human, mouse, rat, rabbit, or camelid species, etc.

In some specific embodiments, the present invention provides an anti-CD 89-anti-CD 20 bispecific antibody, wherein the first antigen or epitope is CD89 and the second antigen or epitope is CD20, or the first antigen or epitope is CD20 and the second antigen or epitope is CD 89. Preferably, the sequence of the first strand is SEQ ID NO:1, the sequences of VH and CH1 of the second chain are SEQ ID nos: 2; the sequence of the scFv region of the second chain is SEQ ID No: 3; and one of the sequence of the Fc region of the second chain and the sequence of the Fc region of the third chain is SEQ ID No: 4 or 49, and the other is SEQ ID No: 5 or 50. In particular, the sequence of the Fc region of the second chain is SEQ ID No: 4, and the sequence of the Fc region of said third chain is SEQ ID No: 5. in particular, the sequence of the Fc region of the second chain is SEQ ID No: 49, and the sequence of the Fc region of said third chain is SEQ ID No: 50.

in some specific embodiments, the present invention provides an anti-CD 19-anti-CD 3 bispecific antibody, wherein the first antigen or epitope is CD19 and the second antigen or epitope is CD3, or the first antigen or epitope is CD3 and the second antigen or epitope is CD 19. Preferably, the sequence of the first strand is SEQ ID NO: 6, the sequences of VH and CH1 of the second chain are SEQ ID No: 7; the sequence of the scFv region of the second chain is SEQ ID No: 8; and one of the sequence of the Fc region of the second chain and the sequence of the Fc region of the third chain is SEQ ID No: 4 or 49, and the other is SEQ ID No: 5 or 50. In particular, the sequence of the Fc region of the second chain is SEQ ID No: 4, and the sequence of the Fc region of said third chain is SEQ ID No: 5. in particular, the sequence of the Fc region of the second chain is SEQ ID No: 49, and the sequence of the Fc region of said third chain is SEQ ID No: 50.

in some specific embodiments, the Fc region of the second chain is linked to the scFv region by a peptide linker, preferably the peptide linker is 0, 1, 2, or 3 GGGGS.

In some specific embodiments, the light chain variable region and the heavy chain variable region in the scFv region of the second chain are linked by a peptide linker, preferably the peptide linker is 0, 1, 2, or 3 GGGGS.

In some specific embodiments, the first antigen or epitope in an antibody of the invention is CD89, and the VL of the first chain comprises the following CDRs: the sequence is shown as SEQ ID NO:9, the sequence of CDR1 shown in SEQ ID NO:10, the sequence of CDR2 shown in SEQ ID NO:11, and the VH of the second chain comprises the following CDRs: the sequence is shown as SEQ ID NO:12, the sequence of CDR1 shown in SEQ ID NO:13, the sequence of CDR2 shown in SEQ ID NO:14, CDR3 shown in fig. 14.

In some specific embodiments, the first antigen or epitope in an antibody of the invention is CD19, and the VL of the first chain comprises the following CDRs: the sequence is shown as SEQ ID NO: 15, the sequence of CDR1 shown in SEQ ID NO: 16, the sequence of CDR2 shown in SEQ ID NO: 17, and the VH of the second chain comprises the following CDRs: the sequence is shown as SEQ ID NO: 18, the sequence of CDR1 shown in SEQ ID NO: 19, the sequence of CDR2 shown in SEQ ID NO: 20, CDR3 shown.

In some specific embodiments, the second antigen or epitope in the antibody of the invention is CD20, and the scFv sequence of the second chain is as set forth in SEQ ID No: 3, respectively.

In some specific embodiments, the second antigen or epitope in the antibody of the invention is CD3, and the scFv sequence of the second chain is as set forth in SEQ ID No: shown in fig. 8.

In another aspect, the invention provides a method for preparing an antibody of the invention, comprising the steps of:

a) transforming a host cell with:

a first vector comprising a nucleic acid molecule encoding the first strand,

a second vector comprising a nucleic acid molecule encoding said second strand, and

a third vector comprising a nucleic acid molecule encoding the third strand;

b) culturing the host cell under conditions that allow synthesis of the antibody; and

c) recovering the antibody from the culture.

In some embodiments, the first, second and third vectors are different vectors. In some embodiments, the first vector, the second vector and the third vector are the same vector.

In another aspect, the invention provides a method for preparing an antibody of the invention, comprising the steps of:

a) transforming a host cell with:

a vector comprising a nucleic acid sequence encoding the first strand, a nucleic acid sequence encoding the second strand, and a nucleic acid sequence encoding the third strand;

b) culturing the host cell under conditions that allow synthesis of the antibody; and

c) recovering the antibody from the culture.

In yet another aspect, the invention provides a host cell comprising:

a first vector comprising a nucleic acid molecule encoding the first strand,

a second vector comprising a nucleic acid molecule encoding said second strand, and

a third vector comprising a nucleic acid molecule encoding the third strand.

In yet another aspect, the invention provides a host cell comprising:

a vector comprising a nucleic acid sequence encoding the first strand, a nucleic acid sequence encoding the second strand, and a nucleic acid sequence encoding the third strand.

In some embodiments of the invention, the vector is an expression vector, preferably a plasmid, virus or other vector.

In some embodiments of the invention, the host cell is a prokaryotic cell or a eukaryotic cell. In particular, the prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, or the like. The eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma and the like, insect cells such as Spodoptera frugiperda and the like, plant cells such as tobacco and the like, and mammalian cells such as 293 cells, 293F cells, CHO cells, NSO cells, BHK cells, COS cells, myeloma cells and the like. In some embodiments, the host cell of the invention is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell or a COS cell.

In yet another aspect, the invention provides an antibody capable of specifically binding CD89, wherein the antibody comprises: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, wherein

The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: as shown in figure 12 of the drawings,

the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: as shown in figure 13, the first and second,

the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: as shown in figure 14, the first and second,

the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: as shown in figure 9, the first and second,

the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:10, and

the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: shown at 11.

In some embodiments, the anti-CD 89 antibodies of the invention comprise an amino acid sequence set forth in SEQ ID NO:23, and the amino acid sequence is as shown in SEQ ID NO:24, or a light chain variable region as shown.

In yet another aspect, the invention provides an antibody capable of specifically binding CD19, wherein the antibody comprises: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDRL, light chain CDR2, light chain CDR3, wherein

The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: as shown at 18, the flow of air is,

the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: as shown in the drawing (19), the,

the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: as shown at 20, the flow of the gas,

the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: as shown in the drawing 15, the flow rate of the gas,

the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 16, and

the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: shown at 17.

In some embodiments, the anti-CD19 antibodies of the invention comprise an amino acid sequence set forth in SEQ ID NO: 38 and the amino acid sequence is as shown in SEQ ID NO: 37, or a light chain variable region as shown in fig.

In yet another aspect, the invention provides a pharmaceutical composition comprising an antibody of the invention and at least one pharmaceutically acceptable excipient.

In a further aspect, the invention provides the use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease.

In a further aspect, the invention provides an asymmetric bispecific antibody as described above for use in the treatment of a disease.

In yet another aspect, the present invention provides a method for treating a disease, comprising: administering a therapeutically effective amount of an antibody of the invention to a subject in need thereof.

In some embodiments, the disease is a cancer or tumor, preferably wherein the first antigen or epitope is expressed on the surface of a cancer cell or tumor cell, e.g., a B lymphocyte tumor (such as non-hodgkin's lymphoma), a leukemia (such as chronic lymphocytic leukemia), lung cancer, stomach cancer, liver cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, head and neck cancer, and cervical cancer.

In some embodiments, the disease is a disease characterized by expression of B cells. In certain embodiments, the disease is non-responsive to treatment with at least one of an anti-CD19 antibody and an anti-CD20 antibody.

In still other embodiments, the disease is an autoimmune disease. In particular, the autoimmune disease is one or more of: multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, psoriasis, vasculitis, IgA nephritis, uveitis, Crohn's disease, type 1 diabetes, and the like.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Notwithstanding that the numerical ranges and parameter approximations set forth the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference that is said to be "incorporated herein" is to be understood as being incorporated in its entirety.

It should also be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly and clearly dictates otherwise. The term "or" may be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

The term "asymmetric" as used herein means that the antibody of the present invention cannot be divided into two parts that are symmetric to each other. In particular, certain natural antibodies (e.g., IgG) comprise two identical heavy chains and two identical light chains, wherein the portions of one heavy chain and one light chain are symmetric to the portions of the other heavy chain and the other light chain. Preferably, the asymmetric antibodies of the invention are heterotrimers composed of three distinct chains.

The term "bispecific" as used herein is intended to encompass any agent having two different binding specificities, e.g., a heteromultimer, a monomer, a protein, a peptide, or a protein or peptide complex, preferably an antibody.

The term "isolated" as used herein means a substance, such as an antibody, that has been identified and separated and/or recovered from a component of its native cell culture environment.

