RU2780591C2 - Monovalent asymmetric bispecific antibodies with tandem fab - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is a monovalent asymmetric bispecific antibody with tandem Fab (MAT-Fab antibody), containing polypeptide chains (a), (b), (c), and (d), where (a) is a heavy polypeptide chain (heavy chain), wherein each heavy chain contains (in the direction from N-end to C-end): VL_A-CL-VH_B-CH1-hinge-CH2-CH3m1, where VL_A is a variable domain of a human immunoglobulin light chain, directly attached to CL, which is a constant domain of the human light chain, wherein VL_A-CL is a component of the immunoglobulin light chain of the first binding unit Fab recognizing the first antigen or epitope, and is directly attached to VH_B, wherein VH_B is a variable domain of the human immunoglobulin heavy chain, directly attached to CH1, which is a constant CH1 domain of the human immunoglobulin heavy chain, wherein VH_B-CH1 is a component of the immunoglobulin heavy chain of the second binding unit Fab recognizing the second antigen or epitope, wherein VH_B-CH1 is directly attached to the hinge-CH2 structure, wherein the hinge-CH2 structure is a hinge-CH2 region of the immunoglobulin heavy chain, and the hinge-CH2 structure is directly attached to CH3m1, which is the first constant CH3 domain of the human immunoglobulin heavy chain, mutated by one or more mutations providing the formation of “knob-into-hole” (KiH) structures with the formation of a structural knob or a structural hole in the specified constant domain CH3m1; (b) is the first light chain of MAT-Fab, containing VH_A-CH1, wherein VH_A is a variable domain of the human immunoglobulin heavy chain, directly attached to CH1, which is a constant CH1 domain of the human immunoglobulin heavy chain, and VH_A-CH1 is a component of the heavy chain of the specified first binding unit Fab; (c) is the second light chain of MAT-Fab, containing VL_B-CL, wherein VL_B is a variable domain of a human immunoglobulin light chain, directly attached to CL, which is CL domain of the human immunoglobulin light chain, and VL_B-CL is a component of the immunoglobulin light chain of the specified second binding unit Fab; and (d) is a polypeptide Fc chain containing hinge-CH2-CH3m2, where the hinge-CH2 structure is the hinge-CH2 region of the immunoglobulin heavy chain, and the hinge-CH2 structure is directly attached to CH3m2, which is the second constant CH3 domain of the human immunoglobulin heavy chain, mutated by one or more mutations providing the formation of “knob-into-hole” (KiH) structures with the formation of a structural knob or a structural hole in the specified constant domain CH3m2; provided that, when CH3m1 domain of the heavy chain is mutated to form a structural knob, CH3m2 Fc chain domain is mutated to form a complementary structural hole, which promotes the pairing of CH3m1 domain with CH3m2 domain; and, when CH3m1 domain of the specified heavy chain is mutated to form a structural hole, CH3m2 Fc chain domain is mutated to form a complementary structural knob, which promotes the pairing of CH3m1 domain with CH3m2 domain; and the specified MAT-Fab antibody optionally contains a mutation in CH3m1 domain and in CH3m2 domain, leading to the introduction of a cysteine residue promoting the formation of a disulfide bond, when CH3m1 domain is paired with CH3m2 domain.

EFFECT: invention makes it possible to increase the efficiency of tumor treatment.

71 cl, 6 dwg, 11 tbl, 1 ex

Description

Field of invention

The present invention relates to engineered bispecific antibodies, compositions thereof, and methods for making and using such bispecific antibodies.

State of the art

Over the past fifty years, attempts to design new forms of antibodies have led to the demonstration and existence of various bispecific and polyspecific binding formats. Various formats include, for example, single chain Fv antibodies (scFv, Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)), tetravalent IgG-scFv hybrids (Coloma and Morrison, Nat. Biotechnol., 15: 159-163 (1997)), diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)), tandem scFv molecules (see e.g. Bargou et al., Science 321, 974-977 (2008)), quadrivalent IgG-like dual variable domain antibodies ("DVD-Ig", Wu et al., Nat. Biotechnol., 25: 1290-1297 (2007) ), quadrivalent "Fab in tandem" immunoglobulins ("FIT-Ig"), (WO 2015/103072, Epimab Biotheraupeutics), bivalent hybrid rat/mouse bispecific IgG (Lindhofer et al., J. Immunol., 155: 219-225 (1995)) and bispecific cross-MAT binding proteins (see, for example, WO 2013/026831 (Roche Glycart AG); WO 2014/167022 (Engmab AG)). Of particular interest for possible use in the treatment of diseases is the design and production of various engineered bispecific antibodies capable of binding two different epitopes or antigens, thereby eliminating the need for combination therapeutics. See, for example, reviews by Spiess et al., Molec. Immunol., 67: 95-106 (2015), Riethmüller, Cancer Immun., 12: 12-18 (2012), Kontermann, Acta Pharmacologica Sinica, 26 (1): 1-9 (2005), Marvin et al., Acta Pharmacologica Sinica, 26(6): 649-658 (2005).

There is growing interest in the development of bispecific antibodies for the treatment of various types of cancer. Of particular interest is the potential use of bispecific antibodies to retarget T cells to kill various tumor cells. In an example of this "T cell retargeting" approach, a bispecific antibody can be constructed that binds to an antigen on the surface of a target cancer cell as well as to an activating component of the T cell receptor (TCR) complex on an immature T cell, such as CD3. Simultaneous binding of the bispecific antibody to both cell types provides a temporary link (intercellular synapse) between the target cell and the T cell, which leads to the activation of cytotoxic T lymphocytes (CTL) that attack the target cancer cells. Thus, T cells are artificially retargeted to specific target cancer cells, regardless of the presentation of the peptide antigen by the target cell or the specificity of the T cell normally required for MHC-limited CTL activation. In this context, it is especially important that CTLs are activated only when they are presented with a bispecific antibody by a target cell, i.e. by mimicking an immunological synapse, rather than simply binding an antibody to a T-cell antigen.

A bispecific antibody format that remains of interest in retargeting T cells to tumor cells is the "bispecific T cell activator" or "BiTE" antibody, for example, containing two scFv antibodies linked by a standard glycine-serine (G4S) linker, with one scFv fragment containing a tumor antigen binding site (e.g., tumor antigen 17-1A) and the other scFv fragment containing a CD3 antigen binding site on a T cell (Mack et al., Proc. Natl. Acad. Sci. USA, 92: 7021-7025 (1995)). The US Food and Drug Administration (FDA) has approved the anti-CD3x anti-CD19 BiTE antibody, blinatumomab, for the treatment of a rare form of B-cell acute lymphoblastic leukemia (ALL). Other bispecific antibody formats being investigated for possible use in retargeting T cells to attach to cancer cells are tetravalent tandem diabodies ("TandAb," Kipriyanov et al., J. Mol. Biol., 293: 41-56 (1999 ); Arndt et al., Blood, 94: 2562-2568 (1999)) and dual affinity retargeting proteins ("DART", Johnson et al., J. Mol. Biol., 399: 436-449 (2010)) . In addition, bispecific antibodies are being explored for use in retargeting cytotoxic effector cells, eg, NK cells and macrophages, to tumor cells (eg, Weiner et al., Cancer Res., 55: 4586-4593 (1995).

It is currently unclear which approach to use to retarget T cells or other cells to cancer cells is preferred in the treatment of a human subject. For example, T cell activation can stimulate intense and sustained release of cytokines. Such a “cytokine storm” can have a harmful effect not only on local tissues, but on the patient as a whole. Accordingly, methods for retargeting T cells or other cells to treat cancer may include one or more stepwise actions performed in vitro, ex vivo or in vivo.

Many bispecific formats, such as BiTE, diabodies, DART, and TandAb, use a single chain format for linking different variable domains to peptide linkers to achieve dual specificity. Because these formats do not contain an Fc region, they typically have extremely short in vivo half-lives and are physically unstable (Spiess et al. (2015), op. cit.). Typically, Fc-containing bispecific formats are designed to increase half-life; in addition, they may provide an effector function for Fc. A number of such Fc-containing bispecific formats use "KiH" technology (Ridgway et al, Protein Eng., 9: 617-621 (1996)) to improve assembly and stability of the Fc region, as well as to promote heterodimerization between the heavy chain CH3 domains of two different antibodies, resulting in a bilaterally heterogeneous bispecific antibody, although some formats may have other problems, such as the possibility of light chain mismatches. Incorrect pairing of light chains can lead to inefficient production of a given format. Accordingly, in the course of trying to solve this problem, a large number of different formats have been developed.

As is evident from the above discussion, there is a wide range of bispecific antibody formats developed and studied as possible formats for the development of new therapeutic antibodies. At the same time, at the moment there is not a single format that has an exhaustive set of properties that would make it suitable for the development of new therapeutic antibodies for the treatment of most diseases. Given the growing number of possible uses for bispecific binding proteins and the various outcomes associated with current formats, there is still a need to develop improved formats that could be designed to address the specific challenges associated with developing antibodies for specific diseases. .

The essence of the invention

The present invention satisfies the above need by providing tandem-linked Fab bispecific antibodies designed to bind two different epitopes present on the same target antigen or on two different target antigens. The bispecific antibody of the present invention contains two Fab units, with each Fab unit binding only one of the epitopes or antigens bound by the antibody, and is referred to as a "univalent asymmetric Fab tandem bispecific antibody" or "MAT-Fab bispecific antibody" or simply "MAT- fab. The MAT-Fab bispecific antibody of the present invention is monovalent (one binding site) for each of two different epitopes or two different antigens bound by the MAT-Fab antibody. The MAT-Fab bispecific antibody of the present invention is a tetrameric protein comprising: a "heavy polypeptide chain" (or "MAT-Fab heavy chain"), an "Fc polypeptide chain" (or "MAT-Fab Fc chain"), and two various light chains ("MAT-Fab first light chain" and "MAT-Fab second light chain").

The Fab fragment of an immunoglobulin consists of two components covalently linked to form an antibody binding site. Each of these two components is a variable domain-constant domain chain (VH-CH1 or VL-CL) and therefore each V-C Fab chain can be described as a "half" of the Fab binding unit. The MAT-Fab antibody heavy chain contains half of the first and half of the second Fab unit (V-C) connected in tandem, followed by an Fc region containing a hinge region, a CH2 domain, and a C-terminal CH3 domain. The Fc chain of MAT-Fab contains an N-terminal hinge region attached to a CH2 domain, which in turn is attached to a C-terminal CH3 domain in the same order as in the natural IgG molecule. At the same time, the Fc chain of the MAT-Fab bispecific antibody of the present invention does not contain fragments of Fab units or any other functional domains attached to the N-terminal hinge region or C-terminal CH3 domain, which is important for the formation of functional Fab binding units. . Each of the two MAT-Fab light chains contains the other half (V-C) of each Fab binding unit. The light and heavy chains of the MAT-Fab are designed such that each light chain is linked in its respective V-C region to the heavy chain, forming the desired Fab binding unit and preventing nuisance mismatch of the light chains with the mismatched V-C region of the heavy chain. Since dimerization of the MAT-Fab Fc chain with the MAT-Fab heavy chain Fc region is desirable, it is preferred that the MAT-Fab Fc chain is virtually identical to the corresponding MAT-Fab heavy chain region, except for mutations that form ridge-to-trough structures. » (KiH) and designed to stimulate the preferential pairing of CH3 domains of the heavy chain and the Fc chain of MAT-Fab compared to the homodimerization of two MAT-Fab heavy chains or two MAT-Fab Fc light chains. Additional beneficial modifications can optionally be made to the CH3 domains of the heavy chain and the Fc chain of MAT-Fab by introducing a cysteine residue to promote the formation of an additional disulfide bond or by introducing one or more salt bridges, such additions resulting in improved heterodimer stability. The salt bridge contains a hydrogen bond and enters into an electrostatic interaction, which can, for example, occur between glutamate and lysine residues.

The assembly of a functional bispecific MAT-Fab antibody of the present invention is achieved by linking each MAT-Fab light chain to its corresponding half of the Fab segment in the heavy chain to form a complete Fab binding unit and heterodimerize the heavy chain Fc domain to the Fc domain of the MAT Fc chain. -Fab. Heterodimerization of a heavy chain Fc domain with an Fc chain Fc domain is directed and promoted by one or more known bump-in-hole ("KiH") mutations in which the CH3 domain of one Fc region on the same chain is mutated with the formation of a ledge structure which makes it unfavorable for homodimerization with identical chains containing a ledge. The CH3 domain of the other Fc region of the other strand is mutated to form a structural pit that effectively pairs with the CH3 domain containing the mutation that provides the structural ridge, also preventing homodimerization with identical pit containing strands (Ridgway et al., Protein Eng., 9: 617-621 (1996); Atwell et al., J. Mol. Biol., 270: 26-35 (1997)). Thus, the assembly of four polypeptide chains results in a bispecific antibody that is univalent for each antigen epitope and structurally asymmetric in that all Fab units are formed by light chains linked to a single heavy chain.

The present invention provides a monovalent asymmetric Fab tandem bispecific antibody ("MAT-Fab antibody", "MAT-Fab") containing four polypeptide chains (a), (b), (c) and (d):

(a) a heavy polypeptide chain ("heavy chain"), with each heavy chain containing (from N-terminus to C-terminus): VL _A -CL-VH _B -CH1-hinge-CH2-CH3m1, where:

VL _A - human immunoglobulin light chain variable domain attached directly to CL, which is a human immunoglobulin light chain constant domain, where VL _A -CL is one half of the first Fab binding unit (recognizing antigen or epitope "A") and attached directly to VH _B , wherein VH _B is a human immunoglobulin heavy chain variable domain attached directly to CH1, which is a human immunoglobulin heavy chain constant CH1 domain, wherein VH _B -CH1 is one half of the second Fab binding unit (recognizing an antigen or epitope "B"), where VH _B -CH1 is attached directly to the hinge-CH2 structure, wherein the hinge-CH2 structure is the immunoglobulin heavy chain hinge-CH2 region, and the hinge-CH2 structure is attached directly to CH3m1, which is the first constant CH3 domain human immunoglobulin heavy chain, mutated p by one or more ridge-to-cavity (KiH) mutations to form a structural ridge or structural trough in said CH3m1 constant domain;

(b) a first light chain containing VH _A -CH1, where VH _A is the human immunoglobulin heavy chain variable domain attached directly to CH1, which is the human immunoglobulin heavy chain constant CH1 domain, and VH _A -CH1 is the other half said first Fab linking unit;

(c) a second light chain containing VL _B -CL, where VL _B is a human immunoglobulin light chain variable domain attached directly to CL, which is a human immunoglobulin light chain constant domain, and VL _B -CL is the other half of said second linking unit Fab;

and

(d) An Fc chain containing hinge-CH2-CH3m2, wherein the hinge-CH2 structure is the hinge-CH2 region of an immunoglobulin heavy chain, and the hinge-CH2 structure is attached to CH3m2, which is the second CH3 constant domain of a human immunoglobulin heavy chain , mutated by one or more mutations that provide the formation of structures "ledge-in-trough" (KiH) with the formation of a structural ledge or structural cavity in the specified constant domain CH3m2;

provided that:

when the CH3m1 domain of the heavy chain is mutated to form a structural overhang, the CH3m2 domain of the Fc chain is mutated to form a structural pit, which favors the pairing of the CH3m1 domain with the CH3m2 domain; and

when the CH3m1 domain of the heavy chain is mutated to form a structural pit, the CH3m2 domain of the Fc chain is mutated to form a structural bulge, which facilitates the pairing of the CH3m1 domain with the CH3m2 domain; and optionally containing (containing or not containing) a mutation in both the CH3m1 domain of the heavy chain and the CH3m2 domain of the Fc chain, leading to the introduction of a cysteine residue that promotes the formation of a disulfide bond when the CH3m1 domain is paired with the CH3m2 domain.

An additional option is to construct one or more salt bridges between the CH3m1 and CH3m2 domains by mutating one or both of the domains such that a residue in one of the domains can form a hydrogen bond and electrostatically interact (bond) with a residue in the other domain. For example, but not limited to, a salt bridge can be introduced by changing (mutating) an amino acid residue in the CH3m1 domain to a glutamate or aspartate residue and changing (mutating) a residue in the CH3m2 domain to a lysine or arginine residue such that a glutamate or aspartate residue in the CH3m1 domain could form a hydrogen bond and interact electrostatically with a lysine or arginine residue in the CH3m2 domain.

The aforementioned CH3 complementary mutations in the CH3m1 domain of the heavy chain and the CH3m2 domain of the Fc chain promote heterodimer formation compared to homodimerization, ie. the corresponding mutations in the CH3 domain are designed to promote preferential pairing of the Fc chain of MAT-Fab and the heavy chain of MAT-Fab compared to the homodimerization of two Fc chains and two heavy chains.

The above-described structural feature of the bispecific MAT-Fab antibody is that all adjacent variable and constant domains of the immunoglobulin heavy and light chains are directly connected to each other without the involvement of an intermediate amino acid or peptide linker. Such direct attachment of adjacent domains of an immunoglobulin (also referred to as "direct fusion" of a domain to an adjacent domain) eliminates potentially immunogenic sites that could be formed by introducing one or more intermediate amino acids, creating a peptide linker that would be heterogeneous or "foreign" for a given subject. and could be recognized as such by the subject's immune system. Contrary to common understanding in the field of antibody design and production, the absence of linkers between the CL domain and the VH _B domain of the MAT-Fab heavy chain and, accordingly, between the tandem Fab binding units does not adversely affect the binding activity of any of the Fab binding units in the MAT antibody. -Fab.

Consistent with the structure of the MAT-Fab bispecific antibody described above, the CH3m1 domain of the heavy chain or the CH3m2 domain of the Fc chain contain knuckle-in-gland (KiH) mutations, resulting in the formation of a structural overhang that facilitates pairing of one of the CH3 domains ( ie CH3m1 or CH3m2) containing a structural ridge with another CH3 domain (ie CH3m2 or CH3m1) containing a structural pit. Preferably, one or more mutations are designed to form a structural ridge in the CH3m1 domain of the heavy chain to mate with a CH3m2 domain that contains a complementary structural pit. Examples of mutations resulting in an amino acid residue substitution to form a structural overhang in the CH3 domain of the MAT-Fab antibody described herein include, but are not limited to, substitution of a threonine residue for a tyrosine residue or substitution of a threonine residue for a tryptophan residue.

Examples of specific mutations resulting in an amino acid residue substitution to form a structural overhang in the CH3 domain of the MAT-Fab described herein include substitution of a threonine-366 residue with a tyrosine residue (T366Y) or a replacement of a threonine-366 residue with a tryptophan residue, but are not limited to them (T366W). Ridgway et al., Protein Eng. 9: 617-621 (1996); Atwell et al., J. Mol. Biol., 270: 26-35 (1997).

Consistent with the structure of the MAT-Fab bispecific antibody described above, the CH3m1 domain of the heavy chain or the CH3m2 domain of the Fc chain contains knuckle-in-gland (KiH) mutations, resulting in the formation of a structural trough that facilitates pairing of one of the CH3 domains ( ie CH3m1 or CH3m2) containing a structural pit with another CH3 domain (ie CH3m2 or CH3m1) containing a structural ridge.

Preferably, one or more mutations are designed to form a structural dent in the CH3m2 domain of the Fc polypeptide chain for mating with a CH3m1 domain that contains a structural ridge. Examples of mutations resulting in the substitution of one or more residues to form a structural pit in the CH3 domain of the MAT-Fab bispecific antibody described herein include substitution of a tyrosine residue with a threonine residue, and a combination of substitution of a threonine residue with a serine residue, substitution of a leucine residue with a alanine and replacement of a tyrosine residue with a valine residue, but are not limited to them. A preferred mutation to form a structural pit in the CH3 domain of the MAT-Fab antibody of the present invention is the replacement of a tyrosine-407 residue with a threonine residue (Y407T). Ridgway et al., Protein Eng. 9: 617-621 (1996). A preferred combination of mutations to form a structural trough in the CH3 domain of the MAT-Fab antibody of the present invention comprises replacing a threonine-366 residue with a serine residue (T366S), replacing a leucine-368 residue with an alanine residue (L368A), and replacing a tyrosine-407 residue with a valine (Y407V). Atwell et al., J. Mol. Biol., 270: 26-35 (1997).

In yet another embodiment, an additional mutation can be introduced to produce a cysteine residue, such as replacing a serine residue with a cysteine residue or replacing a tyrosine residue with a cysteine residue in the CH3m1 domain of the heavy chain and in the CH3m2 domain of the Fc polypeptide chain to form an additional disulfide bond upon domain pairing CH3m1 of the heavy chain with the CH3m2 domain of the Fc polypeptide chain of the MAT-Fab bispecific antibody of the present invention. A specific non-limiting example of cysteine insertion is the replacement of a serine-354 residue with a cysteine (S354C) and a replacement of a tyrosine-349 residue with a cysteine (Y349C) in complementary chains. Merchant et al., Nat. Biotechnol., 16: 677-681 (1998).

In a preferred embodiment, the MAT-Fab bispecific antibody of the present invention can bind one or two target antigens selected from the group consisting of: cytokines, cell surface proteins, enzymes, and receptors.

In yet another embodiment, the MAT-Fab bispecific antibody described herein can modulate the biological function of one or two target antigens. More preferably, the MAT-Fab bispecific antibody described herein can inhibit or neutralize one or more target antigens.

In one embodiment of the present invention, the MAT-25 Fab bispecific antibody described herein can bind two different cytokines. Such cytokines are selected from the group consisting of: lymphokines, monokines and polypeptide hormones.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind at least one target antigen expressed on a cell surface. More preferably, the MAT-Fab bispecific antibody of the present invention binds two cell surface antigens. These two cell surface antigens may be on the same cell or on two cells of the same type. At the same time, more preferably, the MAT-Fab bispecific antibody of the present invention binds an antigen expressed on the surface of a first cell and binds a second antigen expressed on the surface of a second cell, the first and second cells being of different cell types. The MAT-Fab bispecific antibody described herein preferentially binds a first cell surface antigen expressed on an effector cell of the immune system, and also binds a second cell surface antigen expressed on the surface of a cell considered harmful to an individual, whereby elimination or substantial reduction is desirable. populations of such cells.

Effector cells include T cells, NK cells, monocytes, neutrophils, and macrophages.

Cells that are or may be considered harmful to the individual in need of treatment and therefore can be bound by the MAT-Fab antibody described herein include cancer cells, cells that react with self antigens, and cells infected with viruses. Accordingly, in a particularly preferred embodiment, the MAT-Fab bispecific antibody of the present invention binds an antigen expressed on the surface of an effector cell and binds an antigen expressed on the surface of a cancer cell, a self-reactive cell, or a cell infected with viruses.

In a preferred embodiment, the MAT-Fab bispecific antibody of the present invention binds a pair of target antigens selected from the group of antigen pairs consisting of: CD20 and CD3, CD3 and CD19, CD3 and Fc-gamma-RIIIA, CD3 and TPBG, CD3 and Epha10 , CD3 and IL-5Rα, CD3 and TASCTD-2, CD3 and CLEC12A, CD3 and prominin-1, CD3 and IL-23R, CD3 and ROR1, CD3 and IL-3Rα, CD3 and PSA, CD3 and CD8, CD3 and glypican -3, CD3 and FAP, CD3 and EphA2, CD3 and ENPP3, CD3 and CD33, CD3 and CD133, CD3 and EpCAM, CD3 and CD19, CD3 and Her2, CD3 and CEA, CD3 and GD2, CD3 and PSMA, CD3 and BCMA , CD3 and A33, CD3 and B7-H3, CD3 and EGFR, CD3 and P-cadherin, CD3 and HMW-MAA, CD3 and TIM-3, CD3 and CD38, CD3 and TAG-72, CD3 and SSTR, CD3 and FRA , CD16 and CD30, CD64 and Her2, CD 137 and CD20, CD138 and CD20, CD19 and CD20, CD38 and CD20, CD20 and CD22, CD40 and CD20, CD47 and CD20, CD 137 and EGFR, CD137 and Her-2, CD 137 and PD-1, CD 137 and PDL-1, PD-1 and PD-L1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM -3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF and VEGF, the first epitope a VEGF and the second (other) VEGF epitope, VEGF and Ang2, EGFR and cMet, PDGF and VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1, Lag-3 and PD-1, Lag-3 and PD-L1, Lag-3 and CTLA-4, ICOS and CTLA-4, CD138 and CD40, CD38 and CD138, CD38 and CD40, CD-8 and IL-6, CSPGs and RGM A , CTLA-4 and BTN02, CTLA-4 and PD-1, IGF1 and IGF2, IGF1/2 and Erb2B, IGF-IR and EGFR, EGFR and CD13, IGF-IR and ErbB3, EGFR-2 and IGFR, Her2 first epitope and second (other) epitope Her2, factor IXa and Met, factor X and Met, VEGFR-2 and Met, VEGF-A and angiopoietin-2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-1β , MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A, PDL-1 and CTLA-4, PD-1 and TIM-3, RGM A and RGM B, Te38 and TNFα, TNFα and Blys , TNFα and CD22, TNFα and CTLA-4, TNFα and GP130, TNFα and IL-12p40, and TNFα and RANK ligand.

In one embodiment, the MAT-Fab bispecific antibody described herein binds CD3 on an effector cell. Examples of effector cells include, but are not limited to, T cells, natural killer (NK) cells, monocytes, neutrophils, and macrophages.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention binds a surface antigen expressed on an effector cell. The surface antigen is preferably selected from the group consisting of: CD3, CD16 (also referred to as "FcγRIII") and CD64 (also referred to as "FcγRI"). More preferably, the MAT-Fab antibody binds CD3 expressed on a T cell, CD16 expressed on a natural killer (NK cell) or CD64 expressed on a macrophage, neutrophil or monocyte.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention binds a surface antigen that is a tumor-associated antigen. Preferred embodiments of MAT-Fab can recognize tumor-associated antigens, e.g., selected without limitation from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 (“Her2”), embryonic tumor antigen (“CEA”), adhesion molecule epithelial cells ("EpCAM") and orphan receptor-1 protein like receptor tyrosine kinase (ROR1).

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention binds an antigen on an effector cell, which activates the effector cell, and also binds a malignant B cell surface antigen.

The MAT-Fab bispecific antibody of the present invention preferably binds an antigen on an effector cell that activates the effector cell and also binds a malignant B cell surface antigen associated with a cancer selected from the group consisting of: acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia/lymphoma involving precursor cells, B-cell neoplasms involving mature cells, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma , follicular lymphoma, skin follicle central cell lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, macro Waldenström's globulinemia and anaplastic large cell lymphoma.

In a preferred embodiment, the MAT-Fab bispecific antibody binds to CD3 and CD20 target antigens.

In yet another embodiment, the MAT-Fab bispecific antibody described herein binds a surface antigen expressed on an effector cell and a tumor associated antigen expressed on a tumor cell. In a preferred embodiment, the MAT-Fab bispecific antibody described herein binds CD3 on a T cell and CD20 on a malignant B cell. More preferably, the MAT-Fab bispecific antibody binds CD20 through its outer (N-terminal) Fab binding unit and CD3 through its inner (C-terminal) Fab binding unit.

In a preferred embodiment, the MAT-Fab bispecific antibody binds CD20 and CD3 and contains four polypeptide chains containing the amino acid sequences shown in Tables 1-4 or the amino acid sequences shown in Tables 5-8.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind one or two cytokines, cytokine related proteins, or cytokine receptors.

In another embodiment, the MAT-Fab bispecific antibody of the present invention can bind one or more chemokines, chemokine receptors, or chemokine-related proteins.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind CD3 on T cells as well as an antigen or epitope derived from a viral envelope protein present on the surface of virus-infected cells, e.g., HIV-infected CD4+ T cells. .

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention may also have receptors, including lymphokine receptors, monokine receptors, and polypeptide hormone receptors.

In one embodiment, the MAT-Fab bispecific antibody described above is glycosylated. Glycosylation preferably follows the human glycosylation pattern.

In yet another embodiment, the present invention provides one or more isolated nucleic acids encoding one, two, three, or all four polypeptide chains of the MAT-Fab bispecific antibody described herein. In a preferred embodiment, said one or more nucleic acids encode four polypeptide chains containing the amino acid sequences shown in Tables 1-4 or the amino acid sequences shown in Tables 5-8 below.

In yet another embodiment, the present invention provides a vector containing one or more isolated nucleic acids encoding one, two, three, or all four polypeptides of the MAT-Fab bispecific antibody described herein. The vector may be an autonomously replicating vector or a vector that inserts the isolated nucleic acid present in said vector into the genome of the host cell. In addition, isolated nucleic acids encoding one, two, three, or all four polypeptide chains of a MAT-Fab antibody can be inserted into a vector to perform various genetic assays, to express the MAT-Fab antibody, or to investigate or improve one or more properties of the MAT antibody. -Fab described in this document.

In yet another embodiment, the vector of the present invention can be used to replicate said isolated nucleic acid to produce more nucleic acid encoding one or more polypeptides of the MAT-Fab bispecific antibody described herein.

In yet another embodiment, the vector of the present invention can be used to express an isolated nucleic acid encoding one, two, three, or all four polypeptide chains of a MAT-Fab bispecific antibody described herein. Preferred vectors for cloning and expression of the nucleic acids described herein include pcDNA, pTT (Durocher et al, Nucleic Acids Res., 30(2e9): 1-9 (2002)), pTT3 (pTT with additional multiple cloning sites) , pEFBOS (Mizushima and Nagata, Nucleic Acids Res., 18(17): 5322 (1990)), pBV, pJV, pcDNA3.1

TOPO, pEF6 TOPO and pBJ, but are not limited to.

In addition, the present invention provides an isolated host cell containing the above-described vector. Such an isolated host cell containing the vector described herein may be an isolated prokaryotic cell or an isolated eukaryotic cell.

In an embodiment of the present invention, the isolated prokaryotic host cell containing the vector described herein is a bacterial host cell. The bacterial host cell may be a Gram positive, Gram negative, or Gram variable bacterial cell.

The bacterial host cell containing the vector described herein is preferably a gram-negative bacterium. Even more preferably, the bacterial host cell containing the vector described herein is an Escherichia coli cell.

In an embodiment of the present invention, the isolated host cell containing the vector described herein is a eukaryotic host cell. Preferred isolated eukaryotic host cells containing the vector described herein may include a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algae host cell, a nematode host cell, a a protozoan host or a fish host cell. The isolated mammalian host cell containing the vector described herein is preferably selected from the group consisting of:. Chinese Hamster Ovary (CHO) Cells, COS Cells, Vero Cells, SP2/0 Cells, NS/0 Myeloma Cells, Human Embryonic Kidney (HEK293) Cells, Baby Hamster Kidney (BHK) Cells, HeLa Cells, Human B Cells, Cells CV-1/EBNA, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells. Isolated fungal host cells containing the vector described herein are preferably selected from the group consisting of:. Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida. More preferably, the Saccharomyces host cell containing the vector described herein is a Saccharomyces cerevisiae cell.

In addition, a method is provided for the production of a MAT-Fab bispecific antibody described herein, comprising culturing an isolated host cell containing a vector containing a nucleic acid encoding all four polypeptide chains of a MAT-Fab bispecific antibody molecule under conditions sufficient to produce the bispecific antibody. MATFab.

Another aspect of the present invention is a bispecific MAT-Fab antibody obtained using the method described above.

The MAT-Fab bispecific antibody described herein can be conjugated to another compound, eg, within the C-terminus or at the C-terminus of either or both CH3m1 and CH3m2 paired domains, similarly to other conjugated antibodies. Compounds that can be conjugated to a MAT-Fab bispecific antibody include, but are not limited to, a cytotoxic agent, an imaging agent, and a therapeutic agent. Preferred imaging agents that can be conjugated to a MAT-Fab bispecific antibody include, but are not limited to, a radioactive label, an enzyme, a fluorescent label, a luminescent label, a magnetic label, biotin, streptavidin, and avidin. Radiolabels that can be conjugated to the MAT-Fab bispecific antibody described herein include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm, but are not limited to them. Preferred cytotoxic or therapeutic compounds that can be conjugated to the MAT-Fab bispecific antibody described herein include, but are not limited to, an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an antiangiogenic agent, an antimitotic agent, an anthracycline, a toxin, and an antiapoptotic agent. them.

In yet another embodiment, the MAT-Fab bispecific antibody described herein may be a crystallized MAT-Fab antibody that retains binding activity for epitopes or antigens bound by the non-crystallized MAT-Fab antibody. Such crystallized MAT-Fab bispecific antibodies can also provide controlled release of MAT-Fab without a carrier when administered to an individual. A crystallized MAT-Fab bispecific antibody may also have an increased in vivo half-life when administered to an individual compared to the non-crystallized form.

In one embodiment, the present invention provides a composition for releasing a crystallized MAT-Fab bispecific antibody, said composition comprising the crystallized MAT-Fab bispecific antibody described herein, an accessory ingredient, and at least one polymeric carrier. The auxiliary ingredient is preferably selected from the group consisting of: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxy polyethylene glycol and polyethylene glycol. The polymeric carrier is preferably a polymer selected from one or more of the groups consisting of: polyacrylic acid, polycyanoacrylates, polyamino acids, polyanhydrides, polydepsipeptides, polyesters, polylactic acid, lactic-glycolic acid or PLGA, poly-b-hydroxybutyrate, polycaprolactone, polydioxanone ; polyethylene glycol, polyhydroxypropyl methacrylamide, polyorganophosphazene, poly(ortho-ethers), polyvinyl alcohol, polyvinylpyrrolidone, copolymers of maleic anhydride and alkyl vinyl ether, pluronyl polyols, albumin, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides , their mixtures and copolymers.

In yet another embodiment, a method of treating a mammal is provided, comprising the step of administering to the mammal an effective amount of a composition comprising a crystallized MAT-Fab bispecific antibody as described above.

In yet another embodiment, the present invention provides a method of treating a disease or disorder in an individual, comprising administering to said individual a bispecific MAT-Fab antibody that binds one or two epitopes or antigens considered harmful to the individual, wherein the binding of said one or two epitopes or antigens by the bispecific antibody MAT-Fab provides treatment for said disease or disorder.

The MAT-Fab bispecific antibody described herein is particularly useful in a method of treating a disorder involving retargeting (or "recruiting") effector cells (e.g., T cells, NK cells, monocytes, neutrophils, macrophages) to attack specific cells. -targets that are considered harmful to the human subject, and therefore it is desirable to eliminate or significantly reduce the population of harmful target cells. Preferred examples of such harmful target cells are tumor cells, eg blood (including lymph) tumor cells and solid tumor cells (Satta et al., Future Oncol., 9(4): 527-539 (2013)); cells associated with an autoimmune disease, such as B cells responsive to self antigens (Zocher et al., Mol. Immunol., 41(5): 511-518 (2004)); as well as virus-infected cells (e.g., HIV-infected CD4+ T cells (Sung et al., J. Clin. Invest., 125(11): 4077-4090 (2015)). -Fab binds an antigen expressed on the surface of an effector cell and an antigen expressed on the surface of a target cell that is harmful to a human subject, and the binding of the MAT-Fab antibody to the effector cell antigen and to the antigen of the harmful target cell mediates a desirable or favorable intercellular interaction, for example, MAT-Fab bispecific binding activates an effector cell that attacks the target malicious cell Simultaneous binding of the MAT-Fab bispecific antibody described herein to a single target antigen on the effector cell and a single target antigen on the harmful cell- target can activate the effector cell, resulting in an attack on the target cell in the advantageous absence of mass Intense and unwanted release of cytokines (“cytokine storm”), which is otherwise possible when effector cell antigens dimerize with antibodies that simultaneously bind two or more effector cell antigens (i.e. cross-linking of effector cell antigens).

Accordingly, the present invention provides a method for treating a disorder in a human subject, comprising the step of administering to the human subject a bispecific MAT-Fab antibody described herein that binds an antigen on an effector cell and an antigen associated with the disorder and expressed on a target cell that is harmful for a human subject, wherein binding of the MAT-Fab bispecific antibody to both the effector cell and the harmful target cell results in a therapeutically beneficial cell-to-cell interaction between the effector cell and the target cell. For the treatment of many disorders, such a favorable intercellular interaction must include the activation of the effector cell, leading to the attack of the harmful target cell. In other situations, the beneficial effect may be to opsonize and/or kill one or both of the associated target cells.

The method of retargeting an effector cell resulting in an attack of a malicious target cell according to the present invention preferably comprises contacting the effector cell and the malicious target cell with a MAT-Fab bispecific antibody described herein that binds an antigen expressed on the effector cell and selected from the group , consisting of: CD3, CD16 (also called "FcγRIII") and CD64 (also called "FcγRI"). More preferably, the method comprises a MAT-Fab antibody that binds CD3 expressed on a T cell, CD16 expressed on a natural killer (NK cell) or CD64 expressed on a macrophage, neutrophil or monocyte.

In one embodiment, the present invention provides a method of treating a tumor in a human subject in need of treatment, comprising administering to the human subject a MAT-Fab antibody that binds an antigen on an effector cell and also binds an antigen on a target tumor cell, wherein the binding of the MAT-Fab antibody A Fab with an effector cell and a tumor target cell activates the effector cell and leads to an attack on the tumor cell. The antigen on the effector cell is preferably CD3 expressed on a T cell.