The term "antibody" as used herein encompasses full-length antibodies (e.g., an IgG1 or IgG4 antibody), various functional fragments thereof (e.g., may comprise only antigen-binding portions, such as Fab, F (ab')₂Or scFv fragments) as well as modified antibodies (e.g., humanized, glycosylated, etc.). In some applications, it may be useful to modify to remove undesired glycosylation sites, or antibodies that do not have a fucose moiety on the oligosaccharide chain, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to alter Complement Dependent Cytotoxicity (CDC).

The term "functional fragment" as used herein is intended to mean a fragment that retains the function of a full-length antibody, such as an antigen-binding fragment, and in particular refers to antibody fragments that: such as Fv, scFv (sc refers to single chain), Fab, F (ab ') 2, Fab', scFv-Fc fragment or diabody (diabody), or any fragment that should be able to increase half-life by chemical modification, such as the addition of a polyalkylene glycol such as polyethylene glycol ("pegylation"), or by incorporation into liposomes (pegylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG or Fab' -PEG) ("PEG" is polyethylene glycol).

The term "CDR region" or "CDR" as used herein refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest, 5th ed, u.s.department of Health and Human Services, NIH, 1991, and later). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

The term "Fc region" or "Fc portion" as used herein is a term well known to those skilled in the art and is defined based on the papain cleavage of antibodies. The Fc portion of the antibody is directly involved in complement activation, Clq binding, C3 activation and Fc receptor binding. Although the effect of antibodies on the complement system depends on the specific conditions, binding to Clq is caused by defined binding sites in the Fc portion. Such binding sites are known in the art and described, for example, in Lukas, T.J., et al, J.Immunol (J.Immunol) 127(1981)2555-2560Jrunhouse, R., and Cebra, J.J., mol.Immunol (molecular immunology) 16(1979) 907-917; burton, D.R. et al, Nature 288(1980) 338-344; thommesen, J.E., et al, mol.Immunol. (molecular immunology) 37(2000) 995-1004; idusogie, e.e., et al, j.immunol. (journal of immunology) 164(2000) 4178-; hezareh, M.et al, J.Virol (J.Virol.) 75(2001) 12161-12168; morgan, A. et al, Immunology 86(1995) 319-324; and EP 0307434. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331 and P329 (EU catalogue numbering according to Kabat, see below). Antibodies of the subclasses IgGl, IgG2 and IgG3 generally show complement activation, Clq binding and C3 activation, whereas IgG4 does not activate the complement system, does not bind Clq and does not activate C3. Preferably, the Fc portion is a human Fc portion.

The term "Fab region" as used herein refers to the region consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") or the VL and CL domains of the light chain ("Fab light chain") or both.

As used herein, the term "scFv" or "single chain antibody fragment" refers to a single chain of an antibody heavy chain variable region and an antibody light chain variable region linearly linked together by a linker (e.g., a short peptide of 10-25 amino acids), which exhibits specific binding to an antigen.

The term "peptide linker" as used in the present invention denotes an antibody fragment (e.g. single chain Fv, full length antibody, VH domain and/or VL domain, Fab, (Fab) for conjugating different antigen binding sites and/or eventually comprising different antigen binding sites₂And an Fc portion), preferably having an amino acid sequence of synthetic origin. The peptide linker may comprise one or more of the amino acid sequences listed in table 1 below, as well as other arbitrarily selected amino acids.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope in an in vitro assay, preferably in a cell-based ELISA using CHO cells expressing wild-type antigen. Combined finger 10^-8M or less, preferably 10^-13M to 10^-9Binding affinity (KD,. of M). Binding of antibodies to antigen or FcyRIII can be studied by BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). Binding affinity is defined by the terms ka (the association rate constant of an antibody in an antibody/antigen complex), kD (dissociation constant) and kD (kD/ka).

As used herein, "therapeutically effective amount" or "effective amount" refers to a dosage sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. Prescription of treatment (e.g., decisions on dosage, etc.) is ultimately the responsibility of and depends on general practitioners and other physicians, often taking into account the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herol, geranium, crane, etc.), domestic animals (e.g., ducks, geese, etc.) or laboratory animals (e.g., orangutan, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, squirrels, etc.).

The compositions of the present invention can be administered by a variety of methods known in the art. The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the desired result. In order to administer a compound of the invention by a particular route of administration, it may be desirable to coat the compound with, or co-administer the compound with, a material that avoids its inactivation. For example, the compound can be administered to a subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline solutions and aqueous buffers. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

The compositions of the present invention may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Avoidance of the presence of microorganisms can be ensured both by the above sterilization procedure and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the invention (which may be used in a suitable hydrated form) and/or the pharmaceutical compositions of the invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The bispecific antibody of the present invention contains an Fc fragment, so that the protein can be isolated and purified by protein A affinity chromatography after cell expression, which is one of the advantages of the bispecific antibody of the present invention. After affinity chromatography, the product can be further purified by ion exchange, molecular sieve, hydrophobic chromatography, etc. The purity of the bispecific antibody can be identified by polyacrylamide gel electrophoresis, HPLC and the like. The molecular weight of the protein of interest is approximately 133Kd.

The inventors have surprisingly found that the asymmetric bispecific antibodies of the invention exhibit particularly superior properties compared to other bispecific antibodies, including but not limited to: the two different specificities are each monovalent binding (i.e., one antibody molecule binds only one epitope, such as an epitope on an immune effector cell, which can prevent the immune effector cells from coupling and killing each other); high levels of expression in cells; longer half-life in vivo; has higher biological activity; more efficient killing of tumor cells (e.g., B cells); more effective recruitment of immune effector cells (e.g., T cells and/or B cells); and less side effects.

In particular, the antibody structures of the present invention exhibit superior performance compared to bispecific antibodies of the bispecific T cell recruit (BiTE) type (e.g., Blinatumomab) of interest in bispecific antibody technology.

In order to construct bispecific antibodies using CD89 positive cells as immune effector cells, the inventors first prepared an anti-CD 89 monoclonal antibody. The antibody is obtained by conventional hybridoma technology, and then the DNA sequences of the heavy chain and light chain variable regions of the antibody are determined by antibody gene cloning, so as to obtain the amino acid sequences of the heavy chain and light chain variable regions of the antibody (shown as SEQ ID NO: 21, 22, 23 and 24 respectively). Then, based on this, the present inventors constructed bispecific antibody with BiTE structure (anti-CD 20-anti-CD 89 bispecific antibody) and asymmetric bispecific antibody with structure of the present invention, respectively, wherein the CD20 antibody uses the sequence of Rituxan, and a linker sequence similar to Blinatumomab is used between two scfvs. As a result, it was found that the anti-CD 20-anti-CD 89 bispecific antibody using BiTE structure was not expressed in HEK293 cells and CHO, or its expression level was extremely low, and it was difficult to perform further experiments, and even if a plurality of different linker sequences and transfection methods were tried, the desired expression level could not be obtained. In contrast, the asymmetric bispecific antibody of the present invention can be well expressed in HEK293 cells and CHO cells, and has excellent biological activity. Importantly, the antibodies of the invention have a more excellent half-life in vivo.

To examine the versatility of the novel bispecific antibody structure, we constructed bispecific antibodies targeting CD19 and CD3, which were constructed as anti-CD19 Fab-anti-CD 3scFv structures. We found that the bispecific antibody with this structure can be well expressed in 293 cells and CHO cells, thus proving that the asymmetric bispecific antibody of the invention has the characteristic of easy expression. Furthermore, compared with the anti-CD19 scFv-CD3scFv bispecific antibody (i.e. BiTE), we found that the AsBs Ab of our invention not only has the characteristics of easy expression, but also has the characteristics of long half-life and easy purification, and meanwhile, it retains the advantage that BiTE binds with immune effector cells and tumor cells in a monovalent manner. Therefore, the molecular structure of the AsBs Ab bispecific antibody can solve the defects of the BiTE and has high application value.

The following examples are provided to demonstrate and further illustrate some preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.

Examples

Example 1 anti-CD 89 monoclonal antibody preparation

To prepare bispecific antibodies with CD89 cells as immune effector cells, we must first obtain a monoclonal antibody with high affinity for CD 89.

We immunized BALB/c mice (Shanghai Spikex laboratories) with human CD89 recombinant protein (Sino Biologicals), splenic lymphocytes from the immunized mice were fused with SP2/0 myeloma cells (ATCC) to prepare hybridomas, and the fused cells were subjected to colony culture in 96-well cell culture plates, and positive clones were detected by ELISA coated with CD 89. And taking the hybridoma cells with positive clones for amplification culture and performing subclone culture. Several anti-CD 89 antibody hybridoma cell lines were thus obtained, which demonstrated high affinity for CD89 by ELISA and flow cytometry. Determining the anti-CD 89 monoclonal antibody hybridoma cell strain. The heavy and light chain variable region DNA sequences (SEQ ID NOS: 21, 22) of anti-CD 89 were then obtained by cloning antibody genes of hybridoma cells, and thereby the variable region amino acid sequences (SEQ ID NOS: 23, 24) of the heavy and light chains were obtained, and the six CDR region sequences (SEQ ID NOS: 9, 10, 11, 12, 13, 14) of the light and heavy chains of the antibodies were determined by sequence analysis.

Example 2 construction of anti-CD 89-anti-CD 20 asymmetric bispecific antibodies

Among the three polypeptide chains of the bispecific antibody we constituted, the first chain comprised anti-CD 89 antibody VL and human Kappa CL, CD89 antibody VL was PCR-amplified from the antibody gene obtained in example 1 using anti-CD 89 upstream and downstream primers (SEQ ID NOS: 25, 26); human Kappa CL was PCR amplified from plasmid pFUSE2-CLIg-hK (Invivogen) using CL upstream and downstream primers (SEQ ID NOS: 27, 28), and the entire anti-CD 89VL-CL chimeric light chain was synthesized by overlap PCR and cloned into expression plasmid pcDNA 3.1. The second polypeptide chain is anti-CD 89VH, human IgG constant region, and anti-CD20 scFv, anti-CD 89 antibody VH was amplified by PCR from the antibody gene obtained in example 1 using respective upstream and downstream primers (SEQ ID NO: 29, 30), and human IgG constant region was amplified by upstream and downstream primers (SEQ ID NO: 31, 32) from a related plasmid (pcDNA 3.1-hIgG1Fc-Hole, artificial gene synthesis, King of Suzhou, SEQ ID NO: 51), in which Fc contains a notch mutation (T366S, L368A, Y407V, corresponding to 146, 148 and 18 of SEQ ID NO: 4 in the present applicationPosition 7), anti-CD20 scFv was modified from (SEQ ID NO: 33. 34) related plasmid (pcDNA3.1-anti-CD20-scFv, Artificial Gene Synthesis, Kinzonly, Suzhou, SEQ ID NO: 52) amplification, and finally Synthesis of the complete CD89 VH-human IgG CH (concave) -linker sequence (G) by overlap PCR₄S)₃anti-CD20 scFv cloned into the expression plasmid pcDNA 3.1. The third strand is a human Fc containing a hinge region, containing a bulge mutation (T366W, corresponding to position 146 of SEQ ID NO: 5 in the present application), amplified from a plasmid (pcDNA 3.1-hIgG1Fc-Knob, artificial gene synthesis, King of Suzhou, SEQ ID NO: 53) using upstream and downstream primers (SEQ ID NO: 35, 36) and cloned into the expression vector pcDNA 3.1. And two enzyme cutting sites of AgeI and SalI are respectively added in front of and behind the three target sequences and the vector. Carrying out double enzyme digestion on the expression vector by using AgeI and SalI enzymes, and connecting the DNA fragments obtained in the previous step according to the sequence design sequence by a seamless cloning method. Ligase kit by seamless cloning

And (3) processing, and connecting the target fragment into a eukaryotic expression vector pcDNA3.1. And transforming the connected expression vector into DH5 alpha competent cells, then selecting clones, extracting plasmids, carrying out enzyme digestion identification, selecting positive clones, and screening out correct vector clones, thereby obtaining the vector capable of expressing the anti-CD 89-anti-CD 20 asymmetric bispecific antibody.

Furthermore, the inventors have constructed an anti-CD 89-anti-CD 20 asymmetric bispecific antibody (CD89-CD20mut) containing a mutation at position 297 (Asn to Ala) of the Fc region, the mutation being aimed at causing the Fc region to lose ADCC effector function. The recombinant plasmid obtained as described above was subjected to gene-localization mutation to make Asn at position 297 of the Fc region of both the second chain heavy chain (concave hole) and third chain Fc (convex) fragments of the vector to be mutated to Ala (corresponding to position 77 of SEQ ID NOS: 4 and 5 in this application) so that the function of ADCC effect was lost. Then, PCR identification of the bacterial suspension was carried out (as shown in FIG. 2), and DNA sequencing was carried out to select a clone having the correct DNA sequence.

Example 3 construction of anti-CD 19-anti-CD 3 asymmetric bispecific antibodies

In this bispecific antibody, FabPart is an anti-CD19 antibody whose antibody VL and VH sequences (SEQ ID NOS: 37, 38) are derived from a published murine anti-CD19 antibody; the scFv portion is an anti-CD3 antibody whose svFV sequence (SEQ ID NO: 8) is derived from a published scFv antibody. The first polypeptide chain comprises anti-CD19 antibody VL and human Kappa CL, VL is amplified from the relevant plasmid (pcDNA3.1-anti-CD19-scFv, Artificial Gene Synthesis, Kinzhi, Suzhou, SEQ ID NO: 54) by PCR with upstream and downstream primers (SEQ ID NO: 39, 40); human Kappa CL was obtained by PCR using CL upstream and downstream primers (SEQ ID NOS: 41, 42), and the entire anti-CD19 VL-CL chimeric light chain was synthesized by overlap PCR and cloned into the expression plasmid pcDNA3.1. The second polypeptide chain is anti-CD19 VH, human IgG constant region, and anti-CD 3scFv, anti-CD19 antibody VH was amplified by PCR from the relevant plasmid (pcDNA3.1-anti-CD19-scFv, artificial gene synthesis, Kirginia, SEQ ID NO: 54) with corresponding upstream and downstream primers (SEQ ID NO: 43, 44), human IgG constant region was amplified from the relevant plasmid (pcDNA 3.1-hIgG1Fc-Hole, SEQ ID NO: 51) with upstream and downstream primers (SEQ ID NO: 45, 46), Fc of which contained a notch mutation, anti-CD 3scFv was amplified from the relevant plasmid (pcDNA3.1-anti-CD 3-scFv, artificial gene synthesis, Kirginia, SEQ ID NO: 55) with upstream and downstream primers (SEQ ID NO: 47, 48), and finally complete CD19 VH-human IgG CH (notch) -joining sequence (G) was synthesized by overlap PCR₄S)₃anti-CD 3scFv cloned into an expression plasmid. The third strand is identical to the third strand in example 2. The desired sequence was cloned into an expression plasmid by the same method as in example 2, and DNA sequencing was performed to obtain a clone having the correct DNA sequence.

Example 4 bispecific antibody expression and identification

The plasmids encoding three polypeptide chains of the bispecific antibody constructed as described above were transformed into competent bacteria DH5 α, respectively, the bacteria were amplified by bacterial culture and plasmid DNA was purified. The purified plasmid DNA was transiently transfected into 293F cells by co-transfection. The GFP plasmid was used as a positive control, and 293F cells were simultaneously infected to observe the infection efficiency. We transfected 293 cells with anti-CD 89-anti-CD 20 bispecific antibody plasmid and anti-CD 19-anti-CD 3 bispecific antibody plasmid, respectively, and 48 hours after transfection, the expression level of recombinant antibodies was measured by ELISA double antibody sandwich method, which proved that both bispecific antibodies could be well expressed. After 72 hours of transfection, the culture supernatant of the transfected cells was collected and the antibody Protein was purified by Protein A affinity chromatography. Before loading, the sample solution was filtered through a 0.45um filter to remove impurities such as cells, polymers, etc., and 1/10 volumes of binding buffer were added to make the sample the same PH as the binding buffer. After the sample run out, the column was washed with 5ml (5-10 volumes) of conjugate buffer. The purified two bispecific antibodies were analyzed by SDS-PAGE and showed a 133kD band on non-reducing electrophoresis and 77kD and 26-29kD bands on reducing electrophoresis, identical to the predicted molecular weights (FIG. 3).

Example 5 flow cytometry to identify the binding of antibodies to individual targets

To identify the binding capacity of the two bispecific antibodies anti-CD 89-anti-CD 20 and anti-CD 19-anti-CD 3 purified by protein a, we performed flow cytometry experiments with Raji cells (ATCC) positive for CD19 and CD20, CD89 positive PMN cells isolated from human peripheral blood, and Jurkat cells (ATCC) positive for CD 3. Blowing and beating Raji, PMN and Jurkat cells into single cell suspension by using a pipette respectively, re-suspending the cells by using PBS, counting, and taking 1x10⁶Adding bispecific antibody with different dilutions into cells, performing ice bath for 45 minutes, washing twice with PBS, adding FITC-goat anti-human IgG (H + L) secondary antibody for labeling, performing ice bath for 45 minutes, and washing twice with PBS; after resuspension in 500ul PBS, cells were added to the flow tube, detected with a flow cytometer and fluorescence intensity calculated. The negative control group was an irrelevant antibody control. The results show that the two bispecific antibodies have good affinity for their respective antigens (fig. 4, fig. 5).