In a preferred embodiment, a method of treating a cancer characterized by the presence of tumor cells in a human subject in need of treatment comprises retargeting an effector cell resulting in an attack on the target tumor cell, comprising the step of contacting the effector cell and the target tumor cell with a bispecific MAT-Fab antibody. described herein and binding an antigen on an effector cell and an antigen on a target tumor cell, wherein the antigen on the target tumor cell is selected from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 (“Her2”), embryonic tumor antigen ("CEA"), epithelial cell adhesion molecule (EpCAM) and orphan receptor-1 protein like receptor tyrosine kinase (ROR1).

In yet another embodiment, the present invention provides a method of treating a B cell-associated tumor in a human subject, comprising administering to an individual in need of such treatment a MAT-Fab bispecific antibody that binds an antigen on an effector T cell that activates T- cell, and binding antigen on malignant B-cells. The MAT-Fab bispecific antibody of the present invention preferably binds antigen on malignant B cells in a cancer selected from the group consisting of: acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia/lymphoma involving cells progenitors, B-cell neoplasms involving mature cells, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, skin follicular central cell lymphoma, B-cell marginal zone lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma.

In a particularly preferred embodiment, a method provided for treating a B cell associated tumor in a human subject comprises administering a MAT-Fab bispecific antibody described herein that binds CD3 on a T cell and CD20 on a malignant B cell. More preferably, the MAT-Fab bispecific antibody binds CD20 through its outer (N-terminal) Fab-binding unit and CD3 through its inner (C-terminal) Fab-binding unit.

In yet another embodiment, a method for treating a disorder of the present invention may comprise contacting a MAT-Fab antibody with effector cells and deleterious target cells in an ex vivo procedure in which effector cells isolated from a human subject in need of treatment are contacted. with the MAT-Fab antibody outside the human body, and after some time necessary for the binding of the MAT-Fab antibody to the effector cells, the effector cells associated with the MAT-Fab antibody are administered to the human subject in such a way that the complexes of the MAT-Fab antibody, associated with effector cells can be targeted to bind harmful target cells, such as tumor cells, in the body of a human subject. In yet another embodiment, effector cells and tumor cells isolated from a human subject in need of treatment are contacted with a MAT-Fab antibody outside the human body, and after some time, necessary for the MAT-Fab antibody to bind to effector cells and tumor cells. cells, effector and tumor cells associated with the MAT-Fab antibody are administered to a human subject.

In yet another embodiment, a MAT-Fab bispecific antibody can be engineered to deliver a cytotoxic agent to a harmful target cell, such as a tumor cell. In this embodiment, one Fab binding unit of the MAT-Fab antibody contains a target antigen binding site on the harmful target cell, and the other Fab binding unit contains a cytotoxic agent binding site. Such an engineered MAT-Fab of the present invention may be mixed or otherwise contacted with a cytotoxic agent to which it binds via its engineered Fab binding unit. The MAT-Fab carrying the bound cytotoxic agent can then be brought into contact with the noxious tumor cell to deliver the cytotoxic agent to the noxious tumor cell. Such a delivery system is particularly effective if the MAT-Fab binds to the malicious tumor cell and is then internalized into the cell along with the associated cytotoxic agent such that the cytotoxic agent can be released into the malicious tumor cell.

The present invention provides pharmaceutical compositions comprising the MAT-Fab bispecific antibody described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the MAT-Fab bispecific antibody may also contain an additional agent selected from the group consisting of a therapeutic agent, an imaging agent, and a cytotoxic agent. In addition, in accordance with the ex vivo methods described herein, the pharmaceutical composition according to the present invention may contain a bispecific MAT-Fab antibody as part of a complex with an effector cell or a cytotoxic agent.

In yet another embodiment, a preferred pharmaceutical composition comprising the MAT-Fab bispecific antibody described herein may further comprise one or more other therapeutically active compounds for treating a disorder. Examples of preferred additional therapeutically active compounds that may be included in the pharmaceutical composition of the present invention include an antibiotic, an antiviral compound, an anticancer compound (other than a MAT-Fab bispecific antibody), a sedative, a stimulant, a local anesthetic, a corticosteroid, an antihistamine, a non-steroidal anti-inflammatory agent. drug (NSAIDs) and their respective combinations, but are not limited to them.

In yet another embodiment, the pharmaceutical composition described above can be formulated for administration to an individual via at least one route selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intracerebroventricular, intraintestinal , intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostate, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal and transdermal administration.

In another embodiment, the MAT-Fab bispecific antibody described herein can be used in one of the various immunological detection assays or purification formats available in the art for detecting, quantifying, or isolating a target antigen or cells expressing target antigen. Such assays and formats include immunoblot-based assays (eg, Western blotting); immunoaffinity chromatography, for example by adsorbing or binding a MAT-Fab bispecific antibody to a chromatographic resin or beads; immunoprecipitation assays; immunochips; analyzes based on tissue immunohistochemistry; flow cytometry (including cell sorting with fluorescence activation); sandwich immunoassays; immunochips, where the MAT-Fab antibody is immobilized or bound to a support; radioimmunoassay (RIA); enzyme immunoassay (ELISA); enzyme-linked immunosorbent assay (solid-phase ELISA); competitive immunoassays with inhibition; fluorescent polarization immunoassay (FPIA); Enzymatic Enhanced Immunoassay (EMIT) technique; resonant bioluminescence energy transfer (BRET); and homogeneous chemiluminescent assay, but are not limited to. The present invention provides methods using mass spectrometry that include MALDI (Matrix Activated Laser Desorption Ionization) or SELDI (Surface Enhanced Laser Desorption Ionization) involving a MAT-Fab antibody that binds a target antigen or epitope on an antigen or fragment. antigen, but are not limited to.

In addition, the present invention provides a method for detecting an antigen in a sample (e.g., mixture, composition, solution, or biological sample) comprising contacting the sample with a MAT-Fab bispecific antibody of the present invention that binds a target antigen (or epitope) present or suspected present in the sample. Biological samples that can be used as a sample for the immunological detection assay of the present invention include, without limitation, whole blood, plasma, serum, various tissue extracts, tears, saliva, urine, and other bodily fluids.

The MAT-Fab bispecific antibody can be directly or indirectly labeled with a detectable compound to facilitate detection of bound or unbound MAT-Fab bispecific antibody. In a preferred embodiment, a MAT-Fab bispecific antibody suitable for a detection assay is designed such that one of the Fab binding units binds a detectable signal generating compound and the other Fab binding unit binds a target antigen (or epitope) present or suspected to be present. in the sample. Suitable detectable substances are available in the art and include, without limitation, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. A preferred enzyme that can be used in the immunological detection assay of the present invention is an enzyme capable of providing a detectable signal upon contact with one or more reagents. Such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include, without limitation, streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials that can be used in the immunological detection assay of the present invention include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. An example of a luminescent material that can be used in the immunological detection assay of the present invention is luminol. Examples of suitable radioactive isotopes that can be used in the immunological detection assay of the present invention include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm, but not limited to them.

Due to the presence of a dimerized Fc region, the MAT-Fab bispecific antibody of the present invention can also be labeled for the Fc region in a manner similar to natural antibody molecules, eg, IgG antibodies.

Brief description of the drawings

Figure 1A is a diagram showing the domain structure of a bispecific MAT-Fab antibody. Figure 1B is a diagram describing the order of the structural genes inserted into the expression vector constructs for recombinant expression of each of the polypeptide chains that assemble to form the MAT-Fab binding protein of the present invention. Figure 1C is a diagram describing the four expressed polypeptide chains (1-4) to produce the MAT-Fab binding protein shown in FIG. 1A, showing the order of the immunoglobulin-like domains from the N-terminus to the C-terminus of each strand.

Figure 2 shows the profile and associated data analysis bispecific antibodies MAT-Fab KiH1, obtained as described in example 1, using size exclusion chromatography (SEC). SEC analysis revealed that 98.19% of the MAT-Fab KiH1 antibody preparation after one-step purification by protein A chromatography was a single substance, indicating homogeneity of the tetrameric protein product.

Figure 3 shows the profile and associated analysis of the bispecific antibody MAT-Fab KiH2, obtained as described in example 1, using size exclusion chromatography (SEC). SEC analysis revealed that 95.7% of the MAT-Fab KiH2 antibody preparation after one-step purification by protein A chromatography was a single substance, indicating homogeneity of the tetrameric protein product.

Figure 4 shows the results of the analysis of the ability of the bispecific antibodies MAT-Fab KiH1 and MAT-Fab KiH2 to bind CD20 expressed on the surface of Raji cells using fluorescence activated cell sorting (FACS) as described in example 1.

Figure 5 shows the results of the analysis of the ability of the bispecific antibodies MAT-Fab KiH1 and MAT-Fab KiH2 to bind CD3 expressed on the surface of Jurkat cells using fluorescence activated cell sorting (FACS) as described in example 1.

Figure 6 shows the results of the analysis of the ability of MAT-Fab KiH1 and MAT-Fab KiH2 bispecific antibodies to bind CD20 on Raji cells (B cells) and induce T cell-mediated apoptosis in B cells on day 2 in the B cell depletion assay . Chart legend: Ofatumumab is a fully human anti-CD20 monoclonal antibody; "CD3 mAb" is a monoclonal antibody against CD3; MAT-Fab KiH1 and MAT-Fab KiH2 are as described in Example 1; and "Ofatumumab/CD3 mAb" is a mixture of equal amounts of anti-CD20 ofatumumab and anti-CD3 mAb. Both MAT-Fab bispecific antibodies could induce T cell mediated B cell apoptosis on day 2 of the assay.

Detailed description of the invention

For many immune receptors, cell activation is accomplished by cross-linking in a monovalent binding interaction. The cross-linking mechanism is usually mediated by antibody/antigen immune complexes or effector cell-target cell interaction. For example, low affinity activating Fc gamma receptors (FcγR) such as CD16 (FcγRIIIa) and CD32a (FcγRIIa) mediate cell killing by monovalently binding to the Fc region of an antibody. While monovalent binding does not result in activation of cell signaling pathways, when the effector cell interacts with the target cell, cross-linking and clustering of receptors on the cell surface occurs, leading to activation. Activation of CD3 on T cells occurs by interaction of its associated T cell receptor (TCR) in the avid intercellular synapse with the antigen-carrying major histocompatibility complex (MHC) on antigen-presenting cells. Bivalent antibodies that target CD3 can cause a massive release of cytokines (often referred to as a "cytokine storm"), and the subsequent toxic effects present a problem for the development of anti-CD3 antibodies as therapeutic agents. In contrast, univalent binding of CD3 in bispecific formats results in a significantly reduced level of T cell activation. For bivalent monospecific antibodies, a consequence of this biological phenomenon is that bivalent receptor cross-linking can lead to non-specific activation of the effector cell in the absence of target cells. Thus, if the goal of therapy is immune receptor co-interaction, univalent binding is usually greatly favored over bivalent binding. This regimen is incompatible with conventional bivalent antibodies and most multispecific but multivalent antibody formats, such as dual variable domain immunoglobulins (DVD-Igs) and Fab-in-tandem immunoglobulins (FIT-Igs).

The present invention provides a solution to such problems as described above by providing a bispecific antibody format comprising a ridge-in-gap (KiH) Fc region and heavy chain heterodimerization of a tandem Fab with a truncated Fc chain that allows simultaneous monovalent binding of two different antigens or epitopes The bispecific antibody of the present invention is particularly well suited for T cell retargeting as a cancer treatment mechanism.

The present invention provides engineered bispecific antibodies comprising two Fab binding units attached to each other in tandem, in which one Fab binding unit binds an epitope or antigen that is different from the epitope or antigen bound by the other Fab binding unit. Thus, a bispecific antibody of the present invention is said to be "monovalent" for each epitope or antigen it binds. In addition, although the bispecific antibody of the present invention contains a dimerized immunoglobulin Fc constant region (i.e., a dimerized hinge-CH2-CH3 structure), each half of the tandem Fab binding units resides on a single polypeptide arm extending from only one of the dimerized Fc chains. -region (see Fig. 1A). Accordingly, the bispecific antibody of the present invention is "univalent" in relation to the binding sites for each binding epitope or antigen, is "asymmetric" in relation to the location of the Fab units relative to the dimerized Fc region; and contains "tandem" Fab linking units attached directly to each other in the direction from the N-terminus to the C-terminus through a conventional heavy chain. Accordingly, the bispecific antibody of the present invention is also referred to as "univalent asymmetric Fab tandem bispecific antibody". Alternative synonymous terms for a bispecific antibody of the present invention are "MAT-Fab bispecific antibody", "MAT-Fab antibody", or simply "MAT-Fab".

As used herein, the terms "crystal" and "crystallized" refer to an antibody, including a MAT-Fab bispecific antibody of the present invention, in the form of a crystal. A crystal is one of the forms of the solid state of matter, different from other forms, such as an amorphous solid state or a liquid crystalline state. Crystals are composed of regular repeating three-dimensional lattices of atoms, ions, molecules (eg proteins, eg antibodies) or molecular aggregates (eg antigen/antibody complexes). These three-dimensional lattices are ordered according to specific mathematical relationships well known in the art. The basic unit or building block that repeats itself in the structure of a crystal is called a triclinic unit. Repeating triclinic units, ordered according to a well-defined crystallographic symmetry, form the "unit cell" of a crystal. Repeating elementary cells with a regular displacement in all three dimensions form a crystal. See Giegé et al., Chapter 1, in Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed., Ducruix and Giegé, eds. (Oxford University Press, New York, 1999) pp. 1-16. Crystallized MAT-Fab bispecific antibodies of the present invention can be prepared according to methods known in the art, such as those described by Shenoy et al. in International Publication No. WO 2002/072636, incorporated herein by reference.

Unless otherwise specified herein, scientific and technical terms used in connection with the present invention have the meanings accepted among specialists in this field. However, in case of latent ambiguity, the definitions given in the present invention take precedence over any dictionary or external definition. In addition, unless otherwise provided by the context, the use of terms in the singular includes the plural, and the use of terms in the plural includes the singular. In this application, the use of "or" means "and/or", unless otherwise indicated. In addition, the use of the term "including", as well as other forms such as "includes" and "included", is not limiting. In addition, terms such as "element" or "component" include both elements and components containing a single block, and elements and components that include more than one subunit, unless otherwise indicated in an unambiguous manner.

In general, the nomenclatures and operating procedures relating to cell and tissue culture, molecular biology, immunology, microbiology, protein and nucleic acid genetics and chemistry, and hybridization described herein are known and widely used in the art. The methods and procedures of the present invention are generally performed in accordance with conventional methods well known in the art and described in various general or more specific sources cited and discussed throughout the text of this specification, unless otherwise indicated. Enzymatic reactions and purification procedures are performed in accordance with procedures generally accepted in the art, manufacturer's specifications, or as described herein. The nomenclatures, laboratory procedures, and techniques in the fields of analytical chemistry, organic synthesis, and medicinal and pharmaceutical chemistry described herein are known and widely used in the art. Standard techniques include techniques used for chemical synthesis, manufacture, formulation and delivery of pharmaceuticals, and patient treatment.

In this document, the terms "disorder" and "disease" are synonymous and refer to the pathophysiological condition.

As used herein, the term "a disorder (or disease) in which an antigen on a cell (or antigen activity) is deleterious" includes a disorder in which the presence of an antigen in a human subject suffering from the disorder is shown to cause or is suspected to cause the pathophysiology of the disorder is or is suspected to be a contributing factor to the aggravation of the disorder. Accordingly, a disease in which an antigen or antigen activity is deleterious is a disorder in which a decrease in the level of the antigen or antigen activity is expected to alleviate one or more symptoms of the disorder or the progression of the disorder. Such disorders can be confirmed, for example, by an increase in the concentration of antigen in the blood or other body fluid of a human subject suffering from the disorder.

The terms "tumor" and "cancer" are synonymous unless otherwise noted. The cancer or tumor may be a hemoblastosis or a hematological neoplasm (including from lymphoid cells) or a solid cancer or tumor.

The terms "individual" and "subject" refer to a human subject.

As used herein, the terms "Fab", "Fab fragment" or "Fab binding unit" are synonymous and refer to an immunoglobulin unit that binds an epitope (antigen) and is formed by the association of a polypeptide chain containing an immunoglobulin light chain variable domain (VL domain), attached to an immunoglobulin light chain constant domain (CL domain), and a polypeptide chain containing an immunoglobulin heavy chain variable domain (VH domain) attached to an immunoglobulin heavy chain constant domain (CH1 domain), wherein the VL domain pairs with VH- domain to form an epitope (antigen)-binding site, and the CL domain pairs with the CH1 domain. Thus, in this document, the terms "Fab", "Fab fragment" and "Fab binding unit" cover natural Fab fragments obtained by papain hydrolysis of natural immunoglobulin, for example, IgG, and papain hydrolysis allows you to get two Fab fragments containing a fragment of heavy chain containing VH-CH1, and a light chain fragment containing VL-CL. As used herein, these terms also cover recombinant Fabs in which the selected VL domain is attached to the selected CL domain and the selected VH domain is attached to the selected CH1 domain. In addition, in this document, these terms also cover the "cross Fab" type, in which VH-CH1 is present as a light chain polypeptide, and VL-CL is present on a larger heavy chain. In particular, in the MAT-Fab bispecific antibody of the present invention, the N-terminal (outer) binding unit of the Fab refers to this type of "cross-Fab" in which VL-CL is present in the heavy polypeptide chain and is paired with a light chain containing VH-CH1 . In contrast, the internal (C-terminal) binding unit of Fab contains VH-CH1 in the heavy chain, which pairs with a light chain containing VL-CL. This order of external and internal Fab binding units in the MAT-Fab bispecific antibody of the present invention advantageously promotes the correct association of each VL-CL structure with the corresponding VH-CH1 structure to form two functional Fab binding units and prevents non-functional associations.

As used herein, a composition or method described as "comprising" one or more of the named elements or steps is mutable, meaning that said elements or steps are important, but other elements or steps can be added within the scope of said composition or method. In addition, to avoid verbosity, it should be understood that any composition or method described as "comprising" (or "which contain") one or more of the named elements or steps also describes the corresponding, more limited, composition or method, "mainly consisting of" (or "consisting primarily of") the same specified elements or steps, which means that the composition or method includes these important elements or steps, and may also include additional elements or steps that do not significantly affect the main and new characteristics of said composition or method. In addition, it should be understood that in this document any composition or method described as "comprising" or "consisting primarily of" one or more of the specified elements or steps, also describes the corresponding, more limited and strictly defined compositions or methods, " consisting of" (or "which consist of") the specified elements or steps and excludes any unspecified elements or steps. In any composition or method described herein, any of these essential elements or steps may be substituted with known or described equivalents.