Example 6 detection of the Effect of anti-CD 89-anti-CD 20 bispecific antibodies on killing tumor cells

To determine the killing effect of the anti-CD 89-anti-CD 20 bispecific antibody on CD20 positive cells, we tested Raji cells using the CytoTox 96 kit. The CytoTox 96 kit quantitatively detects the level of Lactate Dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis in a manner substantially identical to that of [51Cr ] released in a radioactive assay, in proportion to the number of dead cells. A fixed number of Raji cells were added to a 96-well plate, effector cells human PMNs or PBMCs were added, bispecific antibody was diluted with different dilution gradients of 0.1% BSA/PBS (w/v) were added, lysate (Lysis Solution) (10X) was added, and after 4 hours of incubation, centrifugation was carried out at 250Xg for 4 minutes. Transfer 50. mu.l of supernatant from each well to a 96-well plate using a row gun, add 50. mu.l of substrate to each well, cover the plate with aluminum foil paper or opaque box and incubate for 30 minutes at room temperature in the dark. The results are shown in FIG. 6.

The anti-CD 89-anti-CD 20 bispecific antibody was very effective in killing tumor cells in the presence of PMNs (mostly neutrophils), whereas the control rituximab (CD20-Ig) was not able to kill tumor cells by PMN-mediated killing (PMN cells lack NK cells), while the anti-CD 89-anti-CD 20 bispecific antibody was not effective in killing tumor cells by Fc-mediated ADCC, since Fc mutation of the anti-CD 89-anti-CD 20 bispecific antibody (CD89-CD20mut) to abrogate ADCC function still killed tumor cells well (fig. 6), indicating that the anti-CD 89-anti-CD 20 bispecific antibody recruits PMN effector cells (mostly neutrophils) by CD 89.

The anti-CD 89-anti-CD 20 bispecific antibody of the invention kills tumor cells in the presence of PBMC, but after Fc mutation (CD89-CD20mut) to remove ADCC function, the bispecific antibody loses its killing ability against tumors, indicating that the killing ability by PBMC is Fc-mediated ADCC effect. Similarly, the control rituximab (CD20-Ig) was effective in killing tumor cells via ADCC.

The inventor successfully constructs the bispecific antibody which kills tumor cells by using a PMN (most of neutrophils) mediated cell killing function based on a CD89 surface antigen for the first time, which is a breakthrough in the field of immunotherapy and develops a new subdivided field.

Example 7 detection of T cell killing Capacity in vitro mediated by anti-CD 19-anti-CD 3 bispecific antibody

The killing effect of the anti-CD 19-anti-CD 3 bispecific antibody on CD19 positive cells and the killing capability of the antibody mediated T cells are detected by using a CytoTox 96 non-radioactive cytotoxicity kit. The T cells used were derived from PBMC cells isolated from fresh human peripheral blood and collected for use after 4-5 days of activation with IL-2(10U/ml) and CD3 antibodies. The tumor cells are Raji cells highly expressing CD19 antigen. The target-effect ratio of the tumor target cells to the effector T cells is 1: 10. The working concentration of bispecific antibody was diluted in a ten-fold gradient. The results show that the anti-CD 19-anti-CD 3 bispecific antibody was able to mediate very efficiently the killing effect of T cells on CD19 positive tumor cells with an EC50 value of about 2ng/ml (fig. 7).

The experimental result shows that the asymmetric bispecific antibody structure of the invention can be not only used for two specific targets of anti-CD 89 and anti-CD20, but also be widely applied to a plurality of different targets as a novel bispecific antibody platform. In particular, the asymmetric bispecific antibody structure of the present invention overcomes many of the drawbacks of the prior art (e.g., BiTE technology), and the flexibility and adaptability of its spatial structure enables it to construct a variety of bispecific antibodies against different targets for a variety of different uses.

Example 8 serum half-life assay of anti-CD 89-anti-CD 20 bispecific antibodies

The pharmacokinetics of the anti-CD 89-anti-CD 20 bispecific antibody in animals is studied by taking the anti-CD 89-anti-CD 20 bispecific antibody as an example. 6-8 weeks old C57BL/6 female mice were treated with 5 mice each and 100. mu.g of the antibody per mouse was injected via tail vein with rituximab or bispecific antibody. And orbital bleeds were performed at 2, 4, 8, 24, 48, 72, 96, 120, 144 hours after injection to detect the IgG levels in vivo by ELISA double antibody sandwich method (figure 8). The results unexpectedly found that the bispecific antibody of the present invention has a longer in vivo half-life compared to BiTE (half-life of only 2.1 hours), which is similar to that of rituximab, and thus, the antibody of the present invention combines the advantages of the long half-life of conventional mab and the dual specificity of the existing bispecific antibody, providing a more powerful weapon for clinical treatment.

Example 9 in vivo experiment for tumor treatment with anti-CD 89-anti-CD 20 bispecific antibody

CD20-LLC cells (mouse Lewis lung cancer cell line stably expressing human CD20 antigen) in logarithmic growth phase were inoculated into the right side of the mouseHind limb subcutaneous, 1x10 inoculation per mouse⁶Cells, after inoculation until the tumor volume reaches about 100-150mm³At the time, the mice were randomly divided into groups of 6-8 mice each, and each group was administered once a week by tail vein injection. The treatment group was given a dose of 10mg/kg of drug in a volume of 0.2ml, and the control group was given the same volume of PBS. The length and width of the mouse tumor was measured every two days with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was plotted until the tumors of the PBS group grew to about 1000mm³. The results showed that the anti-CD 89-anti-CD 20 bispecific antibody had very low tumor killing efficiency in the wild type mouse model test, which was substantially comparable to the PBS control group, since there was no CD89 receptor in the wild type mouse, and thus there was substantially no anti-tumor effect of anti-CD 89-anti-CD 20, and on the contrary, rituximab had good anti-tumor ability and was able to inhibit tumor growth (fig. 9). To determine the anti-tumor effect of the anti-CD 89-anti-CD 20 bispecific antibody, we further studied in FC α RI (CD89) transgenic mouse models and found that the anti-CD 89-anti-CD 20 bispecific antibody has the ability to significantly inhibit tumor growth, and surprisingly, its anti-tumor effect was even significantly better than that of the commercial rituximab (CD20-IgG) (fig. 9).

Example 10 in vivo experiment for tumor treatment with anti-CD 19-anti-CD 3 bispecific antibody

Raji cells in logarithmic growth phase were inoculated subcutaneously on the right hindlimb of nude mice, 3x10 per mouse⁶Cell suspension, 6-8 mice per group. Simultaneously, the human T lymphocytes amplified in vitro in advance are injected into mice by tail vein, 1x10⁷Each mouse. The anti-CD 19-anti-CD 3 bispecific antibody was co-administered once every 3 days for 3 times (days 0, 3, 6) at a dose of 5 mg/kg. All adopt tail vein injection for administration. PBS group is control group, T cell group is injected with T cell only, length and width of mouse tumor are measured with vernier caliper about every two days, tumor volume is calculated, and tumor growth curve is drawn from 0 day of treatment administration day until PBS group tumor grows to about 1000mm³. The results show that anti-CD 19-anti-CD 3 bispecific antibodies activate T cells in vivo in a nude mouse-human T cell model assayUnder the action of cells, the killing efficiency to tumors is very high. The tumor reaches 100-150mm 15 days after inoculation³Significantly later than the PBS group. Mice injected with T cells alone, which received no antibody drug treatment, had tumor growth substantially comparable to the PBS control group (figure 10).

The foregoing description is of the preferred embodiments only, which are by way of example only and do not limit the combination of features necessary to practice the invention. The headings provided are not meant to limit the various embodiments of the invention. Terms such as "comprising," "including," and "including" are not intended to be limiting. Furthermore, unless otherwise indicated, the absence of a numerical modification includes the plural, and "or", "or" means "and/or". Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

All publications and patents mentioned in this application are herein incorporated by reference. Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

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[13]Reterink，T.J.，E.W.Levarht，N.KJ et al.Transforming growth fator-beta 1(TGF-beta 1)down-regulates IgA Fc-recepter(CD89)expression on human monocytes.[J].Clin.Exp Immunol.1996.103：161-166.

[14]Sehiell，rC.，A.SPittle，M.Willheim et al.Influence of suramim on the experssion of Fc receptor on human monocytes and U937cells，and on their phagocytic properties.[J].Immunology 1994.81：598-604.

[15]Solinas G，Germano G，Mantovani A，Allavena P.Tumor-associated macrophages(TAM)as major players of the cancer-related inflammation.[J].J Leukoc Biol.2009，86(5)：1065-73.

[16]Sica A，Schioppa T，Mantovani A，Allavena P.Tumour-associated macrophages are a distinct M2polarised population promoting tumour progression：potential targets of anti-cancer therapy.[J].Eur J Cancer.2006，42(6)：717-27.

[17]Bakema JE，Ganzevles SH，Fluitsma DM，etc.Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy.[J].Immunol.2011，187(2)：726-32.

[18]Ridgway，et al.，′Knobs-into-holes′engineering of antibody CH3domains for heavy chain heterodimerization.Protein Engineering vol.9no.7pp.617-621，1996

SEQ ID NO:1 (anti-CD 89 light chain antibodies VL and CL) amino acid sequence

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerLeuGlyGluArgValSerLeuThrCysArgAlaSerGlnAspIleGlySerSerLeuAsnTrpLeuGlnGlnGluProAspGlyThrIleLysArgLeuIleTyrAlaThrSerSerLeuAspSerGlyValProGluArgPheSerGlySerArgSerGlySerHisTyrSerLeuThrIleSerSerLeuGluSerGluAspPheValAspTyrTyrCysLeuGlnTyrAlaSerTyrProTrpThrPheGlyGlyGlyThrLysLeuGluIleLysArgThrValAlaAlaProSerValPheIlePheProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGluCys

SEQ ID NO:2 the second chain of heavy chain antibodies of anti-CD 89VH and CH1 amino acid sequences

GlnIleGlnLeuValGlnSerGlyProGluLeuLysLysProGlyGluThrValLysIleSerCysLysAlaSerGlyTyrValPheThrAsnTyrGlyMetAsnTrpValLysGlnThrProGlyLysGlyLeuLysTrpMetGlyTrpIleAsnThrTyrThrGlyArgProThrSerAlaAspAspPheLysGlyArgPheAlaPheSerLeuGluThrSerAlaSerThrAlaTyrLeuGlnIleAsnAsnLeuLysAsnGluAspThrAlaThrTyrPheCysSerSerGlnGlyPheSerPheThrSerTrpGlyGlnGlyThrLeuValThrValSerAlaAlaSerThrLysGlyProSerValPheProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPheProGluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysValAspLysLysValGluProLysSerCys

SEQ ID NO: 3 (ScFv against CD 20) amino acid sequence of the scFv region of the second chain

GlnAlaTyrLeuGlnGlnSerGlyAlaGluLeuValArgProGlyAlaSerValLysMetSerCysLysAlaSerGlyTyrThrPheThrSerTyrAsnMetHisTrpValLysGlnThrProArgGlnGlyLeuGluTrpIleGlyAlaIleTyrProGlyAsnGlyAspThrSerTyrAsnGlnLysPheLysGlyLysAlaThrLeuThrValAspLysSerSerSerThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrPheCysAlaArgValValTyrTyrSerAsnSerTyrTrpTyrPheAspValTrpGlyThrGlyThrThrValThrValSerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlnIleValLeuSerGlnSerProAlaIleLeuSerAlaSerProGlyGluLysValThrMetThrCysArgAlaSerSerSerValSerTyrMetHisTrpTyrGlnGlnLysProGlySerSerProLysProTrpIleTyrAlaProSerAsnLeuAlaSerGlyValProAlaArgPheSerGlySerGlySerGlyThrSerTyrSerLeuThrIleSerArgValGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnTrpSerPheAsnProProThrPheGlyAlaGlyThrLysLeuGluLeu

SEQ ID NO: 4 (c) the nucleic acid sequence of the second chain Fc region (Fc-Hole fragment)

AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeuMetIleSerArgThrProGluValThrCysValValValAspValSerHisGluAspProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAlaSerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeuSerCysAlaValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePheLeuValSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys

SEQ ID NO: 5 the amino acid sequence of the Fc region (Fc-Knob fragment) of the third chain

AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeuMetIleSerArgThrProGluValThrCysValValValAspValSerHisGluAspProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAlaSerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProlleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeuTrpCysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys

SEQ ID NO: 6 (anti-CD 19 light chain antibodies VL and CL) amino acid sequence of the first chain

AspIleGlnLeuThrGlnSerProAlaSerLeuAlaValSerLeuGlyGlnArgAlaThrIleSerCysLysAlaSerGlnSerValAspTyrAspGlyAspSerTyrLeuAsnTrpTyrGlnGlnIleProGlyGlnProProLysLeuLeuIleTyrAspAlaSerAsnLeuValSerGlyIleProProArgPheSerGlySerGlySerGlyThrAspPheThrLeuAsnIleHisProValGluLysValAspAlaAlaThrTyrHisCysGlnGlnSerThrGluAspProTrpThrPheGlyGlyGlyThrLysLeuGluIleLysArgThrValAlaAlaProSerValPheIlePheProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGluCys

SEQ ID NO: 7 the second chain anti-CD19 heavy chain antibody VH and CH1 amino acid sequences

GlnValGlnLeuGlnGlnSerGlyAlaGluLeuValArgProGlySerSerValLysIleSerCysLysAlaSerGlyTyrAlaPheSerSerTyrTrpMetAsnTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIleGlyGlnIleTrpProGlyAspGlyAspThrAsnTyrAsnGlyLysPheLysGlyLysAlaThrLeuThrAlaAspGluSerSerSerThrAlaTyrMetGlnLeuSerSerLeuAlaSerGluAspSerAlaValTyrPheCysAlaArgArgGluThrThrThrValGlyArgTyrTyrTyrAlaMetAspTyrTrpGlyGlnGlyThrThrValThrValSerSerAlaSerThrLysGlyProSerValPheProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPheProGluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValProSerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysValAspLysLysValGluProLysSerCys

SEQ ID NO: 8 (ScFv against CD 3) nucleic acid sequence of scFv region of the second chain

AspIleLysLeuGlnGlnSerGlyAlaGluLeuAlaArgProGlyAlaSerValLysMetSerCysLysThrSerGlyTyrThrPheThrArgTyrThrMetHisTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIleGlyTyrIleAsnProSerArgGlyTyrThrAsnTyrAsnGlnLysPheLysAspLysAlaThrLeuThrThrAspLysSerSerSerThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCysAlaArgTyrTyrAspAspHisTyrCysLeuAspTyrTrpGlyGlnGlyThrThrLeuThrValSerSerValGluGlyGlySerGlyGlySerGlyGlySerGlyGlySerGlyGlyValAspAspIleGlnLeuThrGlnSerProAlaIleMetSerAlaSerProGlyGluLysValThrMetThrCysArgAlaSerSerSerValSerTyrMetAsnTrpTyrGlnGlnLysSerGlyThrSerProLysArgTrpIleTyrAspThrSerLysValAlaSerGlyValProTyrArgPheSerGlySerGlySerGlyThrSerTyrSerLeuThrIleSerSerMetGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnTrpSerSerAsnProLeuThrPheGlyAlaGlyThrLysLeuGluLeuLys

SEQ ID NO:9 anti-CD 89 antibody VL CDR1

GlnAspIleGlySerSer

SEQ ID NO:10 anti-CD 89 antibody VL CDR2

AlaThrSer

SEQ ID NO:11 CD89 antibody VL CDR3

LeuGlnTyrAlaSerTyrProTrpThr

SEQ ID NO:12 anti-CD 89 antibody VH CDR1

GlyTyrValPheThrAsnTyrGly

SEQ ID NO:13 anti-CD 89 antibody VH CDR2

IleAsnThrTyrThrGlyArgPro

SEQ ID NO:14 anti-CD 89 antibody VH CDR3

SerSerGlnGlyPheSerPheThrSer

SEQ ID NO: 15 anti-CD19 antibody VL CDR1

GlnSerValAspTyrAspGlyAspSerTyr

SEQ ID NO: 16 anti-CD19 antibody VL CDR2

AspAlaSer

SEQ ID NO: 17 anti-CD19 antibody VL CDR3

GlnGlnSerThrGluAspProTrpThr

SEQ ID NO: 18 anti-CD19 antibody VH CDR1

GlyTyrAlaPheSerSerTyrTrp

SEQ ID NO: 19 anti-CD19 antibody VH CDR2

IleTrpProGlyAspGlyAspThr

SEQ ID NO: 20 anti-CD19 antibody VH CDR3

AlaArgArgGluThrThrThrValGlyArgTyrTyrTyrAlaMetAspTyr

SEQ ID NO: 21 anti-CD 89 heavy chain variable region DNA sequence

SEQ ID NO: 22 anti-CD 89 light chain variable region DNA sequence

SEQ ID NO:23 anti-CD 89 heavy chain variable region amino acid sequence

GlnIleGlnLeuValGlnSerGlyProGluLeuLysLysProGlyGluThrValLysIleSerCysLysAlaSerGlyTyrValPheThrAsnTyrGlyMetAsnTrpValLysGlnThrProGlyLysGlyLeuLysTrpMetGlyTrpIleAsnThrTyrThrGlyArgProThrSerAlaAspAspPheLysGlyArgPheAlaPheSerLeuGluThrSerAlaSerThrAlaTyrLeuGlnIleAsnAsnLeuLysAsnGluAspThrAlaThrTyrPheCysSerSerGlnGlyPheSerPheThrSerTrpGlyGlnGlyThrLeuValThrValSerAla