The element or step "selected from the group consisting of..." followed by the elements or steps listed below refers to one or more of the elements or steps in the list below, including combinations of two or more of the listed elements or steps.

Organization and production of bispecific antibodies MAT-Fab

The monovalent asymmetric Fab tandem bispecific antibody (MAT-Fab bispecific antibody) of the present invention contains:

(a) a heavy polypeptide chain (“heavy chain”), wherein said heavy chain contains (from N-terminus to C-terminus): VL _A -CL-VH _B -CH1-hinge-CH2-CH3m1, where: VL _A is a human immunoglobulin light chain variable domain fused to CL, which is a human light chain constant domain, where VL _A -CL is one half of the first Fab binding unit (capable of binding antigen or epitope "A") and is fused directly to VH _B , wherein VH _B is a human immunoglobulin heavy chain variable domain fused to CH1, which is a human immunoglobulin heavy chain constant CH1 domain, wherein VH _B -CH1 is one half of the second Fab binding unit (capable of binding antigen or epitope "B" ), wherein VH _B -CH1 is attached to a hinge-CH2 structure, wherein the hinge-CH2 structure is the immunoglobulin heavy chain hinge-CH2 region, and the hinge-CH2 structure is attached to CH3m1, which which is the first human immunoglobulin heavy chain CH3 constant domain mutated with one or more ridge-in-hollow (KiH) mutations to form a structural ridge or structural trough in said CH3m1 constant domain;

(b) a first light chain containing VH _A -CH1, where VH _A is a human immunoglobulin heavy chain variable domain fused to CH1, which is a human immunoglobulin heavy chain constant CH1 domain, and VH _A -CH1 is the other half said first Fab binding unit (capable of binding to antigen or epitope "A");

(c) a second light chain comprising VL _B -CL, where VL _B is a human immunoglobulin light chain variable domain fused to CL, which is a human immunoglobulin light chain constant domain, and VL _B -CL is the other half of said second a Fab binding unit (capable of binding to an antigen or epitope "B"); and

(d) An Fc chain containing hinge-CH2-CH3m2, wherein the hinge-CH2 structure is the hinge-CH2 region of the immunoglobulin heavy chain constant region, and the hinge-CH2 structure is attached to CH3m2, which is the second heavy chain constant CH3 domain of a human immunoglobulin mutated with one or more ridge-in-the-hole (KiH) mutations to form a structural ridge or structural pit in said CH3m2 constant domain complementary to the corresponding structural pit or structural ridge of the heavy chain CH3m1 domain (a) ;

provided that:

when the CH3m1 domain of the heavy chain (a) is mutated to form a structural overhang, the CH3m2 domain of the Fc chain (d) is mutated to form a complementary structural pit, which facilitates pairing of the CH3m1 domain with the CH3m2 domain; or

when the CH3m1 domain of the heavy chain (a) is mutated to form a structural pit, the CH3m2 domain of the Fc chain (d) is mutated to form a complementary structural ridge, which facilitates the pairing of the CH3m1 domain with the CH3m2 domain; and

optionally contains (i.e., may or may not contain) a mutation in both the CH3m1 domain and the CH3m2 domain, leading to the introduction of a cysteine residue that promotes the formation of a disulfide bond when the CH3m1 domain is paired with the CH3m2 domain.

The heavy chain Fc regions and the Fc chain may optionally be modified to modulate Fc effector function. For example, mutation ₂₃₄ LeuLeu ₂₃₅ in the CH2 domain, eg ₂₃₄ AlaAla ₂₃₅ (EC numbering), is known to impair or abolish ADCC/CDC effector functions. Such a MAT-Fab change is shown below in the examples where a LeuLeu→AlaAla substitution was made at residues 18-19 of the hinge-CH2 region of the heavy chain and Fc chain of MAT-Fab (KiH1), and of the heavy chain and Fc chain of MAT-Fab (KiH2 ) (i.e. see ₄₇₈ AlaAla ₄₇₉ in SEQ ID NO:1 in Table 1 and ₄₇₈ AlaAla ₄₇₉ in SEQ ID NO:5 in Table 5; and ₄₀ AlaAla ₄₁ in SEQ ID NO:4 in Table 4 and ₄₀ AlaAla ₄₁ in SEQ ID NO:8 in Table 8).

A feature of the structure of the bispecific MAT-Fab antibody described herein is that all adjacent variable and constant domains of the heavy and light chains of an immunoglobulin are directly connected to each other without the involvement of an intermediate synthetic amino acid or peptide linker. This direct attachment of contiguous immunoglobulin domains eliminates potentially immunogenic sites that may be generated by the introduction of one or more amino acid intermediates. In the art, it is generally believed that intermediate flexible peptide linkers (e.g., the GGGGS linker and its repeats) in engineered antibody molecules with tandem-linked binding domains are needed to allow adjacent antigen binding sites to bind simultaneously and to avoid mutual steric interference. Contrary to such beliefs, the absence of linkers in the MAT-Fab format does not adversely affect the binding activity of any of the Fab binding units of the MAT-Fab antibody. In particular, it has been found that the addition of linkers between the CL and VH _B of the heavy chain polypeptide does not increase the binding activity of any of the Fab binding units. This means that the structural organization of the MAT-Fab antibody described above provides, by itself, optimal flexibility and three-dimensional configuration, as reflected in the accessibility and ability of each Fab binding unit to bind a given antigen or epitope.

In accordance with the structure of the MAT-Fab bispecific antibody described above, the CH3m1 domain of the heavy chain of MAT-Fab or the CH3m2 domain of the Fc chain of MAT-Fab contain keyhole-in-hole (KiH) mutations, resulting in the formation of a structural ridge that promotes mating. one of the CH3 domains (ie CH3m1 or CH3m2) containing a structural ridge with another CH3 domain (ie CH3m2 or CH3m1) containing a structural pit. Preferably, the mutation is designed to form a structural overhang in the CH3m1 domain of the heavy chain for mating with the CH3m2 domain of the Fc chain, which contains a complementary structural pit. Examples of mutations resulting in the replacement of one or more amino acid residues to form a structural overhang in the CH3 domain of the MAT-Fab antibody described herein include, but are not limited to, the replacement of a threonine residue with a tyrosine residue or the replacement of a threonine residue with a tryptophan residue. Examples of specific mutations resulting in an amino acid residue substitution to form a structural overhang in the CH3 domain of the MAT-Fab described herein include substitution of a threonine-366 residue with a tyrosine residue (T366Y) or a replacement of a threonine-366 residue with a tryptophan residue, but are not limited to them (T366W). Ridgway et al., Protein Eng. 9: 617-621 (1996); Atwell et al., J. Mol. Biol., 270: 26-35 (1997). Such a specific change in MAT-Fab composition is shown below in the examples where a Thr→Tyr substitution was made at residue 21 of the MAT-Fab CH3 domain (KiH1) and a Thr→Trp substitution at residue 21 of the MAT-Fab CH3 domain (KiH2) (t i.e. see Tyr610 in SEQ ID NO:1 in Table 1 and Trp610 in SEQ ID NO:5 in Table 5).

Consistent with the structure of the MAT-Fab bispecific antibody described above, either the CH3m1 domain of the MAT-Fab heavy chain or the CH3m2 domain of the Fc chain contain knuckle-in-gland (KiH) mutations that result in the formation of a structural trough that promotes pairing of one of the CH3s. -domain containing a structural cavity, with another CH3-domain containing a structural ledge. A large number of different lip-to-trough mutations are known in the art in the CH3 domains of the Fc regions of antibodies. See, for example, Ridgway et al., Protein Eng., 9: 617-621 (1996); Atwell et al., J. Mol. Biol., 270: 26-35 (1997); Klein et al., mAbs, 4(6): 653-663 (2012). Examples of mutations resulting in the substitution of one or more residues in the CH3 domain to form a structural trough in the CH3 domain of the bispecific MAT-Fab antibody described herein include substitution of a tyrosine residue with a threonine residue and a combination of substitution of a threonine residue with a serine residue, substitutions a leucine residue to an alanine residue; and replacement of a tyrosine residue by a valine residue, but are not limited to. A preferred mutation to form a structural pit in the CH3 domain of the MAT-Fab antibody of the present invention is the replacement of a tyrosine-407 residue with a threonine residue (Y407T). Ridgway et al., Protein Eng. 9: 617-621 (1996). Such a change in the MAT-Fab composition is shown below in the examples where a Tyr→Thr substitution was made at residue 62 of the MAT-Fab CH3 domain (KiH1) (i.e., see Thr213 in SEQ ID NO:4 in Table 4). A preferred combination of mutations to form a structural trough in the CH3 domain of the MAT-Fab antibody of the present invention comprises replacing a threonine-366 residue with a serine residue (T366S), replacing a leucine-368 residue with an alanine residue (L368A), and replacing a tyrosine-407 residue with a valine (Y407V). Atwell et al., J. Mol. Biol., 270: 26-35 (1997). Such a specific change in MAT-Fab composition is shown below in the examples where a Thr→Ser substitution at residue 21 of the MAT-Fab CH3 domain (KiH2), a Leu→Ala substitution at residue 23 of the MAT-Fab CH3 domain (KiH2), and a substitution Tyr→Val at residue 62 of the CH3 domain of MAT-Fab (KiH2) (i.e., see Ser172, Ala ₁₇₄ and Val ₂₁₃ in SEQ ID NO:8 in Table 8). Preferably, one or more mutations are designed to form a structural pit in the CH3m2 domain of the Fc polypeptide chain for mating with a CH3m1 domain that contains a structural ridge.

The CH3m1 and CH3m2 domains of the MAT-Fab antibody described herein can be further mutated to introduce a cysteine residue to form an additional disulfide bond when the CH3m1 domain of the heavy chain of MAT-Fab is paired with the CH3m2 domain of the Fc chain of MAT-Fab and, thereby stabilizing the heterodimer of the Fc region of the MAT-Fab antibody. Specific examples of such mutations include, without limitation, the replacement of serine-354 with cysteine (S354C) and tyrosine-349 with cysteine (Y349C). Merchant et al., Nat.

Biotechnol., 16: 677-681 (1998). Such Cys substitutions in MAT-Fab are shown below in the examples where the Ser→Cys substitution at residue 9 of the MAT-Fab heavy chain CH3 domain (KiH2) and the Tyr→Cys substitution at residue 4 of the CH3 domain of the MAT-Fc Fc chain were made. Fab (KiH2) (i.e., see Cys ₅₉₈ in SEQ ID NO:5 and Cys ₁₅₅ in SEQ ID NO:8 in Table 8).

An additional option is to construct one or more salt bridges between the CH3m1 and CH3m2 domains of the MAT-Fab described herein by mutating one or both of the domains such that a residue in one of the domains can form a hydrogen bond and electrostatically interact (bind) with the rest in another domain. For example, a salt bridge can be introduced by changing (mutating) an amino acid residue in the CH3m1 domain to a glutamate or aspartate residue and changing (mutating) a residue in the CH3m2 domain to a lysine or arginine residue such that the glutamate or aspartate residue in the CH3m1 domain can form a hydrogen bond and interact electrostatically with a lysine or arginine residue in the CH3m2 domain.

In addition to the modifications to the MAT-Fab format constant region discussed above, each of the heavy chain and/or Fc chain constant regions of the MAT-Fab bispecific antibody described above optionally contains (i.e., may or may not contain) one or more mutations that reduce or abolish at least one Fc effector function, such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Such mutations include, but are not limited to, leucine-234 to alanine (L234A) and leucine-235 to alanine (L235A) (Canfield et al., J. Exp. Med., 173(6):1483-91 (1991 )). The MAT-Fab bispecific antibody preferably contains amino acid modifications in the CH2 domains of the heavy chain and the Fc chain to change from leucine-234 to alanine (L234A, EU numbering) and to change from leucine-235 to alanine (L235A, EU numbering). Such a change in the composition of MAT-Fab is shown below in the examples where the LeuLeu → AlaAla substitution was made at residues 18-19 of the hinge-CH2 region of MAT-Fab (KiH1) and MAT-Fab (KiH2) (i.e. see ₄₇₈ AlaAla ₄₇₉ in SEQ ID NO: 1 in Table 1 and ₄₇₈ AlaAla ₄₇₉ in SEQ ID NO: 5 in Table 5).

The MAT-Fab bispecific antibodies described herein can bind each antigen epitope with high affinity. Specifically, the present invention provides an approach to constructing a bispecific MAT-Fab antibody using two parent antibodies, where one parent antibody binds a first antigen epitope and the other parent antibody binds a second antigen epitope.

Heavy and light chain variable domains (VH, VL) and immunoglobulin heavy and light chain constant domains (e.g., CL, CH1, CH2, CH3) for use in the MAT-Fab bispecific antibody of the present invention can be obtained or prepared from known or produced immunoglobulins or their genetically modified (mutated) versions. For example, Fab binding units, individual variable domains, and individual constant domains are readily derived from "parent" antibodies that bind epitopes or target antigens to be bound by a bispecific MAT-Fab antibody. Individual immunoglobulin constant domains can also be derived from parent antibodies that do not bind to the same epitope or antigen to which the MAT-Fab is to bind, since such constant domains can be attached to variable domains derived from a different parent antibody that binds the desired epitope. or the target antigen to which the MAT-Fab antibody is to bind. Other sources of Fab binding units or individual immunoglobulin variable and constant domains include recombinant parental antibodies or binding proteins that bind one or more epitopes or antigens that the MAT-Fab bispecific antibody is intended to bind. Parent antibodies that can be used as sources of Fab binding units or individual variable and constant domains for use in the production of the MAT-Fab antibody of the present invention include clinically approved therapeutic antibodies.

The antigen-binding variable domains and Fab binding units of the MAT-Fab bispecific antibody described herein can be derived from parental antibodies, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and any recombinant antibodies. Such parent antibodies may be naturally occurring or may be produced using recombinant technology.

Many methods of producing antibodies are known to those skilled in the art, including the use of hybridoma techniques, the antibody selective lymphocyte method (SLAM), the use of libraries (e.g., phage, yeast, or RNA-protein hybrid displays or other libraries), immunization of an animal, non-human and carrying at least some human immunoglobulin loci, and obtaining chimeric, humanized and CDR-grafted antibodies, but not limited to this. See, for example, Neuberger, M.S., et al., Nature 314 (1985) 268-270; Riechmann, L., et al., Nature 332 (1988) 323-327; and EP 2443154 B1 (2013).

In addition, variable domains can be generated using affinity maturation techniques.

In accordance with the present invention, a method for making a MAT-Fab antibody comprises selecting parent antibodies having at least one property desired for a MAT-Fab antibody. Said desirable property is preferably one or more properties used to describe the characteristics of a MAT-Fab antibody, e.g. antigen or epitope specificity, antigen or epitope affinity, activity, biological function, epitope recognition, stability, solubility, production efficiency, reduced immunogenicity , pharmacokinetics, bioavailability, tissue cross-reactivity or orthologous antigen binding.

Variable domains can be obtained from parent antibodies using recombinant DNA techniques. In one embodiment, the variable domain is a mouse heavy or light chain variable domain. In yet another embodiment, the variable domain is a transplanted CDR or a humanized heavy or light chain variable domain. In yet another embodiment, the variable domain used in the MAT-Fab antibody of the present invention is a human heavy or light chain variable domain.

One or more constant domains can be joined to each other or to variable domains using recombinant DNA techniques, PCR, or other methods available in the art suitable for recombining immunoglobulin domains. In one embodiment, the heavy chain variable domain sequence is fused to the heavy chain constant domain and the light chain variable domain sequence is fused to the light chain constant domain. In another embodiment, the constant domains are human immunoglobulin heavy chain constant domains and human immunoglobulin light chain constant domains, respectively.

In addition, the variable and constant domains can be modified to improve the properties of the putative MAT-Fab bispecific antibody. For example, as noted above, the CH3 domains of the Fc regions of the MAT-Fab antibody of the present invention carry lip-to-bottom mutations that promote proper association of the MAT-Fab heavy chain Fc regions and the MAT-Fab Fc polypeptide chain. Fab. In addition, the CH3 domains can be further mutated to introduce cysteine residues that form an additional disulfide bond when two CH3 domains are linked to form a stable Fc heterodimer of the MAT-Fab antibody. In addition, the Fc region of a MAT-Fab antibody may contain one or more amino acid modifications to increase or decrease Fc function, including antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), phagocytosis, opsonization, or cell or receptor binding, but not limited to them.

As noted above, each of the two Fc domains of the MAT-Fab bispecific antibody described herein contains knuckle-in-gland (KiH) mutations favoring association of the Fc region of the MAT-Fab heavy polypeptide chain with the Fc chain MATFab. At the same time, additional properties or modifications of the Fc regions may be desirable. For example, the Fc region of a MAT-Fab antibody can be derived from an Fc region of an immunoglobulin selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In yet another embodiment, one or more amino acids in the Fc region can be modified to increase or decrease the affinity of the Fc region of the FcγR bispecific MAT-Fab antibody (Fc receptor) relative to the unmodified Fc region. For example, modification(s) of the Fc region may alter affinity for FcγRI, FcγRII and/or FcγRIII.

In another embodiment, one or more amino acids in the Fc region can be modified to modulate the functional or physical half-life of the MAT-Fab bispecific antibody in vivo.

The structural organization of each of the polypeptide chains of the bispecific MAT-Fab antibody of the present invention allows for the correct intracellular assembly of the MAT-Fab antibody using the natural mechanisms of expression, folding, and secretion of the protein present in the cell, without the need for post-production processing techniques to obtain a functional MAT-Fab antibody. Fab. Particularly preferred is the use of an isolated mammalian host cell containing one or more expression vectors encoding the four polypeptide chains of the desired MAT-Fab bispecific antibody described herein for the production and expression of a functional MAT-Fab antibody.

Target antigen binding

The MAT-Fab bispecific antibody of the present invention is capable of binding one or two epitopes or target antigens. Typically, an anti-MAT-Fab bispecific antibody is designed to bind an epitope on one antigen and an epitope on the other antigen, allowing the MAT-Fab to be an artificial link between the two antigens to achieve a specific antigen-binding effect. An anti-MAT-Fab antibody of the present invention can be designed to bind a cytokine, a polypeptide ligand, a cell surface receptor, a non-receptor cell surface protein, an enzyme, a complex of two or more of the above, or a combination thereof.

In yet another embodiment, the MAT-Fab bispecific antibody described herein can modulate the biological function of one or two target antigens. More preferably, the MAT-Fab bispecific antibody described herein can neutralize one or more target antigens.