SEQ ID NO:24 anti-CD 89 light chain variable region amino acid sequence

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerLeuGlyGluArgValSerLeuThrCysArgAlaSerGlnAspIleGlySerSerLeuAsnTrpLeuGlnGlnGluProAspGlyThrIleLysArgLeuIleTyrAlaThrSerSerLeuAspSerGlyValProGluArgPheSerGlySerArgSerGlySerHisTyrSerLeuThrIleSerSerLeuGluSerGluAspPheValAspTyrTyrCysLeuGlnTyrAlaSerTyrProTrpThrPheGlyGlyGlyThrLysLeuGluIleLys

SEQ ID NO: 25 anti-CD 89VL fragment upstream primer

TAAGCTTGGTACCGAGCTCGGATCCGCCGCCACCATGGTCAGCTACTGGGACACC

SEQ ID NO: 26 anti-CD 89VL fragment downstream primer

GATGGTGCAGCCACAGTTCGTTTGATTTCCAGCTTGGTGC

SEQ ID NO: upstream primer of 27 CL fragment

GCACCAAGCTGGAAATCAAACGAACTGTGGCTGCACCATC

SEQ ID NO: downstream primer of 28 CL fragment

GCGGGCCCTCTAGACTCGAGCGGCCGCGTCGACCTAACACTCTCCCCTGTTGAAGCTCT

SEQ ID NO: upstream primer of 29 CD89VH fragment

TAAGCTTGGTACCGAGCTCGGATCCGCCGCCACCATGGTCAGCTACTGGGACACC

SEQ ID NO: downstream primer of 30 CD89VH fragment

GATGGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAG

SEQ ID NO: 31 human IgG constant region upstream primer

CTCTGGTCACTGTCTCTGCAGCCTCCACCAAGGGCCCATC

SEQ ID NO: 32 human IgG constant region downstream primer

TTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTG

SEQ ID NO: upstream primer of 33 CD20ScFv fragment

GTGGTGGTAGCGGTGGCGGTGGTAGTCAGGCTTATTTGCAACAGTCTGGCGCG

SEQ ID NO: downstream primer of 34 CD20ScFv fragment

CCAAGCTGGAGCTGGAATAGGTCGACGCGGCCGCTCGAGTCTAGAGGGCCCGCG

SEQ ID NO: 35 human Fc (bulge) upstream primer containing hinge region

AAGCTTGGTACCGAGCTCGGATCCGCCGCCACCATGGTCAGCTACTGGGACAC

SEQ ID NO: 36 human Fc (bulge) downstream primer with hinge region

CGCGGGCCCTCTAGACTCGAGCGGCCGCGTCGACCTATTTACCCGGAGACAGGGAGAG

SEQ ID NO: 37 anti-CD19 light chain variable region amino acid sequence

AspIleGlnLeuThrGlnSerProAlaSerLeuAlaValSerLeuGlyGlnArgAlaThrIleSerCysLysAlaSerGlnSerValAspTyrAspGlyAspSerTyrLeuAsnTrpTyrGlnGlnIleProGlyGlnProProLysLeuLeuIleTyrAspAlaSerAsnLeuValSerGlyIleProProArgPheSerGlySerGlySerGlyThrAspPheThrLeuAsnIleHisProValGluLysValAspAlaAlaThrTyrHisCysGlnGlnSerThrGluAspProTrpThrPheGlyGlyGlyThrLysLeuGluIleLys

SEQ ID NO: 38 anti-CD19 heavy chain variable region amino acid sequence

GlnValGlnLeuGlnGlnSerGlyAlaGluLeuValArgProGlySerSerValLysIleSerCysLysAlaSerGlyTyrAlaPheSerSerTyrTrpMetAsnTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIleGlyGlnIleTrpProGlyAspGlyAspThrAsnTyrAsnGlyLysPheLysGlyLysAlaThrLeuThrAlaAspGluSerSerSerThrAlaTyrMetGlnLeuSerSerLeuAlaSerGluAspSerAlaValTyrPheCysAlaArgArgGluThrThrThrValGlyArgTyrTyrTyrAlaMetAspTyrTrpGlyGlnGlyThrThrValThrValSerSer

SEQ ID NO: 39 anti-CD19 VL fragment upstream primer

GTCTGCTTCTCACAGGATCTAGTTCCGGAGATATCCAACTGACCCAGAGC

SEQ ID NO: 40 anti-CD19 VL fragment downstream primer

GATGGTGCAGCCACAGTTCGCTTGATTTCCAGTTTTGTGC

SEQ ID NO: upstream primer of 41 CL fragment

AAACTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTC

SEQ ID NO: downstream primer of 42 CL fragment

GCGGGCCCTCTAGACTCGAGCGGCCGCGTCGACCTAACACTCTCCCCTGTTGAAGCTCT

SEQ ID NO: 43 anti-CD19 VH fragment upstream primer

TCTGCTTCTCACAGGATCTAGTTCCGGACAGGTGCAGCTCCAGCAAAGC

SEQ ID NO: downstream primer of 44 anti-CD19 VH fragment

GATGGGCCCTTGGTGGAGGCGCTGCTCACTGTCACTGTGG

SEQ ID NO: upstream primer of 45 human IgG constant region fragment

GGGAACCACAGTGACAGTGAGCAGCGCCTCCACCAAGGGCCCATCGGT

SEQ ID NO: downstream primer of 46 human IgG constant region fragment

TTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTG

SEQ ID NO: 47 upstream primer of anti-CD 3ScFv fragment

GGTAGTGACATCAAACTCCAACAGAGCGGAGCCGAAC

SEQ ID NO: 48 anti-CD 3ScFv fragment downstream primer

GCGGGCCCTCTAGACTCGAGCGGCCGCGTCGACCTACTTCAGCTCCAGCTTGGTGCCG

SEQ ID NO: 49 sequence of the Fc region (Fc-notch fragment) of said second chain which does not contain the Asn to Ala mutation at position 297 of the Fc region

AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeuMetIleSerArgThrProGluValThrCysValValValAspValSerHisGluAspProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeuSerCysAlaValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePheLeuValSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys

SEQ ID NO: 50 sequence of the Fc region (Fc-bulge fragment) of said third chain which does not contain the Asn to Ala mutation at position 297 of the Fc region

AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysProLysAspThrLeuMetlleSerArgThrProGluValThrCysValValValAspValSerHisGluAspProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSerLeuTrpCysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerProGlyLys

SEQ ID NO: 51 DNA sequence of artificial gene synthesized IgG constant region (containing Fc-concave fragment)

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCAGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTCCTGCGCGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ ID NO: 52 DNA sequence of anti-CD20-scFv artificially synthesized by gene

SEQ ID NO: 53 DNA sequence of an artificially genetically synthesized IgG constant region (comprising Fc-bulge fragment)

SEQ ID NO: 54 DNA sequence of anti-CD19-scFv artificially synthesized by gene

GATATCCAACTGACCCAGAGCCCCGCTAGCCTGGCCGTCAGCCTGGGCCAGAGGGCCACCATTTCCTGCAAGGCTAGCCAGAGCGTCGACTACGACGGCGACTCCTACCTGAACTGGTACCAGCAGATTCCTGGCCAGCCTCCCAAGCTGCTGATCTATGACGCCTCCAATCTGGTGAGCGGCATCCCCCCCAGATTTTCCGGCAGCGGCTCCGGCACCGATTTTACCCTGAACATCCACCCCGTCGAGAAAGTGGATGCCGCCACCTACCACTGCCAGCAGAGCACAGAGGATCCCTGGACCTTCGGAGGCGGCACAAAACTGGAAATCAAGGGCGGCGGCGGAAGCGGAGGAGGAGGATCCGGAGGAGGCGGAAGCCAGGTGCAGCTCCAGCAAAGCGGCGCCGAGCTGGTGAGACCCGGAAGCTCCGTGAAAATCAGCTGCAAGGCCTCCGGCTACGCCTTCTCCTCCTACTGGATGAACTGGGTGAAGCAGAGACCTGGACAAGGCCTCGAGTGGATCGGACAGATCTGGCCCGGCGACGGAGACACCAACTACAATGGCAAATTTAAAGGAAAAGCCACACTGACCGCTGACGAGAGCTCCTCCACAGCCTATATGCAACTGAGCTCCCTGGCCTCCGAGGATAGCGCCGTCTACTTCTGCGCTAGGAGAGAGACCACCACCGTGGGCAGATATTACTACGCCATGGATTACTGGGGCCAGGGAACCACAGTGACAGTGAGCAGC

SEQ ID NO: 55 DNA sequence of anti-CD3-scFv artificially synthesized by gene

Sequence listing

<110> Shanghai city Hospital of same economic nature

<120> an asymmetric bispecific antibody

<130> MP1703081

<160> 55

<170> PatentIn version 3.3

<210> 1

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 1

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Glu Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser His Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 2

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 2 VH CH1

<400> 2

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Arg Pro Thr Ser Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ser Ser Gln Gly Phe Ser Phe Thr Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215

<210> 3

<211> 241

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 2 scFv

<400> 3

Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Thr Gly Thr Thr Val Thr Val Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro

130 135 140

Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

145 150 155 160

Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Ser Ser Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly

180 185 190

Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

195 200 205

Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu

225 230 235 240

Leu

<210> 4

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 2 Fc

<400> 4

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 5

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 3 Fc

<400> 5

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 6

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 1 VL CL CD19

<400> 6

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 7

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 2 VH CH1 CD19

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys

225

<210> 8

<211> 243

<212> PRT

<213> Artificial Sequence

<220>

<223> Chain 2 scFv CD3

<400> 8

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys

<210> 9

<211> 6

<212> PRT

<213> Mus musculus

<400> 9

Gln Asp Ile Gly Ser Ser

1 5

<210> 10

<211> 3

<212> PRT

<213> Mus musculus

<400> 10

Ala Thr Ser

1

<210> 11

<211> 9

<212> PRT

<213> Mus musculus

<400> 11

Leu Gln Tyr Ala Ser Tyr Pro Trp Thr

1 5

<210> 12

<211> 8

<212> PRT

<213> Mus musculus

<400> 12

Gly Tyr Val Phe Thr Asn Tyr Gly

1 5

<210> 13

<211> 8

<212> PRT

<213> Mus musculus

<400> 13

Ile Asn Thr Tyr Thr Gly Arg Pro

1 5

<210> 14

<211> 9

<212> PRT

<213> Mus musculus

<400> 14

Ser Ser Gln Gly Phe Ser Phe Thr Ser

1 5

<210> 15

<211> 10

<212> PRT

<213> Mus musculus

<400> 15

Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr

1 5 10

<210> 16

<211> 3

<212> PRT

<213> Mus musculus

<400> 16

Asp Ala Ser

1

<210> 17

<211> 9

<212> PRT

<213> Mus musculus

<400> 17

Gln Gln Ser Thr Glu Asp Pro Trp Thr

1 5

<210> 18

<211> 8

<212> PRT

<213> Mus musculus

<400> 18

Gly Tyr Ala Phe Ser Ser Tyr Trp

1 5

<210> 19

<211> 8

<212> PRT

<213> Mus musculus

<400> 19

Ile Trp Pro Gly Asp Gly Asp Thr

1 5

<210> 20

<211> 17

<212> PRT

<213> Mus musculus

<400> 20

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

1 5 10 15

Tyr

<210> 21

<211> 426

<212> DNA

<213> Mus musculus

<400> 21

atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60

acaggatcta gttccggaca gatccagttg gtgcaatctg gacctgagct gaagaagccc 120

ggagagacag tcaagatctc ctgcaaggct tcggggtatg tcttcacaaa ctatggaatg 180

aactgggtga agcagactcc aggaaagggt ttaaagtgga tgggctggat aaacacctac 240

actggcaggc caacatctgc tgatgacttc aagggacggt ttgccttctc tttggaaacc 300

tctgccagca ctgcctattt gcagatcaac aacctcaaaa atgaggacac ggctacatat 360

ttctgttcaa gccaggggtt ttcgtttact tcctggggcc aggggactct ggtcactgtc 420

tctgca 426

<210> 22

<211> 399

<212> DNA

<213> Mus musculus

<400> 22

atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60

acaggatcta gttccggaga catccagatg acccagtctc catcctcctt atctgcctct 120

ctgggagaaa gagtcagtct cacttgtcgg gcaagtcagg acattggtag tagtttaaac 180

tggcttcagc aggaaccaga tggaactatt aaacgcctga tctacgccac atccagttta 240

gattctggtg tccccgaaag gttcagtggc agtaggtctg ggtcacatta ttctctcacc 300

atcagcagcc ttgagtctga agattttgta gactattact gtctacaata tgctagttat 360

ccgtggacgt tcggtggagg caccaagctg gaaatcaaa 399

<210> 23

<211> 116

<212> PRT

<213> Mus musculus

<400> 23

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Arg Pro Thr Ser Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ser Ser Gln Gly Phe Ser Phe Thr Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 24

<211> 107

<212> PRT

<213> Mus musculus

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Glu Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser His Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> CD89 VL upstream primer

<400> 25

taagcttggt accgagctcg gatccgccgc caccatggtc agctactggg acacc 55

<210> 26

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> CD89 VL downstream primer

<400> 26

gatggtgcag ccacagttcg tttgatttcc agcttggtgc 40

<210> 27

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> CL upstream primer

<400> 27

gcaccaagct ggaaatcaaa cgaactgtgg ctgcaccatc 40

<210> 28

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> CL down stream primer

<400> 28

gcgggccctc tagactcgag cggccgcgtc gacctaacac tctcccctgt tgaagctct 59

<210> 29

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> CD89 VH upstream primer

<400> 29

taagcttggt accgagctcg gatccgccgc caccatggtc agctactggg acacc 55

<210> 30

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> CD89 VH downstream primer

<400> 30

gatgggccct tggtggaggc tgcagagaca gtgaccagag 40

<210> 31

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> human IgG constant upstream primer

<400> 31

ctctggtcac tgtctctgca gcctccacca agggcccatc 40

<210> 32

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> human IgG constant downstream primerr

<400> 32

tttacccgga gacagggaga ggctcttctg cgtg 34

<210> 33

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> CD20 scFv upstream primer

<400> 33

gtggtggtag cggtggcggt ggtagtcagg cttatttgca acagtctggc gcg 53

<210> 34

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> CD20 scFv downstream primer

<400> 34

ccaagctgga gctggaatag gtcgacgcgg ccgctcgagt ctagagggcc cgcg 54

<210> 35

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> human Fc knob upstream primer

<400> 35

aagcttggta ccgagctcgg atccgccgcc accatggtca gctactggga cac 53

<210> 36

<211> 58

<212> DNA

<213> Artificial Sequence

<220>

<223> human Fc-knob downstream primer

<400> 36

cgcgggccct ctagactcga gcggccgcgt cgacctattt acccggagac agggagag 58

<210> 37

<211> 111

<212> PRT

<213> Mus musculus

<400> 37

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 38

<211> 124

<212> PRT

<213> Mus musculus

<400> 38

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 39

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 VL upstream primer

<400> 39

gtctgcttct cacaggatct agttccggag atatccaact gacccagagc 50

<210> 40

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 VL downstream primer

<400> 40

gatggtgcag ccacagttcg cttgatttcc agttttgtgc 40

<210> 41

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> CL upstream primer

<400> 41

aaactggaaa tcaagcgaac tgtggctgca ccatctgtct tc 42

<210> 42

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> CL downstream primer

<400> 42

gcgggccctc tagactcgag cggccgcgtc gacctaacac tctcccctgt tgaagctct 59

<210> 43

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 VH upstream primer

<400> 43

tctgcttctc acaggatcta gttccggaca ggtgcagctc cagcaaagc 49

<210> 44

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 VH downstream primer

<400> 44

gatgggccct tggtggaggc gctgctcact gtcactgtgg 40

<210> 45

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> human IgG constant upstream primer

<400> 45

gggaaccaca gtgacagtga gcagcgcctc caccaagggc ccatcggt 48

<210> 46

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> human IgG constant downstream primer

<400> 46

tttacccgga gacagggaga ggctcttctg cgtg 34

<210> 47

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3 scFv upstream primer

<400> 47

ggtagtgaca tcaaactcca acagagcgga gccgaac 37

<210> 48

<211> 58

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3 scFv downstream primer

<400> 48

gcgggccctc tagactcgag cggccgcgtc gacctacttc agctccagct tggtgccg 58

<210> 49

<211> 227

<212> PRT

<213> Artificial

<220>

<223> Fc-hole

<400> 49

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 50

<211> 227

<212> PRT

<213> Artificial

<220>

<223> Fc-knob

<400> 50

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 51

<211> 990

<212> DNA

<213> Artificial

<220>

<223> IgG constant Fc-hole

<400> 51

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcag ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgtcctgc gcggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctcgtc agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa 990