In addition, the MAT-Fab bispecific antibody described herein can bind two different cytokines. Such cytokines may be selected from the group consisting of: lymphokines, monokines, and polypeptide hormones.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind at least one target antigen expressed on a cell surface. More preferably, the MAT-Fab bispecific antibody of the present invention binds two cell surface antigens. These two cell surface antigens may be on the same cell or on two cells of the same type. At the same time, more preferably, the MAT-Fab bispecific antibody of the present invention binds an antigen expressed on the surface of a first cell and binds a second antigen expressed on the surface of a second cell, the first and second cells being of different cell types. The MAT-Fab bispecific antibody described herein preferably binds a first cell surface antigen expressed on an effector cell of the immune system and also binds a second cell surface antigen expressed on the surface of a cell considered harmful to the individual, whereby elimination or downsizing is desirable. populations of such cells. Effector cells that the MAT-Fab bispecific antibody of the present invention can bind include T cells, natural killer (NK) cells, monocytes, neutrophils, and macrophages. Examples of cells that are considered harmful to an individual and that the MAT-Fab bispecific antibody of the present invention can bind include tumor cells, cells that react with self antigens, and cells infected with a virus.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention binds a surface antigen expressed on an effector cell. The surface antigen is preferably selected from the group consisting of: CD3, CD16 (also referred to as "FcγRIII") and CD64 (also referred to as "FcγRI"). More preferably, the MAT-Fab antibody binds CD3 expressed on T cells, CD16 expressed on natural killer (NK) cells, or CD64 expressed on macrophages, neutrophils or monocytes.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention binds a surface antigen that is a tumor-associated antigen. Preferred tumor-associated antigens bound by the MAT-Fab antibody of the present invention are selected from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 ("Her2"), embryonic tumor antigen ("CEA"), epithelial cell adhesion molecule (EpCAM) and orphan receptor-1 protein like receptor tyrosine kinase (ROR 1).

In one embodiment, the MAT-Fab bispecific antibody described herein binds CD3 on an effector cell.

In one embodiment, the MAT-Fab bispecific antibody described herein binds CD20 present on a cancer cell.

In yet another embodiment, the MAT-Fab bispecific antibody described herein binds a surface antigen expressed on an effector cell as described herein and a tumor associated antigen expressed on a tumor cell as described herein. In a preferred embodiment, the MAT-Fab bispecific antibody described herein binds CD3 on a T cell and CD20 on a malignant B cell.

In a preferred embodiment, the MAT-Fab bispecific antibody described herein binds CD3 and CD20. More preferably, the MAT-Fab bispecific antibody binds CD20 through its outer (N-terminal) Fab binding unit and CD3 through its inner (C-terminal) Fab binding unit.

In a preferred embodiment, the MAT-Fab bispecific antibody binds CD20 and CD3 and contains four polypeptide chains containing the amino acid sequences shown in Tables 1-4 (SEQ ID NOs: 1, 2, 3, 4) or the amino acid sequences shown in Tables 5-8 (SEQ ID NOS: 5, 6, 7, 8).

In a preferred embodiment, the MAT-Fab bispecific antibody of the present invention binds a pair of target antigens selected from the group of antigen pairs consisting of: CD20 and CD3, CD3 and CD19, CD3 and Fc-gamma-RIIIA, CD3 and TPBG (trophoblast glycoprotein) , CD3 and Epha10, CD3 and IL-5Rα, CD3 and TASCTD-2, CD3 and CLEC12A, CD3 and prominin-1, CD3 and IL-23R, CD3 and ROR1, CD3 and IL-3Rα, CD3 and PSA antigen), CD3 and CD8, CD3 and glypican-3, CD3 and FAP (fibroblast activation protein), CD3 and EphA2, CD3 and ENPP3, CD3 and CD33, CD3 and CD133, CD3 and EpCAM, CD3 and CD19, CD3 and Her2, CD3 and CEA, CD3 and GD2, CD3 and PSMA (prostate specific membrane antigen), CD3 and BCMA (B cell maturation antigen), CD3 and A33, CD3 and B7-H3, CD3 and EGFR (epidermal growth factor receptor), CD3 and P-cadherin, CD3 and HMW-MAA (high molecular weight human melanoma associated antigen), CD3 and TIM-3, CD3 and CD38, CD3 and TAG-72, CD3 and SSTR (somtostatin receptor), CD3 and FRA, CD16 and C D30, CD64 and Her2, CD 137 and CD20, CD138 and CD20, CD19 and CD20, CD38 and CD20, CD20 and CD22, CD40 and CD20, CD47 and CD20, CD 137 and EGFR, CD137 and Her-2, CD 137 and PD -1, CD 137 and PDL-1, PD-1 and PD-L1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), VEGF first epitope and VEGF second (other) epitope, VEGF and Ang2, EGFR and cMet, PDGF ( platelet growth factor) and VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1, Lag-3 and PD-1, Lag-3 and PD-L1, Lag-3 and CTLA-4, ICOS and CTLA-4, CD138 and CD40, CD38 and CD138, CD38 and CD40, CD-8 and IL-6, CSPGs (chondroitin sulfate proteoglycans) and RGM A (guide signal repulsion molecules A), CTLA -4 and BTN02, CTLA-4 and PD-1, IGF1 and IGF2, IGF1/2 and Erb2B, IGF-IR and EGFR, EGFR and CD13, IGF-IR and ErbB3, EGFR-2 and IGFR, Her2 first epitope and second (other) Her2 epitope, factor IXa and Met, factor X and Met, VEGFR-2 and Met, VEGF-A and angiopoietin- 2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-1β, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A, PDL-1 and CTLA-4, PD -1 and TIM-3, RGM A and RGM B, Te38 and TNFα, TNFα and Blys, TNFα and CD22, TNFα and CTLA-4, TNFα and GP130, TNFα and IL-12p40, and TNFα and RANK ligand.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind one or two cytokines, cytokine related proteins, or cytokine receptors.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention can bind one or more chemokines, chemokine receptors, or chemokine-related proteins.

In yet another embodiment, the MAT-Fab bispecific antibody of the present invention may also have receptors, including lymphokine receptors, monokine receptors, and polypeptide hormone receptors. Glycosylated bispecific MAT-Fab antibodies

The MAT-Fab antibody of the present invention may contain one or more carbohydrate residues. The MAT-Fab bispecific antibody described above is preferably glycosylated. More preferably, the glycosylation follows the human glycosylation pattern.

The production of the nascent protein in vivo may require additional processing, known as post-translational modification. In particular, enzymatic addition of carbohydrate (glycosyl) residues is possible; this process is known as glycosylation.

The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins.

Natural antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain as well as the variable domain. Carbohydrate residues in the Fc domain have a significant effect on the effector function of the Fc domain with minimal effect on antigen binding or antibody half-life (Jefferis, Biotechnol. Prog., 21: 11-16 (2005)). In contrast, variable domain glycosylation can affect the antigen-binding activity of an antibody. Glycosylation in the variable domain may adversely affect antigen binding affinity, probably due to steric interference (Co, M.S., et al., Mol. Immunol., 30: 1361-1367 (1993)), or may result in increased affinity for the antigen ( Wallick et al., J. Exp. Med., 168:1099-1109 (1988); Wright, A., et al., EMBO J., 10:2717-2723 (1991)).

One aspect of the present invention is directed to the creation of glycosylation site mutants, with a mutated O- or N-linked glycosylation site of a MAT-Fab antibody. One of skill in the art can create such mutants using standard well known techniques. Another object of the present invention are mutants at the site of glycosylation, retaining biological activity, but with increased or decreased binding activity.

In yet another embodiment, the glycosylation of the MAT-Fab antibody of the present invention is modified. For example, an aglycosylated MAT-Fab antibody (ie, an antibody without glycosylation) can be made. Glycosylation can be modified, for example, to increase the affinity of the MAT-Fab antibody for one or more antigens. Such carbohydrate modifications can be made, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites in the variable region, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the MAT-Fab antibody for the antigen. This approach is described in more detail in International Publication No. WO 2003/016466 and US Pat. Nos. 5,714,350 and 6,350,861.

Alternatively or in addition, a modified MAT-Fab antibody of the present invention can be prepared having a modified glycosylation pattern, such as a hypofucosylated antibody with a reduced amount of fucosyl residues (see Kanda et al., J. Biotechnol., 130(3): 300 -310 (2007)) or an antibody with an increased number of structures bisected by GlcNAc. These modified glycosylation patterns have been shown to enhance the ADCC activity of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with modified glycosylation mechanisms. Cells with a modified glycosylation mechanism are described in the art and can be used as host cells in which the recombinant antibodies of the present invention can be expressed, thereby producing a glycosylation modified antibody. See, for example, Shields et al., J. Biol. Chem., 277: 26733-26740 (2002); Umana et al., Nat. Biotech., 17: 176-180 (1999) and EP 1176195; international publications No. WO 2003/035835 and WO 1999/54342.

Glycosylation of proteins depends on the amino acid sequence of the protein in question, as well as on the host cell in which the protein is expressed. Different organisms can produce different glycosylation enzymes (eg, glycosyltransferases and glycosidases) and use different substrates (nucleotides and carbohydrates). Due to such factors, the pattern of protein glycosylation and the composition of glycosyl residues may differ depending on the host system in which a particular protein is expressed. The glycosyl residues used in the present invention may include, but are not limited to, glucose, galactose, mannose, fucose, N-acetylglucosamine, and sialic acid. The glycosylated MAT-Fab preferably contains glycosyl residues consistent with the human glycosylation pattern.

It is known to those skilled in the art that different glycosylation patterns can lead to different protein characteristics. For example, the efficacy of a therapeutic protein produced in a host microorganism cell, such as a yeast cell, and glycosylated using an endogenous yeast pathway, may be reduced compared to the same protein expressed in a mammalian cell, such as a CHO cell line. In addition, such glycoproteins may be immunogenic in humans and have a reduced in vivo half-life after administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and stimulate rapid clearance of the protein from the circulation. Other undesirable effects may include changes in the folding, solubility, susceptibility of the protein to proteases, transport, transport, compartmentalization, secretion, recognition of the protein by other proteins or factors, antigenicity or allergenicity of the protein. Accordingly, a MAT-Fab antibody with a specific glycosylation composition and pattern, e.g., a glycosylation composition and pattern identical or at least similar to the glycosylation of antibodies produced in human cells or species-specific cells of the intended animal subject, may be preferred by the practitioner.

Expression of glycosylated MAT-Fab antibodies different from expression in the host cell can be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using methods known in the art, the practitioner can generate a MAT-Fab antibody with a human glycosylation pattern. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that the glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to protein glycosylation in animal cells, in particular human cells (e.g. , US Patent Publication Nos. 2004/0018590 and 2002/0137134).

Nucleic acids, vectors, hosts

The present invention provides one or more isolated nucleic acids encoding one, two, three, or all four polypeptide chains of the MAT-Fab bispecific antibody described herein.

The present invention also provides a vector containing one or more isolated nucleic acids encoding one, two, three, or all four polypeptides of the MAT-Fab bispecific antibody described herein. The vector may be an autonomously replicating vector or a vector that inserts the isolated nucleic acid present in said vector into the genome of the host cell. In addition, isolated nucleic acids encoding one, two, three, or all four polypeptide chains of a MAT-Fab antibody can be inserted into a vector to perform various genetic assays, to express the MAT-Fab antibody, or to investigate or improve one or more properties of the MAT antibody. -Fab described in this document.

The vector of the present invention can be used to replicate an isolated nucleic acid encoding one, two, three, or all four polypeptide chains of the MAT-Fab antibody described herein.

The vector of the present invention can be used to express an isolated nucleic acid encoding a MAT-Fab bispecific antibody described herein, or to express one or more isolated nucleic acids encoding one or more MAT-Fab antibody polypeptide chains.

Preferred vectors for cloning and expressing the nucleic acids described herein include pcDNA, pTT (Durocher et al, Nucleic Acids Res., 30(2e9): 1-9 (2002)), pTT3 (pTT with additional multiple cloning sites) , pEFBOS (Mizushima and Nagata, Nucleic Acids Res., 18(17): 5322 (1990)), but not limited to pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO and pBJ.

In addition, the present invention provides an isolated host cell containing the above-described vector. Such an isolated host cell containing the vector described herein may be an isolated prokaryotic cell or an isolated eukaryotic cell.

An isolated prokaryotic host cell containing the vector described herein may be a bacterial host cell. The bacterial host cell may be a gram positive, gram negative or gram variable bacterial host cell. The bacterial host cell containing the vector described herein is preferably a Gram-negative bacterium. Even more preferably, the bacterial host cell containing the vector described herein is an Escherichia coli cell.

An isolated host cell containing the vector described herein may be a eukaryotic host cell. Preferred isolated eukaryotic host cells containing the vector described herein may include a mammalian host cell, an insect host cell, a plant host cell, a fungal host cell, a eukaryotic algae host cell, a nematode host cell, a a protozoan host or a fish host cell. The isolated mammalian host cell containing the vector described herein is preferably selected from the group consisting of:. Chinese Hamster Ovary (CHO) Cells, COS Cells, Vero Cells, SP2/0 Cells, NS/0 Myeloma Cells, Human Embryonic Kidney (HEK293) Cells, Baby Hamster Kidney (BHK) Cells, HeLa Cells, Human B Cells, Cells CV-1/EBNA, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells. Isolated fungal host cells containing the vector described herein are preferably selected from the group consisting of: Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida. More preferably, the Saccharomyces host cell containing the vector described herein is a Saccharomyces cerevisiae cell.

Further provided is a method for producing a bispecific MAT-Fab antibody described herein, comprising culturing an isolated host cell containing a vector containing a nucleic acid encoding a MAT-Fab antibody under conditions sufficient to produce a MAT-Fab antibody.

Another aspect of the present invention is a bispecific antibody

MAT-Fab obtained using the above method.

Conjugates

Mainly due to the presence of a dimerized Fc region, the MAT-Fab bispecific antibody of the present invention can be conjugated with various agents that are currently conjugated with IgG antibodies. For example, the MAT-Fab bispecific antibody described above can be conjugated with an agent selected from the group consisting of: an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. A preferred imaging agent that can be conjugated to the MAT-Fab bispecific antibody of the present invention is selected from the group consisting of: radioactive label, enzyme, fluorescent label, luminescent label, magnetic label, biotin, streptavidin, or avidin. Radiolabels that can be conjugated to the MAT-Fab bispecific antibody described herein include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm, but are not limited to them. Preferred cytotoxic or therapeutic compounds that can be conjugated to the MAT-Fab bispecific antibody described herein include, but are not limited to, an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an antiangiogenic agent, an antimitotic agent, an anthracycline, a toxin, and an antiapoptotic agent. them. In some cases, the MAT-Fab antibody is conjugated directly to an agent. Alternatively, the MAT-Fab antibody is conjugated to the agent via a linker. Suitable linkers include, but are not limited to, the amino acid and polypeptide linkers described herein. Linkers may be cleavable or non-cleavable.

Crystallized forms

The MAT-Fab bispecific antibody of the present invention can be used in a crystallized form obtained using a large-scale crystallization method such as that described by Shenoy et al. in International Publication No. WO 2002/072636, incorporated herein by reference. Such methods make it possible to obtain antibody molecules that differ significantly from those used in classical X-ray crystallography studies. While crystal quality is very important for accurate measurements in X-ray crystallography studies, this is not the case for crystals produced by large-scale crystallization processes for pharmaceutical applications. Crystals of antibody molecules obtained using large-scale crystallization methods are sufficiently pure for pharmaceutical research and production, retain biological activity, are especially suitable for storage (because antibody crystals are less susceptible to unwanted interactions that can occur in solutions), can be used to obtain parenteral formulations containing very high concentrations of a biologically active antibody molecule can be used to make suitable dosage formulations (containing high concentrations in non-aqueous suspensions), can provide increased in vivo half-life, and can be formulated with (encapsulated in) polymeric carriers for controlled release of antibody molecules in vivo.

Particularly preferred is a crystallized MAT-Fab antibody that retains binding activity as well as any desired biological activity in a non-crystalline form. A crystallized MAT-Fab bispecific antibody can also provide controlled release of MAT-Fab without a carrier when administered to an individual.

A preferred composition for releasing a crystallized MAT-Fab bispecific antibody of the present invention comprises the crystallized MAT-Fab bispecific antibody described herein, an auxiliary ingredient, and at least one polymeric carrier. The auxiliary ingredient is preferably selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxy polyethylene glycol and polyethylene glycol. The polymeric carrier is preferably a polymer selected from one or more of the groups consisting of: polyacrylic acid, polycyanoacrylates, polyamino acids, polyanhydrides, polydepsipeptides, polyesters, polylactic acid, lactic-glycolic acid or PLGA, poly-b-hydroxybutyrate, polycaprolactone, polydioxanone ; polyethylene glycol, polyhydroxypropylmethacrylamide, polyorganophosphazene, poly(ortho-ethers), polyvinyl alcohol, polyvinylpyrrolidone, copolymers of maleic anhydride and alkyl vinyl ether, pluronyl polyols, albumin, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulphated polysaccharides , their mixtures and copolymers.

Use of bispecific MAT-Fab antibodies

The present invention provides a method of treating a disease or disorder in a human subject, comprising administering to said individual a bispecific MAT-Fab antibody that binds one or two target epitopes, one or two target antigens, or a combination of a target epitope and a target antigen considered to be harmful. in a human subject, wherein binding of the epitopes or antigens by the MAT-Fab bispecific antibody provides treatment for said disease or disorder.

A disorder in which an epitope, antigen, or antigen activity is deleterious is meant to include a disorder in which the presence of an epitope (e.g., an epitope of a cell surface protein or soluble protein) or antigen or antigen activity in a human subject suffering from the disorder is shown to be harmful. , causes or is suspected to cause the pathophysiology of the disorder, or is or is suspected to be a factor contributing to the aggravation of the disorder. Accordingly, a disorder in which an epitope, antigen, or antigen activity is deleterious is a disorder in which a reduction in the level of the epitope, antigen, or antigen activity is expected to alleviate one or more symptoms of the disorder or the progression of the disorder, and thereby treat the disorder. Such disorders can be confirmed, for example, by an increase in the concentration of an antigen (or epitope) in the blood or other body fluid of a human subject suffering from the disorder.

In yet another embodiment, the present invention provides a method of treating a human subject suffering from a disorder in which one or two target antigens or epitopes capable of binding to a MAT-Fab bispecific antibody described herein are deleterious to the human subject, wherein said method comprises administering to a human subject a MAT-Fab bispecific antibody described herein such that the activity of one or two target antigens or epitopes in the human subject is inhibited and treatment is achieved.