<210> 52

<211> 723

<212> DNA

<213> Artificial

<220>

<223> CD20 scFv

<400> 52

caggcttatt tgcaacagtc tggcgcggaa cttgtaagac caggggcttc tgtgaagatg 60

agctgcaagg ctagtggata tacattcacg tcctataata tgcactgggt caagcagact 120

ccccggcaag gcctggaatg gatcggagca atctaccctg gtaacggaga tacctcctat 180

aatcagaaat tcaaggggaa agccaccctt accgtggata aatctagtag caccgcctac 240

atgcagctgt cctcactcac atcagaggac tccgccgtct acttctgtgc ccgcgtggtt 300

tactattcaa acagctactg gtactttgac gtttggggga caggcaccac tgtgactgtg 360

agcggtggtg gtggttctgg cggcggcggc tccggtggtg gtggttctca aattgtgctg 420

tcccagtccc cggccatcct ttcagccagt ccaggagaaa aagtcacgat gacctgtaga 480

gcttcctcaa gtgtgtctta tatgcactgg tatcagcaga agccaggatc atctcccaaa 540

ccatggatat acgccccttc caatctcgcc agcggagtcc ctgcacgctt cagcggtagc 600

ggctctggga cttcttacag tctcactatc agtagggtgg aagctgagga cgcagccaca 660

tactattgcc agcaatggag ctttaacccc cccacattcg gcgctggcac caagctggag 720

ctg 723

<210> 53

<211> 681

<212> DNA

<213> Artificial

<220>

<223> IgG constant Fc-knob

<400> 53

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacgc cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 54

<211> 750

<212> DNA

<213> Artificial

<220>

<223> CD19 scFv

<400> 54

gatatccaac tgacccagag ccccgctagc ctggccgtca gcctgggcca gagggccacc 60

atttcctgca aggctagcca gagcgtcgac tacgacggcg actcctacct gaactggtac 120

cagcagattc ctggccagcc tcccaagctg ctgatctatg acgcctccaa tctggtgagc 180

ggcatccccc ccagattttc cggcagcggc tccggcaccg attttaccct gaacatccac 240

cccgtcgaga aagtggatgc cgccacctac cactgccagc agagcacaga ggatccctgg 300

accttcggag gcggcacaaa actggaaatc aagggcggcg gcggaagcgg aggaggagga 360

tccggaggag gcggaagcca ggtgcagctc cagcaaagcg gcgccgagct ggtgagaccc 420

ggaagctccg tgaaaatcag ctgcaaggcc tccggctacg ccttctcctc ctactggatg 480

aactgggtga agcagagacc tggacaaggc ctcgagtgga tcggacagat ctggcccggc 540

gacggagaca ccaactacaa tggcaaattt aaaggaaaag ccacactgac cgctgacgag 600

agctcctcca cagcctatat gcaactgagc tccctggcct ccgaggatag cgccgtctac 660

ttctgcgcta ggagagagac caccaccgtg ggcagatatt actacgccat ggattactgg 720

ggccagggaa ccacagtgac agtgagcagc 750

<210> 55

<211> 729

<212> DNA

<213> Artificial

<220>

<223> CD3 scFv

<400> 55

gacatcaaac tccaacagag cggagccgaa ctggccagac ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta taccttcacc aggtatacca tgcattgggt gaaacagaga 120

cccggacagg gactggagtg gatcggctac atcaacccct ccaggggcta caccaattac 180

aaccagaaat tcaaggacaa ggccaccctg accaccgaca aaagctcctc cacagcttac 240

atgcagctga gctccctgac aagcgaagac agcgctgtgt actactgcgc caggtactac 300

gatgaccatt actgcctgga ctattgggga cagggcacca ccctcacagt gagcagcgtc 360

gagggaggca gcggaggaag cggaggatcc ggaggctccg gaggcgtgga cgatattcag 420

ctgacccaat cccccgccat catgtccgct agccctggcg agaaggtgac catgacatgc 480

agagccagca gcagcgtctc ctacatgaac tggtatcagc agaagtccgg cacaagcccc 540

aagaggtgga tttacgacac cagcaaggtg gcctccggcg tgccctacag gtttagcggc 600

tccggcagcg gaacaagcta ctccctgacc atctcctcca tggaggctga ggacgccgcc 660

acctattact gtcagcagtg gagctccaac cccctgacct tcggagccgg caccaagctg 720

gagctgaag 729

Claims (14)

1. An isolated asymmetric bispecific antibody comprising:

a first chain comprising, in order from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL);

a second chain comprising, in order from N-terminus to C-terminus, a heavy chain variable region, a heavy chain constant region, and a single chain antibody (scFv) region, wherein VL and CL of the first chain and the second chain heavy chains VH and CH1 together comprise an antigen binding fragment (Fab) for a first antigen or epitope; the heavy chain constant region of the second chain comprises the CH1 and Fc regions of an antibody heavy chain; the scFv region of the second chain comprises, in order from N-terminus to C-terminus, a heavy chain variable region and a light chain variable region for a second antigen, or comprises, in order from N-terminus to C-terminus, a light chain variable region and a heavy chain variable region for a second antigen or epitope; and

a third chain comprising a third chain heavy chain Fc region, wherein the third chain heavy chain Fc region comprises an antibody hinge region, CH2, and CH 3;

the sequence of the first chain is SEQ ID NO. 1, and the sequences of the VH and CH1 of the second chain are SEQ ID NO. 2; the sequence of the scFv region of the second chain is SEQ ID NO 3; and one of the sequence of the Fc region of the second chain and the sequence of the Fc region of the third chain is SEQ ID NO. 4 or 49 and the other is SEQ ID NO. 5 or 50.

2. The antibody of claim 1, wherein the second chain comprises a hinge region between CH1 and the Fc region, and the third chain comprises, in order from the N-terminus to the C-terminus, a hinge region of the third chain and a heavy chain Fc region, the hinge region of the second chain and the hinge region of the third chain forming an intermolecular disulfide bond therebetween.

3. The antibody according to claim 2, wherein the light chain constant region of the first chain has a disulfide bond linkage to the heavy chain constant region of the second chain, and/or wherein there are 0, 1 or 2 disulfide bridges between the CH3 domain of the second chain and the CH3 domain of the third chain.

4. The antibody of claim 1, wherein the interface at which the CH3 domain of the second chain and the CH3 domain of the third chain are in contact is modified to reduce homodimer formation, wherein the modification is:

a) replacing the amino acid residue in the CH3 domain of the second strand located within the above-mentioned interface with an amino acid residue having a larger side chain volume, thereby generating a bulge on the second strand side of the interface,

b) replacing an amino acid residue in the CH3 domain of the third strand located within the above-described interface with an amino acid residue having a smaller side chain volume, thereby creating a cavity on the third strand side of the interface,

wherein the protrusion is positioned in the cavity; or

a) Replacing an amino acid residue in the CH3 domain of the second strand located within the above-described interface with an amino acid residue having a smaller side chain volume, thereby creating a cavity on the second strand side of the interface,

b) replacing the amino acid residue in the CH3 domain of the third strand located within the above-mentioned interface with an amino acid residue having a larger side chain volume, thereby generating a bulge on the third strand side of the interface,

wherein the protrusion is positioned in the cavity.

5. The antibody according to claim 4, wherein the amino acid residue with larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and wherein the amino acid residue with a smaller side chain volume is selected from the group consisting of: alanine (a), serine (S), threonine (T), and valine (V).

6. An antibody according to claim 1, wherein the Fc region of the second chain and/or the heavy chain Fc region of the third chain is further modified to remove ADCC effector function.

7. An antibody according to claim 1, wherein the Fc region of the second chain is linked to the scFv region by a peptide linker.

8. The antibody according to claim 1, wherein the first antigen or epitope is CD89 and the VL of the first chain comprises the following CDRs: CDR1 having the sequence shown in SEQ ID NO. 9, CDR2 having the sequence shown in SEQ ID NO. 10, CDR3 having the sequence shown in SEQ ID NO. 11, and the VH of the second chain comprises the following CDRs: CDR1 with the sequence shown in SEQ ID NO. 12, CDR2 with the sequence shown in SEQ ID NO. 13 and CDR3 with the sequence shown in SEQ ID NO. 14.

9. A method for preparing an antibody according to claim 1, comprising the steps of:

a) transforming a host cell with:

a first vector comprising a nucleic acid molecule encoding the first strand,

a second vector comprising a nucleic acid molecule encoding said second strand, and

a third vector comprising a nucleic acid molecule encoding the third strand;

b) culturing the host cell under conditions that allow synthesis of the antibody; and

c) recovering the antibody from the culture.

10. A host cell comprising:

a first vector comprising a nucleic acid molecule encoding the first strand of claim 1,

a second vector comprising a nucleic acid molecule encoding the second strand of claim 1, and

a third vector comprising a nucleic acid molecule encoding the third strand of claim 1;

the vector is a plasmid, virus or other vector;

the host cell is a prokaryotic cell or a eukaryotic cell.

11. An antibody capable of specifically binding CD89, wherein the antibody comprises: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, wherein

The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO. 12,

the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO. 13,

the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 14,

the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO. 9,

the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO. 10, and

the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 11.

12. The antibody according to claim 11, wherein the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID No. 23 and a light chain variable region having the amino acid sequence set forth in SEQ ID No. 24.

13. A pharmaceutical composition comprising the antibody of any one of claims 1-8 and 11-12 and at least one pharmaceutically acceptable excipient.

14. Use of an antibody according to any one of claims 1-8 and 11-12 in the manufacture of a medicament for the treatment of a disease, which disease is a tumor.

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