The MAT-Fab bispecific antibody described herein is particularly useful in a method of treating a disorder involving "retargeting" (or "recruiting") effector cells (e.g., T cells, NK cells, monocytes, neutrophils, macrophages) to attack specific target cells that express the antigen associated with the disorder and are considered harmful to the human subject, and therefore it is desirable to eliminate or significantly reduce the population of harmful target cells. Preferred examples of such harmful target cells are tumor cells (eg, tumor cells in the blood (including lymph) and solid tumor cells), cells responsive to self-antigens, and virus-infected cells. In the retargeting method of the present invention, a MAT-Fab antibody binds an antigen expressed on the surface of an effector cell and an antigen expressed on the surface of a target cell that is harmful to a human subject, wherein the binding of the MAT-Fab antibody to the effector cell antigen and to the antigen of the harmful cell activates an effector cell that attacks the harmful target cell.

Accordingly, the present invention provides a method of treating a disorder in a human subject, comprising the step of administering to the human subject a MAT-Fab bispecific antibody described herein that binds an antigen on an effector cell and an antigen associated with the disorder expressed on a target cell, which is detrimental to a human subject, wherein binding of the MAT-Fab bispecific antibody to both the effector cell and the target cell activates the effector cell, which attacks the detrimental target cell.

Simultaneous binding of the MAT-Fab bispecific antibody described herein to a single target antigen on an effector cell and a single target antigen on a deleterious target cell can activate the effector cell, resulting in an attack on the target cell, while advantageously avoiding a massive deleterious release of cytokines. (cytokine storm). The massive release of cytokines (cytokine storm) can have a harmful effect not only on local tissues, but also on more distant tissues of the patient's body, which can lead to possible complications and adverse side effects. Such a cytokine storm can occur in a natural immune response in which an effector cell, such as a T cell, binds an antigen presenting cell (APC), or in artificial crosslinking of two or more antigens on the surface of the effector cell with a divalent or polyvalent antibody. In contrast to the above, due to the design of the bispecific MAT-Fab antibody according to the present invention, which provides for antigen binding to the effector cell by only one of the Fab binding units, the likelihood of non-specific activation of T cells and subsequent cytokine storm is significantly reduced, while the antibody retains the ability to activate the effector cell , resulting in an attack on the target cell by the other Fab binding unit of the MAT-Fab antibody.

The method of retargeting an effector cell resulting in an attack by a malicious target cell preferably comprises contacting the effector cell and the malicious target cell with a MAT-Fab bispecific antibody described herein that binds an antigen on the malicious target cell and an antigen expressed on the effector cell . Preferred effector cell antigens include CD3, CD16 (also referred to as "FcγRIII") and CD64 (also referred to as "FcγRI"). More preferably, the method comprises a MAT-Fab antibody that binds CD3 expressed on a T cell, CD16 expressed on a natural killer (NK cell) or CD64 expressed on a macrophage, neutrophil or monocyte.

In another embodiment, the present invention provides a method of treating a tumor in a human subject in need of treatment, comprising the step of administering to the human subject a MAT-Fab antibody that binds an antigen on an effector cell and also binds an antigen on a target tumor cell, wherein the binding the MAT-Fab antibody with the effector cell and the target tumor cell activates the effector cell, which attacks the tumor cell. The antigen on the effector cell is preferably CD3 expressed on a T cell.

In a preferred embodiment, a method of treating a tumor in a human subject in need of treatment comprises retargeting an effector cell resulting in an attack on the target tumor cell, comprising the step of administering to the human subject a MAT-Fab bispecific antibody described herein that binds an antigen to the effector cell. cell and an antigen on the target tumor cell, wherein the antigen on the target tumor cell is selected from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 (“Her2”), embryonic tumor antigen (“CEA”), molecule epithelial cell adhesion (EpCAM) and orphan receptor-1 protein like receptor tyrosine kinase (ROR 1).

In yet another embodiment, the present invention provides a method for treating a B cell-associated tumor in a human subject, comprising the step of administering to the human subject in need of such treatment a MAT-Fab bispecific antibody that binds an antigen on an effector cell and an antigen on malignant B cells. The MAT-Fab bispecific antibody of the present invention preferably binds an antigen on malignant B cells in a cancer selected from the group consisting of: acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia/cell-involving lymphoma - precursors, B-cell neoplasms involving mature cells, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, lymphoma from central cells of skin follicles, B-cell marginal zone lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma.

In a particularly preferred embodiment, a method of treating a B cell associated tumor in a human subject comprises administering to the human subject a MAT-Fab bispecific antibody described herein that binds CD3 on a T cell and CD20 on a tumor target B cell. . More preferably, the MAT-Fab bispecific antibody binds CD20 through its outer (N-terminal) Fab binding unit (e.g., see VHA-CH1 and VLA-CL Fab-containing domains in Figure 1A) and CD3 through its inner (C- terminal) Fab binding unit (eg, see Fab-containing domains of VHB-CH1 and VLB-CL in Fig. 1A).

In yet another embodiment, a method for treating a disorder of the present invention may include contacting a MAT-Fab antibody with effector cells and deleterious target cells in a specific type of ex vivo procedure in which effector cells isolated from a human subject in need of treatment , are contacted with the MAT-Fab antibody outside the human body, and after some time necessary for the binding of the MAT-Fab antibody to the effector cells, the effector cells associated with the MAT-Fab antibody are administered to the human subject in such a way that the complexes of the MAT-Fab antibody Fab associated with effector cells can be targeted to bind harmful target cells, such as tumor cells, in the body of a human subject. In yet another embodiment, effector cells and tumor cells isolated from a human subject in need of treatment are contacted with a MAT-Fab antibody outside the human body, and after some time is necessary for the MAT-Fab antibody to bind to the effector cells and tumor cells. cells, effector cells and tumor cells associated with the MAT-Fab antibody are administered to a human subject.

In yet another embodiment, a MAT-Fab bispecific antibody can be engineered to deliver a cytotoxic agent to a harmful target cell, such as a tumor cell. In this embodiment, one Fab binding unit of the MAT-Fab contains a binding site for a target antigen on a harmful target cell, and the other Fab binding unit contains a binding site for a cytotoxic agent. Such an engineered MAT-Fab of the present invention may be mixed or otherwise contacted with a cytotoxic agent to which it binds via its engineered Fab binding unit. The MAT-Fab carrying the bound cytotoxic agent can then be brought into contact with the noxious tumor cell to deliver the cytotoxic agent to the noxious tumor cell. Such a delivery system is particularly effective if the MAT-Fab binds to the malicious tumor cell and is then internalized into the cell along with the associated cytotoxic agent such that the cytotoxic agent can be released into the malicious tumor cell.

In addition, the bispecific MAT-Fabs provided herein can be used for tissue-specific delivery (targeting a tissue marker and disease mediator to enhance local pharmacokinetics and thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and a molecule within the cell), delivery to the brain (eg, targeting the transferrin receptor and a central nervous system (CNS) disease mediator to cross the blood-brain barrier). MAT-Fab antibodies can also be used as a carrier protein to deliver an antigen to a specific site by binding to a non-neutralizing epitope of that antigen, as well as to increase the half-life of the antigen.

In addition, the MAT-Fab bispecific antibody design can be physically attached to or targeted to medical devices implanted in a patient (see Burke et al. (2006) Advanced Drug Deliv. Rev. 58(3): 437-446; Hildebrand et al. (2006) Surface and Coatings Technol. 200: 6318-6324; "Drug/device combinations for local drug therapies and infection prophylaxis," Wu, Peng, et al., (2006) Biomaterials, 27 (11):2450-2467 "Mediation of the cytokine network in the implantation of orthopedic devices," Marques et al., in Biodegradable Systems in Tissue Engineering and Regenerative Medicine, Reis et al., eds. (CRC Press LLC, Boca Raton, 2005) pp. 377-397). Targeting appropriate cell types to the area of a medical implant can stimulate healing and restoration of normal tissue function. Alternatively, MAT-Fab bispecific antibodies can be used to inhibit mediators (including but not limited to cytokines) released upon device implantation. The present invention also provides diagnostic applications, including, but not limited to, diagnostic assay methods, diagnostic kits containing one or more binding proteins, and adaptation of said methods and kits for use in automated and/or semi-automated systems. The proposed methods, kits and adaptations can be used to detect, monitor and/or treat a disease or disorder in an individual. This is detailed below.

The MAT-Fab bispecific antibody described herein is readily adaptable in one of the various immunological detection assays and purification formats available in the art for detecting, quantifying, or isolating a target antigen or cell expressing the target antigen. Such formats include immunoblot-based assays (eg, Western blotting); immunoaffinity chromatography, for example by adsorbing or binding a MAT-Fab bispecific antibody to a chromatographic resin or beads; immunoprecipitation assays; immunochips; analyzes based on tissue immunohistochemistry; flow cytometry (including cell sorting with fluorescence activation); sandwich immunoassays; immunochips, where the MAT-Fab antibody is immobilized or bound to a support; radioimmunoassay (RIA); enzyme immunoassay (ELISA); enzyme-linked immunosorbent assay (solid-phase ELISA); competitive immunoassays with inhibition; fluorescent polarization immunoassay (FPIA); Enzymatic Enhanced Immunoassay (EMIT) technique; resonant bioluminescence energy transfer (BRET); and homogeneous chemiluminescent assay, but are not limited to. The present invention provides methods using mass spectrometry that include MALDI (Matrix Activated Laser Desorption Ionization) or SELDI (Surface Enhanced Laser Desorption Ionization) with a fragment of a MAT-Fab that binds a target antigen or epitope on an antigen or fragment thereof. , but are not limited to them.

In addition, the present invention provides a method for detecting an antigen in a sample (eg, mixture, composition, solution, or biological sample) comprising contacting the sample with a MAT-Fab bispecific antibody of the present invention that binds the target antigen. Biological samples that can be used as a sample for the immunological detection assay of the present invention include, without limitation, whole blood, plasma, serum, various tissue extracts, tears, saliva, urine, and other bodily fluids. The immunoassay of the present invention preferably detects any of the following: a MAT-Fab bispecific antibody bound to a target antigen, a complex of a MAT-Fab bispecific antibody and a target antigen, or a MAT-Fab bispecific antibody remaining in an unbound state. Methods for detecting a binding complex comprising a target antigen and a MAT-Fab antibody preferably use a detection system that uses one or more signal generating molecules (detectable labels) that should generate a signal easily detectable by the naked eye or easily detectable or measurable by an instrument. to detect the signal (for example, with a spectrophotometer). The MAT-Fab bispecific antibody can be directly or indirectly labeled with a detectable signal generating system to facilitate detection of bound or unbound MAT-Fab bispecific antibody. Detectable signals that can be used in detecting a binding complex containing a target antigen and a bispecific MAT-Fab include a fluorescent signal (e.g., generated by a fluorescent dye or a cyanine molecule that can be bound to one of the Fab binding units of the MAT-Fab or another way to attach to the MAT-Fab antibody); a visible color signal (eg, generated by an enzyme or a colored molecule (eg, pigment), which can also be directly or indirectly attached to a MAT-Fab antibody); a radioactive signal (eg, generated by a radioisotope that can be directly or indirectly attached to a MAT-Fab antibody); and a light signal (eg, generated by a chemiluminescent or bioluminescent system), but are not limited to. An example of a bioluminescent system is the luciferin-luciferase system, in which the luciferase can be directly or indirectly coupled to a MAT-Fab antibody or secondary detection antibody to generate a detectable light signal in the presence of a luciferin substrate.

A preferred enzyme that can be used in the immunological detection assay of the present invention is an enzyme capable of providing a detectable signal upon contact with one or more reagents. Such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

Examples of suitable fluorescent materials that can be used in the immunological detection assay of the present invention include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.

An example of a luminescent material that can be used in the immunological detection assay of the present invention is luminol.

Examples of suitable radioactive isotopes that can be used in the immunological detection assay of the present invention include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm, but not limited to them.

Pharmaceutical compositions

The present invention provides pharmaceutical compositions for use in the treatment of a human subject comprising the MAT-Fab bispecific antibody described herein. Such compositions are prepared using techniques and ingredients well known in the art for preparing pharmaceutical compositions for administering therapeutic antibodies to human subjects. A composition containing a MAT-Fab antibody described herein can be formulated for administration by any of a variety of routes or routes of administration. A composition containing the MAT-Fab antibody described herein can be formulated for parenteral or non-parenteral administration. A composition containing a MAT-Fab antibody for use in the treatment of cancer, an autoimmune disease, or an inflammatory disease can be formulated for parenteral administration, such as, but not limited to, intravenous, subcutaneous, intraperitoneal, or intramuscular administration. More preferably, the composition is formulated for intravenous administration. Such parenteral administration is preferably carried out by injection or infusion of the composition.

Compositions containing a MAT-Fab antibody for human administration may contain an effective amount of the MAT-Fab antibody in combination with one or more pharmaceutically acceptable ingredients, e.g., a pharmaceutically acceptable carrier (carrier medium, buffer), an excipient, or other ingredient. By "pharmaceutically acceptable" is meant that the compound, component, or ingredient of the composition is compatible with the physiology of the human subject, and does not adversely affect the effective activity of the MAT-Fab antibody component or the desired property or activity of any other component that may be present in compositions for administration to a human subject. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases it may be preferable to include isotonic agents in the composition, including carbohydrates, polyhydric alcohols such as mannitol or sorbitol; sodium chloride and combinations thereof, but not limited to. Pharmaceutically acceptable carriers may additionally contain minor amounts of excipients, such as humectants or emulsifiers, preservatives or buffers, to increase the half-life or effectiveness of the composition. An excipient is generally any compound or combination of compounds that imparts a desired property to the composition in addition to its primary therapeutic activity. It may be necessary to adjust the pH of the composition, for example, to promote or maintain the solubility of the ingredients, maintain the stability of one or more ingredients of the composition, and/or to curb unwanted growth of microorganisms that could potentially be introduced into it at some points in the procedure.

Compositions containing the MAT-Fab antibody may also contain one or more other ingredients, such as other drugs (eg, an anticancer agent, an antibiotic, an anti-inflammatory compound, and an antiviral agent), excipients, adjuvants, and combinations thereof.

The composition according to the present invention may be in various forms. These forms include, but are not limited to, liquid, semi-solid, and solid dosage forms, dispersions, suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended route of administration. Preferred compositions are in the form of injections or infusions, for example compositions similar to those used to administer therapeutic antibodies approved for use in humans. In a preferred embodiment, the MAT-Fab described herein is administered by intravenous injection or infusion. In yet another embodiment, the MAT-Fab antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound, i. MAT-Fab antibodies, in the required amount in an appropriate solvent, optionally in combination with ingredients that provide the desired property of the composition, followed by filter sterilization in accordance with the requirements. Typically, dispersions are made by incorporating the active compound into a sterile carrier medium containing a basic dispersion medium (eg, sterile water, sterile isotonic saline, etc.) and, optionally, one or more other ingredients that may be required to obtain proper dispersion. In the case of sterile lyophilized powders for the preparation of sterile injectable solutions, preferred methods of manufacture include vacuum drying and spray drying, which make it possible to obtain a powder of the active ingredient in combination with any additional desired ingredient from its solution previously sterilized by filtration. Proper fluidity of the solution can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. Long-term absorption of injectable compositions can be accomplished by including in the composition an absorption retarding agent, for example a monostearate salt and/or gelatin.

The MAT-Fab antibody can be administered using a variety of methods known in the art. The practitioner will appreciate that the route or mode of administration varies depending on the desired results. In some embodiments, the MAT-Fab antibody can be formulated in combination with a carrier that protects the antibody from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid. Various methods for making such compositions are known to those skilled in the art.

The above pharmaceutical composition for administration to an individual can be prepared by at least one method selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intracerebroventricular, intraintestinal, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostate, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal and transdermal administration.

Additional implementation options and features of the present invention will become apparent from the following non-limiting examples.

EXAMPLES

Example 1 Construction, Expression, Purification and Assay of the CD20/CD3 MAT-Fab Bispecific Antibody

To demonstrate the MAT-Fab technology, examples of the bispecific CD20/CD3 MAT-Fab antibody were obtained, differing in the bump-in-trough mutations in the Fc region:

MAT-Fab (KiH1) and MAT-Fab (KiH2).

To obtain MAT-Fab antibodies, the DNA encoding each polypeptide chain was synthesized de novo.

The DNA construct used to generate MAT-Fab antibodies capable of binding CD3 and CD20 encoded the variable and constant domains of the parent monoclonal antibodies (mAbs). Each MAT-Fab consisted of four polypeptides shown in the diagram below:

where "(KiH)" means the presence of one or more mutations that favor or stabilize the heterodimerization of the CH3 domain in the heavy chain and the Fc chain, the "A" antigen is CD20, and the "B" antigen is CD3.

The two constructs, designated MAT-Fab (KiH1) and MAT-Fab (KiH2), differed only in the bump-in-trough mutations in the Fc regions favoring heterodimerization.

Briefly, the parent mAb included two high affinity antibodies, an anti-CD20 mAb (ofatumumab) and an anti-CD3 mAb (US Patent Publication No. 2009/0252683).

MAT-Fab KiH1 and MAT-Fab KiH2 heavy chain constructs

For heavy chain production of the MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies, the DNA construct encoded a 22 amino acid signal peptide fused to the VL-CL light chain of the parent anti-CD20 mAb (ofatumumab) fused to the N-terminus of the VH region. -CH1 of the original anti-CD3 mAb (US Patent Publication No. 2009/0252683) attached to the hinge-CH2 region of the original anti-CD3 mAb attached to a mutated IgG1 CH3 region derived from the original anti-CD3 mAb.

The heavy chain CH3 domain of the MAT-Fab bispecific antibody (KiH1) was mutated to replace a threonine (T) residue with a tyrosine (Y) residue to form a structural overhang at residue 21 of the CH3 domain. See Y ₆₁₀ in SEQ ID NO:1 in Table 1 below (residue in underlined font) for this mutation. The corresponding position of the CH3 domain of the MAT-Fab Fc chain (KiH1) was mutated to replace a tyrosine (Y) residue with a threonine (T) residue to form a structural overhang at residue 62 of the CH3 domain. See T ₂₁₃ in SEQ ID NO:4 in Table 4 below (residue underlined) for this mutation.

The heavy chain CH3 domain of the MAT-Fab bispecific antibody (KiH2) was mutated to replace the threonine (T) residue with tryptophan (W) to form a structural overhang in the CH3 domain. See W610 in SEQ ID NO:5 in Table 5 below (residue underlined) for this mutation. The CH3 domain of the MAT-Fab Fc chain (KiH2) was mutated to replace threonine (T) with serine (S), leucine (L) with alanine (A), and tyrosine (Y) with valine to form a structural trough in the CH3 domain. See S ₁₇₂ , A ₁₇₄ and V ₂₁₃ in SEQ ID NO:8 in Table 8 below (the remainder in underlined font) for these mutations.

The heavy chain CH3 domain of the MAT-Fab bispecific antibody (KiH2) was also mutated to replace a serine (S) residue with a cysteine (C) residue; and the Fc chain of the bispecific MAT-Fab antibody (KiH2) was mutated to replace a tyrosine (Y) residue with a cysteine (C) residue. Insertion of these cysteine residues (C) provides for the formation of a disulfide bond with the complementary mutated CH3 domain of the Fc polypeptide chain, resulting in improved heterodimer stability. See C ₅₉₈ in SEQ ID NO:5 in Table 5 (residue in underline) and C ₁₅₅ in SEQ ID NO:8 in Table 8 below (residue in underline) for these cysteine substitutions.

The heavy chains and Fc chains of both MAT-Fab KiH1 and MAT-Fab KiH2 were also mutated to replace the two leucine residues at positions 18-19 of the hinge-CH2 region with two alanine residues to reduce or abolish ADCC/CDC effector functions (Canfield et al., J. Exp. Med., 173(6): 1483-1491 (1991)). These leucine to alanine substitution mutations are shown at ₄₇₈ AA ₄₇₉ in SEQ ID NO:1 in Table 1, ₄₀ AA ₄₁ in SEQ ID NO: 4 in Table 4, ₄₇₈ AA ₄₇₉ in SEQ ID NO: 5 in Table 5, and ₄₀ AA ₄₁ in SEQ ID NO:8 in Table 8.

MAT-Fab KiH1 and MAT-Fab KiH2 first light chain constructs

To generate the first light chains of the MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies, the DNA construct encoded a 19 amino acid signal peptide fused to the VH-CH1 fragment of the original anti-CD20 mAb (ofatumumab).

MAT-Fab KiH1 and MAT-Fab KiH2 Second Light Chain Constructs

To generate second light chains of MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies, the DNA construct encoded a 20 amino acid signal peptide fused to the light chain VL-CL fragment of the parent anti-CD3 mAb (U.S. Patent Publication No. 2009/0252683) .

MAT-Fab KiH1 and MAT-Fab KiH2 Fc chain constructs

For the production of polypeptide Fc chains of the MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies, the DNA construct encoded a 22 amino acid signal peptide (same as for the heavy chain constructs) attached to the hinge-CH2 region of the original anti-CD3 mAb. , attached to a mutated IgG1 isotype CH3 region containing the mutations discussed above in the discussion of MAT-Fab heavy chain constructs.

The heavy chain CH3 domain of the Fc chain of the bispecific MAT-Fab antibody (KiH2) was mutated to replace the tyrosine (Y) residue with a threonine (T) residue to form a structural trough in the CH3 domain complementary to the structural nub mutation in the heavy CH3 domain. chains (see above).

The CH3 domain of the Fc chain of the bispecific MAT-Fab antibody (KiH2) was mutated to replace a threonine (T) residue with a serine (S) residue, replace a leucine (L) residue with an alanine (A) residue, and replace a tyrosine (Y) residue with a valine residue (V) for the formation of a structural cavity in the CH3 domain. The CH3 domain was also mutated to replace a tyrosine (Y) residue with a cysteine (C) residue. Insertion of this cysteine residue (C) provides for the formation of a disulfide bond with the complementary mutated MAT-Fab heavy chain CH3 domain (KiH2) described above, further stabilizing heterodimerization.

The amino acid sequences of these four polypeptide chains of the MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies are shown in the tables below. Table 1. Heavy chain of the CD20/CD3 MAT-Fab bispecific antibody (KiH1)

12345678901234567890123456789012345678901234567890 Heavy chain MAT-Fab (KiH1) MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATLSCRASQS
VSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLLESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1) signal
subsequence residues 1-22 SEQ ID NO:1 VL region
ofatumumab vs.
CD20 residues 23-129 SEQ ID NO:1 CL ofatumumab residues 130-236 SEQ ID NO:1 VH vs. CD3 residues 237-361 SEQ ID NO:1 CH1 residues 362-460 SEQ ID NO:1 hinge-CH2 residues 461-588 SEQ ID NO:1 mutated
(KiH1)CH3 residues 589-691 SEQ ID NO:1

Table 2. First light chain of the CD20/CD3 bispecific antibody MAT-Fab (KiH1)

12345678901234567890123456789012345678901234567890 First light chain
MAT-Fab (KiH1) MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFND
YAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYL QMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKQVEPKSC2 signal
subsequence residues 1-19 SEQ ID NO:2 VH area
ofatumumab vs.
CD20 residues 20-141 SEQ ID NO:2 CH1 ofatumumab residues 142-244 SEQ ID NO:2

Table 3. Second light chain of the CD20/CD3 MAT-Fab bispecific antibody (KiH1)

12345678901234567890123456789012345678901234567890 Second light chain
MAT-Fab (KiH1) MTWTPLLFLTLLLHCTGSLSELVVTQEPSLTVSPGGTVTLTCRSSTGAVT
(SEQ ID NO:3) signal
subsequence residues 1-20 SEQ ID NO:3 VL-region vs.
CD3 residues 21-129 SEQ ID NO:3 CL vs. CD3 residues 130-235 SEQ ID NO:3

Table 4. Polypeptide Fc chain of CD20/CD3 MAT-Fab (KiH1)

12345678901234567890123456789012345678901234567890 Polypeptide Fc-
chain MAT-Fab
(KiH1) MDMRVPAQLLGLLLWFPGSRCPKSCDKTHTCPPCPAPEAAGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
PGK (SEQ ID NO:4) signal
subsequence residues 1-22 SEQ ID NO:4 hinge-CH2 residues 23-150 SEQ ID NO:4 mutated
(KiH1)CH3 residues 151-253 SEQ ID NO:4

Table 5. Heavy chain of the CD20/CD3 MAT-Fab bispecific antibody (KiH2)

12345678901234567890123456789012345678901234567890 Heavy chain MAT-
Fab (KiH2) MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATLSCRASQS
VSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLLESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:5) signal
subsequence residues 1-22 SEQ ID NO:5 VL region
ofatumumab vs.
CD20 residues 23-129 SEQ ID NO:5 CL ofatumumab residues 130-236 SEQ ID NO:5 VH vs. CD3 residues 237-361 SEQ ID NO:5 CH1 residues 362-460 SEQ ID NO:5 hinge-CH2 residues 461-588 SEQ ID NO:5 mutated
(KiH2)
CH3 residues 589-691 SEQ ID NO:5

Table 6. First light chain of the CD20/CD3 bispecific antibody MAT-Fab (KiH2)

12345678901234567890123456789012345678901234567890 First light chain
MAT-Fab (KiH2) MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFND
YAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYL QMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKQVEPKSC6 signal
subsequence residues 1-19 SEQ ID NO:6 VH area
ofatumumab vs.
CD20 residues 20-122 SEQ ID NO:6 CH1 ofatumumab residues 123-244 SEQ ID NO:6

Table 7 Second light chain of the CD20/CD3 bispecific antibody MAT-Fab (KiH2)

12345678901234567890123456789012345678901234567890 Second light chain
MAT-Fab (KiH2) MTWTPLLFLTLLLHCTGSLSELVVTQEPSLTVSPGGTVTLTCRSSTGAVT
(SEQ ID NO:7) signal
subsequence residues 1-20 SEQ ID NO:7 VL-region vs.
CD3 residues 21-129 SEQ ID NO:7 CL vs. CD3 residues 130-235 SEQ ID NO:7

Table 8. Polypeptide Fc chain of CD20/CD3 MAT-Fab (KiH2)

12345678901234567890123456789012345678901234567890 Polypeptide Fc-
chain MAT-Fab
(KiH2) MDMRVPAQLLGLLLWFPGSRCPKSCDKTHTCPPCPAPEAAGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
PGK (SEQ ID NO:8) signal
subsequence residues 1-22 SEQ ID NO:8 hinge-CH2 residues 23-150 SEQ ID NO:8 mutated
(KiH2)CH3 residues 151-253 SEQ ID NO:8

Expression levels

The above DNA constructs encoding each of the four polypeptide chains for MAT-Fab (KiH1) and MAT-Fab (KiH2) were cloned into the pTT expression vector by standard methods. The resulting recombinant pTT vectors encoding SEQ ID NO:1-4 or encoding SEQ ID NO:5-8 were co-transfected into HEK 293E cells to express the respective MAT-Fab (KiH1) and MAT-Fab (KiH2) bispecific antibodies. Expression levels in cell cultures are shown in the table below.

Table 9

construct Expression level MAT-Fab (KiH1) 20.6 mg/l MAT-Fab (KiH2) 36.7 mg/l

Purification and research of characteristics

After completion of the production phase, cell cultures were harvested and subjected to protein purification by protein A affinity chromatography.

The monomeric fractions of each of the MAT-Fab antibody preparations after one-step purification by protein A chromatography were determined by size exclusion chromatography (SEC). The results of the SEC analysis are shown in figures 2 and 3.

SEC analysis revealed that 98.19% of the MAT-Fab antibody preparation (KiH1) and 95.7% of the MAT-Fab antibody preparation (KiH2) were represented by a single substance ("monomeric fraction").

Fluorescence Activated Cell Sorting (FACS)

The ability of purified MAT-Fab (KiH1) and MAT-Fab (KiH2) to bind target antigens CD20 and CD3 expressed on the cell surface was tested by fluorescence activated cell sorting (FACS) using the protocols described below.

Equipment

Serial number BD FACSVerse: 01131005894

Serial number of Olympus CKX41: 01121005367

Eppendorf 5810R centrifuge serial number: 01121005414

Serial number Thermo Series II with water jacket: 01121005408

Materials and reagents

BD Falcon Round Bottom Tube, P/N 352052 Lot # 3070549 96-well U-bottom Low Evaporation Tissue Culture Plate, P/N 353077 Lot # 34285048

FBS, GIBCO P/N 10099, Lot 1652792

PBS, GIBCO P/N 10010, Lot 1710584

Media RPMI 1640(1x), GIBCO P/N A10491, Lot 1747206

Mouse anti-human IgG1 Alexa Fluor® 488. Invitrogen, cat. no. A-10631, lot

1744792

cell lines

Jurkat, ATCC catalog # TIB-152, is a T-cell leukemia cell line expressing the CD3 surface antigen.

Raji, ATCC Cat # CCL-86 Lot 60131961, is a Burkitt B-cell lymphoma line expressing the CD20 surface antigen.

Antibodies for FACS

Description of antibodies Target Molecular
weight
(kilodalton) mg/ml human IgG1 vs.
RAC low molecular weight compound 150 1.44 MAT-Fab (KiH1) CD20 and CD3 150 2.19 MAT-Fab (KiH2) CD20 and CD3 150 2

FACS procedure

1. Aliquot 5x105 cells in ice-cold PBS.

2. Cells were blocked in 2% PBS/PBS for 30 on ice.

3. Cells were incubated with anti-CD20 antibodies for 1 hour on ice. The initial antibody concentration was (133.33 nM) 20 μg/ml; the antibody was serially diluted in a ratio of 1:5. The volume of each dilution was 200 μl.

4. Washed three times with PBS, centrifuged at 2000 rpm for 5 min at 4°C.

5. Cells were incubated with secondary mouse anti-human Fc IgG1 antibody, 100 µl (10 µg/ml) mouse anti-IgG1 Alexa Fluor® 488 (AF488) (diluted 1:100) for 1 h on ice and kept out of Sveta.

6. Washed three times with PBS, centrifuged at 2000 rpm for 5 min at 4°C.

7. Cells were resuspended in PBS. Mean fluorescence intensity (MFI) was determined on a BD FACSVerse flow cytometer.

results

The results are shown in Figures 4 and 5. Both MAT-Fab bispecific antibodies were able to bind CD20 and CD3 on B cells (CD20 Raji cells) and T cells (CD3 Jurkat cells), respectively.

Functional activity to induce B cell apoptosis in the presence of T cells

The ability of purified MAT-Fab (KiH1) and MAT-Fab (KiH2) to bind target antigens CD20 and CD3 expressed on the cell surface was tested by B cell apoptosis assay using the protocols described below.

Equipment

Serial number BD FACSVerse: 01131005894

Serial number of Olympus CKX41: 01121005367

Eppendorf 5810R centrifuge serial number: 01121005414

Serial number Thermo Series II with water jacket: 01121005408

Materials and reagents

BD Falcon Round Bottom Tube, P/N 352052 Lot # 3070549 96-well U-bottom Low Evaporation Tissue Culture Plate, P/N 353077 Lot # 4292046

FBS, GIBCO P/N 10099, Lot 1652792

FSB, GIBCO Cat. No. 10010, Lot 1710584

RPMI 1640(1×), GIBCO P/N 11875, Lot 1731226 Ficoll Paque Plus, GE HEALTHCARE, P/N GE17144002, Lot 10237843 Mouse anti-human CD19 PE, BD Pharmingen, P/N 555413, batch: 5274713

cell lines

Raji, ATCC Part # CCL-86, Lot 60131961.

Donor number: 00123

Antibodies for B cell apoptosis assay

Name of the antibody batch number Antibody specificity MAT-Fab (KiH1) 160411002 CD20/CD3 Ofatumumab 20140225B CD20 mAb against CD3 Pr2016012817 CD3 MAT-Fab (KiH2) 160429004 CD20/CD3

Procedures

Procedure for isolating mononuclear cells

Preparing a blood sample

1. 100 ml of fresh human blood was taken into test tubes with anticoagulant.

2. An equal volume of RPMI1640 (100 ml) was added to the blood and mixed gently.

3. The ficoll-pack density gradient medium was heated to 18-20°C before use.

4. The ficoll-pack medium vial was inverted several times to ensure thorough mixing.

5. Ficoll-Pak medium (15 ml) was added to a 50 ml centrifuge tube.

6. The diluted blood sample (20 ml) was carefully layered onto the Ficoll-Pak medium solution without mixing the Ficoll-Pak medium solution and the diluted blood sample.

7. Fikoll-pack with a blood sample was centrifuged at 400 g for 30-40 min at a temperature of 18°C to 20°C.

8. The layer of mononuclear cells was transferred from the gradient to a sterile centrifuge tube. Cell isolate washing

1. The volume of transferred mononuclear cells was assessed and at least 3 volumes of PBS were added to the mononuclear cells in the centrifuge tube.

2. Cells and PBS were centrifuged at 400-500 x g for 10 min at 18°C to 20°C, the supernatant was removed.

3. Washing was repeated.

4. The supernatant was removed and the cell pellet was resuspended in assay buffer.

B-cell depletion

The assay medium was RPMI1640 supplemented with 10% PBS

1. Target cells (Raji cells) were harvested in logarithmic growth phase and washed once with assay medium. The cell density was adjusted to 5 x 10 ⁵ cells/ml and 100 μl of the cell suspension was added to each well of the assay plate.

2. The assay antibody was serially diluted and added to each well of the above assay plate in a volume of 50 µl. The final initial concentration of the antibody was 50 mg/ml and subsequently subjected to serial dilution.

3. The MIC density was adjusted to 2.5 x 10 ⁶ cells/ml and 100 μl of the cell suspension was added to each well of the assay plate (5:1 ratio of effector cells to target cells).

4. Tablets for analysis were incubated at 37°C with 5% CO ₂ for 1 day, 2 days and 3 days.

On days 1, 2, and 3, samples were collected by centrifugation at 2000 rpm for 5 min at 4°C and washed once with PBS.

5. Each sample was incubated with 50 µl of PE-conjugated anti-human CD19 detection antibody diluted 1:5 for 30 minutes on ice.

6. Samples were washed with PBS and analyzed with FACSVerse.

results

The results are shown in Figure 6. By day 2 of the cell depletion assay, both MAT-Fab bispecific antibodies could induce T cell mediated B cell apoptosis.

All patents, applications and publications cited in the above text are incorporated herein by reference.

Additional modifications and embodiments of the invention described herein are obvious to those skilled in the art without departing from the description of the present invention or the claims below.

--->

SEQUENCE LIST

<110> EPIMAB BIOTHERAPEUTICS, INC.

WU Chengbin

<120> Univalent asymmetric bispecific antibodies with

tandem fab

<130>EPM-103.1P

<140>

<141>

<150> PCT/US2017/046875

<151> 08/15/2017

<150> PCT/CN2016/109267

<151> 09.12.2016

<150> PCT/CN2016/095546

<151> 08/16/2016

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 691

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hybrid immunoglobulin using sequences

originating from various organisms

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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr

20 25 30

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

35 40 45

Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

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

100 105 110

Arg Ser Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

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

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

275 280 285

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp

290 295 300

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

305 310 315 320

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

325 330 335

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Pro Gly Lys

690

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Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

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Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met

115 120 125

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Pro Lys Ser Cys

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Met Thr Trp Thr Pro Leu Leu Phe Leu Thr Leu Leu Leu His Cys Thr

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Gly Ser Leu Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Val

20 25 30

Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala

35 40 45

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

50 55 60

Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr

65 70 75 80

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

85 90 95

Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu

100 105 110

Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val

115 120 125

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

130 135 140

Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser

145 150 155 160

Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser

165 170 175

Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn

180 185 190

Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp

195 200 205

Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr

210 215 220

Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230 235

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<213> Artificial sequence

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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Pro Lys Ser Cys Asp Lys Thr His Thr Cys

20 25 30

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

35 40 45

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

50 55 60

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

65 70 75 80

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

85 90 95

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

100 105 110

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

115 120 125

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

130 135 140

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

145 150 155 160

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

165 170 175

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

180 185 190

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

195 200 205

Ser Phe Phe Leu Thr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

210 215 220

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

225 230 235 240

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

245 250

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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr

20 25 30

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

35 40 45

Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

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

100 105 110

Arg Ser Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

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

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

275 280 285

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp

290 295 300

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

305 310 315 320

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

325 330 335

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Pro Gly Lys

690

<210> 6

<211> 244

<212> PROTEIN

<213> Homo sapiens

<400> 6

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met

115 120 125

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Pro Lys Ser Cys

<210> 7

<211> 235

<212> PROTEIN

<213> Artificial sequence

<220>

<223> humanized immunoglobulin light chain

<400> 7

Met Thr Trp Thr Pro Leu Leu Phe Leu Thr Leu Leu Leu His Cys Thr

1 5 10 15

Gly Ser Leu Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Val

20 25 30

Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala

35 40 45

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

50 55 60

Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr

65 70 75 80

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

85 90 95

Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu

100 105 110

Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val

115 120 125

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

130 135 140

Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser

145 150 155 160

Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser

165 170 175

Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn

180 185 190

Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp

195 200 205

Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr

210 215 220

Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230 235

<210> 8

<211> 253

<212> PROTEIN

<213> Artificial sequence

<220>

<223> modified immunoglobulin hinge and constant regions

<400> 8

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Pro Lys Ser Cys Asp Lys Thr His Thr Cys

20 25 30

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

35 40 45

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

50 55 60

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

65 70 75 80

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

85 90 95

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

100 105 110

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

115 120 125

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

130 135 140

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

145 150 155 160

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

165 170 175

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

180 185 190

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

195 200 205

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

210 215 220

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

225 230 235 240

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

245 250

<---

Claims (91)

1. Monovalent asymmetric bispecific antibody with tandem Fab (MAT-Fab antibody) containing polypeptide chains (a), (b), (c) and (d), where: (a) is a heavy polypeptide chain (heavy chain), with each heavy chain containing (from N-terminus to C-terminus): VL _A -CL-VH _B -CH1-hinge-CH2-CH3m1, where: VL _A is a human immunoglobulin light chain variable domain directly fused to CL, which is a human light chain constant domain, wherein VL _A -CL is an immunoglobulin light chain component of the first Fab binding unit recognizing the first antigen or epitope and is directly fused with VH _B , wherein VH _B is a human immunoglobulin heavy chain variable domain directly fused to CH1, which is a human immunoglobulin heavy chain constant CH1 domain, wherein VH _B -CH1 is the immunoglobulin heavy chain component of the second Fab binding unit recognizing a second antigen or epitope, wherein VH _B -CH1 is directly fused to the hinge-CH2 structure, wherein the hinge-CH2 structure is the immunoglobulin heavy chain hinge-CH2 region, and the hinge-CH2 structure is directly fused to CH3m1, which is the first constant CH3- immunoglob heavy chain domain a human ulina mutated with one or more ridge-to-cavity (KiH) mutations to form a structural ridge or structural trough in said CH3m1 constant domain; (b) is the first MAT-Fab light chain containing VH _A -CH1, wherein VH _A is a human immunoglobulin heavy chain variable domain directly fused to CH1, which is a human immunoglobulin heavy chain constant CH1 domain, and VH _A -CH1 is the heavy chain component of said first Fab linking unit; (c) is a second MAT-Fab light chain containing VL _B -CL, wherein VL _B is a human immunoglobulin light chain variable domain directly fused to CL, which is a human immunoglobulin light chain CL domain, and VL _B is CL is an immunoglobulin light chain component of said second Fab binding unit; and (d) is an Fc chain polypeptide containing hinge-CH2-CH3m2, where the hinge-CH2 structure is the hinge-CH2 region of the immunoglobulin heavy chain, and the hinge-CH2 structure is directly fused to CH3m2, which is the second constant CH3 domain a human immunoglobulin heavy chain mutated with one or more ridge-to-cavity (KiH) mutations to form a structural ridge or structural trough in said CH3m2 constant domain; provided that: when the CH3m1 domain of the heavy chain is mutated to form a structural overhang, the CH3m2 domain of the Fc chain is mutated to form a complementary structural pit, which facilitates pairing of the CH3m1 domain with the CH3m2 domain; and when the CH3m1 domain of said heavy chain is mutated to form a structural pit, the CH3m2 domain of the Fc chain is mutated to form a complementary structural overhang that facilitates pairing of the CH3m1 domain with the CH3m2 domain; and said MAT-Fab optionally contains a mutation in the CH3m1 domain and in the CH3m2 domain, leading to the introduction of a cysteine residue that promotes the formation of a disulfide bond when the CH3m1 domain is paired with the CH3m2 domain. 2. The MAT-Fab according to claim 1, characterized in that one or more KiH mutations in the CH3m1 domain or the CH3m2 domain, leading to the formation of a structural overhang, is a replacement of a threonine residue with a tyrosine residue or a replacement of a threonine residue with a tryptophan residue. 3. The MAT-Fab antibody according to claim 2, characterized in that the KiH mutation to replace a threonine residue with a tyrosine residue replaces the threonine at position 21 of the CH3 domain with tyrosine. 4. The MAT-Fab antibody according to claim 2, characterized in that the KiH mutation to replace a threonine residue with a tryptophan residue replaces the threonine at position 21 of the CH3 domain with tryptophan. 5. Antibody MAT-Fab according to any one of paragraphs. 2-4, characterized in that the KiH mutation for the formation of a structural overhang is located in the CH3m1 domain of the heavy chain. 6. The MAT-Fab antibody according to claim 1, characterized in that the mutation in the CH3m1 domain or the CH3m2 domain to form a structural cavity is a replacement of a tyrosine residue with a threonine residue, or a combination of replacing a threonine residue with a serine residue, replacing a leucine residue with an alanine residue, and replacing a tyrosine residue with a valine residue. 7. The MAT-Fab according to claim 6, characterized in that the mutation to replace a tyrosine residue with a threonine residue replaces the tyrosine at position 62 of the CH3 domain with a threonine. 8. The MAT-Fab according to claim 6, characterized in that the combination of replacing a threonine residue with a serine residue, replacing a leucine residue with an alanine residue, and replacing a tyrosine residue with a valine residue is a combination of replacing threonine at position 21 of the CH3 domain with serine, replacement of leucine at position 23 of the CH3 domain with alanine; and replacement of tyrosine at position 62 of the CH3 domain with valine. 9. Antibody MAT-Fab according to any one of paragraphs. 6-8, characterized in that one or more KiH mutations for the formation of a structural cavity are located in the CH3m2 domain of the Fc chain. 10. The MAT-Fab according to claim 1, characterized in that each of the CH3m1 domain and the CH3m2 domain further contains a mutation to change the amino acid residue to a cysteine to stimulate the formation of a disulfide bond between the CH3m1 and CH3m2 domains. 11. The MAT-Fab antibody according to claim 10, characterized in that said mutation is a serine to cysteine substitution or a tyrosine to cysteine substitution. 12. The MAT-Fab according to claim 11, characterized in that the replacement of a serine residue with a cysteine residue replaces the serine at position 9 of the CH3 domain with a cysteine. 13. The MAT-Fab according to claim 11, characterized in that the replacement of a tyrosine residue with a cysteine residue replaces the tyrosine at position 4 of the CH3 domain with a cysteine. 14. The MAT-Fab according to claim 10, characterized in that the CH3m1 domain contains a mutation for replacing serine at position 9 of the CH3 domain with cysteine, and the CH3m2 domain contains a mutation for replacing tyrosine at position 4 of the CH3 domain with cysteine. 15. A MAT-Fab according to claim 1, characterized in that each of the CH2 domains of the heavy chain (a) and the Fc chain (d) contains one or more mutations to reduce or eliminate at least one Fc effector function. 16. The MAT-Fab antibody according to claim 15, characterized in that each of the heavy chain CH2 domain and the Fc chain CH2 domain contains two mutations for replacing leucine-234 with alanine and for replacing leucine-235 with alanine (EC numbering ). 17. MAT-Fab antibody according to claim 1, containing: (a) a heavy chain containing the amino acid sequence shown in Table 1, (b) a first light chain containing the amino acid sequence shown in Table 2, (c) a second light chain containing the amino acid sequence shown in Table 3, and (d) Fc chain containing the amino acid sequence shown in Table 4. 18. MAT-Fab antibody according to claim 1, containing: (a) a heavy chain containing the amino acid sequence shown in Table 5, (b) a first light chain containing the amino acid sequence shown in Table 6, (c) a second light chain containing the amino acid sequence shown in Table 7, and (d) Fc chain containing the amino acid sequence shown in table 8. 19. The MAT-Fab antibody according to claim 1, characterized in that the MAT-Fab antibody binds two different epitopes. 20. The MAT-Fab antibody according to claim 1, characterized in that the MAT-Fab antibody binds two different target antigens. 21. The MAT-Fab of claim 20, wherein the two different target antigens are two different target cytokines. 22 The MAT-Fab according to claim 21, characterized in that said two different cytokines are selected from the group consisting of: lymphokines, monokines, and polypeptide hormones. 23. The MAT-Fab antibody according to claim 20, characterized in that said two different target antigens are selected from the group of antigen pairs consisting of: CD20 and CD3, CD3 and CD19, CD3 and Fc-gamma-RIIIA, CD3 and TPBG, CD3 and Epha10, CD3 and IL-5Rα, CD3 and TASCTD-2, CD3 and CLEC12A, CD3 and prominin-1, CD3 and IL-23R, CD3 and ROR1, CD3 and IL-3Rα, CD3 and PSA, CD3 and CD8, CD3 and Glypican-3, CD3 and FAP, CD3 and EphA2, CD3 and ENPP3, CD3 and CD33, CD3 and CD133, CD3 and EpCAM, CD3 and CD19, CD3 and Her2, CD3 and CEA, CD3 and GD2, CD3 and PSMA, CD3 and BCMA, CD3 and A33, CD3 and B7-H3, CD3 and EGFR, CD3 and P-cadherin, CD3 and HMW-MAA, CD3 and TIM-3, CD3 and CD38, CD3 and TAG-72, CD3 and SSTR, CD3 and FRA, CD16 and CD30, CD64 and Her2, CD 137 and CD20, CD138 and CD20, CD19 and CD20, CD38 and CD20, CD20 and CD22, CD40 and CD20, CD47 and CD20, CD 137 and EGFR, CD137 and Her- 2, CD 137 and PD-1, CD 137 and PDL-1, PD-1 and PD-L1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD- 1, TIM-3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF and VEGF, VEGF first epitope and VEGF second (other) epitope, V EGF and Ang2, EGFR and cMet, PDGF and VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1, Lag-3 and PD-1, Lag-3 and PD- L1, Lag-3 and CTLA-4, ICOS and CTLA-4, CD138 and CD40, CD38 and CD138, CD38 and CD40, CD-8 and IL-6, CSPGs and RGM A, CTLA-4 and BTN02, CTLA-4 and PD-1, IGF1 and IGF2, IGF1/2 and Erb2B, IGF-IR and EGFR, EGFR and CD13, IGF-IR and ErbB3, EGFR-2 and IGFR, Her2 first epitope and Her2 second (other) epitope, Factor IXa and Met, factor X and Met, VEGFR-2 and Met, VEGF-A and angiopoietin-2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-lβ, MAG and RGM A, NgR and RGM A , NogoA and RGM A, OMGp and RGM A, PDL-1 and CTLA-4, PD-1 and TIM-3, RGM A and RGM B, Te38 and TNFα, TNFα and Blys, TNFα and CD-22, TNFα and CTLA -4, TNFα and GP130, TNFα and IL-12p40, and TNFα and RANK ligand. 24. The MAT-Fab antibody according to claim 20, characterized in that one of the two different target antigens associated with the MAT-Fab antibody is an antigen expressed on the surface of the effector cell, and the other target antigen associated with the MAT-Fab antibody , is an antigen associated with a disorder expressed on the surface of a target cell considered to be harmful to a human subject. 25. The MAT-Fab antibody according to claim 24, characterized in that the effector cell is selected from the group consisting of: T cell, natural killer (NK cell), monocyte, neutrophil and macrophage. 26. The MAT-Fab antibody according to claim 24, characterized in that the antigen on the effector cell is selected from the group consisting of: CD3, CD16 and CD64. 27. The MAT-Fab antibody according to claim 24, characterized in that the harmful target cell is selected from the group consisting of: a tumor cell, an auto-reactive cell, and a virus-infected cell. 28. The MAT-Fab according to claim 24, characterized in that the antigen associated with the disorder expressed on the surface of the harmful target cell is a tumor-associated antigen expressed on the surface of the tumor cell. 29. The MAT-Fab antibody according to claim 28, characterized in that the tumor-associated antigen is selected from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 (HER2), embryonic tumor antigen (CEA), epithelial cell adhesion molecule (EpCAM) and orphan receptor-1 protein like receptor tyrosine kinase (ROR 1). 30. The MAT-Fab of claim 28, wherein the tumor cell is a malignant B cell. 31. The MAT-Fab antibody according to claim 30, characterized in that the malignant B cell is a cell of a cancerous disorder selected from the group consisting of: acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia /lymphomas involving progenitor cells, B-cell neoplasms involving mature cells, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, follicular central cell lymphoma skin, B-cell marginal zone lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma. 32. The MAT-Fab of claim 28, wherein the antigen on the effector cell is CD3 on a T cell and the tumor associated antigen on the tumor cell is CD20 on a malignant B cell. 33. A MAT-Fab according to claim 1 conjugated to an agent selected from the group consisting of: a therapeutic agent, an imaging agent, and a cytotoxic agent. 34. The MAT-Fab antibody according to claim 33, characterized in that the imaging agent is selected from the group consisting of: radioactive label, enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label, biotin, streptavidin and avidin. 35. MAT-Fab antibody according to claim 34, characterized in that the radioactive label is selected from the group consisting of: ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho and ¹⁵³ Sm. 36. The MAT-Fab antibody according to claim 33, characterized in that the therapeutic or cytotoxic agent is selected from the group consisting of: antimetabolite, alkylating agent, antibiotic, growth factor, cytokine, antiangiogenic agent, antimitotic agent, anthracycline, toxin, and antiapoptotic agent . 37. MAT-Fab antibody according to claim 1 in crystallized form. 38. Composition for releasing crystallized MAT-Fab antibody, containing: (a) a crystallized MAT-Fab according to claim 37; (b) an excipient ingredient and (c) polymer carrier. 39. A composition for releasing a crystallized MAT-Fab antibody according to claim 38, characterized in that the excipient ingredient is selected from the group consisting of: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxy polyethylene glycol and polyethylene glycol. 40. A composition for releasing a crystallized MAT-Fab antibody according to claim 38 or 39, characterized in that the polymeric carrier is preferably a polymer selected from one or more elements of the groups consisting of: polyacrylic acid, polycyanoacrylates, polyamino acids, polyanhydrides, polydepsipeptides, polyesters, polylactic acid, copolymer of lactic and glycolic acids or PLGA, poly-b-hydroxybutyrate, polycaprolactone, polydioxanone; polyethylene glycol, polyhydroxypropylmethacrylamide, polyorganophosphazene, poly(ortho-ethers), polyvinyl alcohol, polyvinylpyrrolidone, copolymers of maleic anhydride and alkyl vinyl ether, pluronyl polyols, albumin, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulphated polysaccharides , their mixtures and copolymers. 41. Pharmaceutical composition for the treatment of tumors, containing the antibody MAT-Fab according to any one of paragraphs. 23-29 and a pharmaceutically acceptable carrier. 42. The pharmaceutical composition according to claim 41, made for administration to an individual by at least one method selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar , intracerebroventricular, intraintestinal, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostate, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, rectal , sublingual, intranasal and transdermal administration. 43. An isolated polynucleotide encoding a MAT-Fab antibody according to claim 1. 44. An expression vector containing the isolated nucleic acid according to claim 43. 45. Expression vector according to claim 44, characterized in that said vector is selected from the group consisting of: pcDNA, pTT pTT3, pEFBOS, pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO and pBJ. 46. Selected host cell to obtain antibodies MAT-Fab according to any one of paragraphs. 1-37 containing the vector of claim 45. 47. Selected host cell to obtain antibodies MAT-Fab according to any one of paragraphs. 1-37 containing the vector of claim 44. 48. An isolated host cell according to claim 47, characterized in that said host cell is a prokaryotic host cell or a eukaryotic host cell. 49. A host cell according to claim 48, characterized in that said host cell is a prokaryotic host cell. 50. A host cell according to claim 49, characterized in that said prokaryotic host cell is a bacterial host cell. 51. A host cell according to claim 50, characterized in that the bacterial host cell is an Escherichia coli cell. 52. A host cell according to claim 48, characterized in that said host cell is a eukaryotic host cell. 53. The host cell according to claim 52, characterized in that the eukaryotic host cell is selected from the group consisting of: mammalian host cell, insect host cell, plant host cell, fungal host cell, eukaryotic algae host cell , a nematode host cell, a protozoan host cell, or a fish host cell. 54. A host cell according to claim 53, characterized in that said host cell is a mammalian host cell. 55. The host cell according to claim 54, characterized in that the mammalian host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells. 56. The host cell of claim 55, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell, a COS cell, or a human embryonic kidney (HEK293) cell. 57. The host cell according to claim 53, characterized in that the eukaryotic host cell is a fungal cell. 58. The host cell according to claim 57, characterized in that the fungal cell is selected from the group consisting of: Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida. 59. A host cell according to claim 58, characterized in that the Saccharomyces host cell is a Saccharomyces cerevisiae cell. 60. The host cell according to claim 53, characterized in that the eukaryotic host cell is an insect cell. 61. The host cell of claim 60, wherein said insect cell is an Sf9 insect cell. 62. Method for the production of antibodies MAT-Fab, including culturing the host cell described in any of paragraphs. 47-61 in culture medium under conditions sufficient to produce the binding protein. 63. A method for treating a tumor in a human subject, comprising the step of administering to the human subject a MAT-Fab antibody according to claim 1 that binds an antigen on an effector cell and binds an antigen associated with a disorder expressed on a target cell harmful to the human subject, moreover, the binding of the MAT-Fab antibody to both the effector cell and the target cell mediates the interaction of the effector cell with said harmful target cell and provides treatment for the disorder, where the effector cell is selected from the group consisting of: T-cell, natural killer (NK -cells), monocyte, neutrophil and macrophage. 64. The method according to p. 63, characterized in that the antigen expressed on the effector cell is selected from the group consisting of: CD3, CD16 and CD64. 65. The method according to p. 63, characterized in that the harmful target cell is selected from the group consisting of: a tumor cell, an auto-reactive cell, and a cell infected with a virus. 66. The method according to claim 63, characterized in that the antigen associated with the disorder expressed on the surface of the harmful target cell is a tumor-associated antigen expressed on the surface of the tumor cell. 67. The method according to p. 66, characterized in that the tumor-associated antigen expressed on the tumor cell is selected from the group consisting of: CD19, CD20, human epidermal growth factor receptor-2 (HER2), embryonic tumor antigen (CEA), molecules epithelial cell adhesion (EpCAM) and orphan receptor-1 protein like receptor tyrosine kinase (ROR 1). 68. The method of claim 66 wherein the tumor cell is a malignant B cell. 69. The method according to p. 68, characterized in that the malignant B-cell is a cell of a cancerous disorder selected from the group consisting of: acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia/lymphoma with Progenitor cell involvement, B-cell neoplasms involving mature cells, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, skin follicular central cell lymphoma, B marginal zone-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma. 70. The method of claim 66 wherein the antigen expressed on the effector cell is CD3 expressed on a T cell and the tumor associated antigen on the tumor cell is CD20 on a malignant B cell. 71. The method according to p. 70, characterized in that the MAT-Fab antibody contains four polypeptide chains containing the amino acid sequences shown in tables 1-4, or the amino acid sequences shown in tables 5-8.

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