Covalent multi-specific antibody

Abstract

To provide a novel bispecific antibody and tri-specific antibody having high stability.SOLUTION: An engineered antibody is provided, comprising: (i) a first polypeptide comprising a second light chain variable domain VL2 binding a second target and a first heavy chain variable domain VH1 binding a first target, wherein the VL2 is linked to the VH1 via a linker; (ii) a second polypeptide comprising a first light chain variable domain VL1 binding a first target and a second heavy chain variable domain VH2 binding a second target, as well as a cysteine-containing hinge domain and a CH2-CH3 domain of IgG, wherein the VL1 is linked to the VH2 via a linker; (iii) a third polypeptide comprising a cysteine-containing hinge domain and a CH2-CH3 domain of IgG; wherein VL1 and VH1 associate to bind the first target which is CD3, and VL2 and VH2 associate to bind the second target which is CD19.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel covalent multispecific antibodies with high stability and their therapeutic uses.

[0002] This application claims priority to a Chinese patent application filed on June 20, 2019, bearing application number 201910535703.7, and entitled "Covalently Linked Multispecific Antibody," the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Monoclonal antibodies (mAbs) have broad diagnostic and therapeutic potential for clinical application in cancer and other diseases. Whether in the form of naked antibodies or conjugates linked to cytotoxic agents (e.g., radioisotopes, drugs, toxins, or prodrug-converting enzymes), monoclonal antibodies play an important role in cancer immunotherapy. Both of these approaches are undergoing therapeutic evaluation, have undergone some development, and have achieved some clinical success. Naked mAbs can achieve clinical responses by inducing cytotoxic effects after binding to cell surface proteins overexpressed on cancer cells. Studies have shown that these therapeutic effects are achieved by controlling tumor growth through programmed cell death (apoptosis) or by inducing antitumor immune responses.

[0004] Due to the unique properties of antibodies, such as specific targeting and mediating effector functions, antibody drugs for targeted immunotherapy of diseases have been developed since the invention of monoclonal antibody technology by Cesar Milstein and Georges JF Kohler in 1975. Currently, more than 60 antibody-based biopharmaceuticals have been approved, with annual global sales exceeding $5 billion. The current successful application of antibody drugs has shaped the pharmaceutical industry and greatly improved public health. In addition to the research and development of antibody drugs for new targets, there is also great potential for the research and development of optimal combination therapies and innovative bispecific antibodies.

[0005] Therapeutic antibodies have been in clinical use for over 20 years. Anti-tumor antibody drugs currently in clinical use include Rituxan (1997), Herceptin (1998), Mylotarg (2000), Campath (2001), Zevalin (2002), Bexxer (2003), Avastin (2004), Erbitux (2004), Vectibix (2006), Arzerra (2009), Benlysta (2011), Yervoy (2011), Adcetris (2011), Perjeta (2012), Kadcyla (2013), Opdivo (2014), Keytruda (2014), and Tecentriq (2016). These antibodies primarily target EGFR, Her2, CD20 or VEGF, and the more recently discovered PD1 or PD-L1.

[0006] Multifunctional antibodies are constructed based on conventional antibodies through complex design and molecular engineering, possessing the ability to bind to multiple antigens. In practical applications, the therapeutic effect of a single multifunctional antibody molecule is equivalent to that of a combination of several conventional antibodies. However, the advantages of multifunctional antibodies go far beyond the simple superposition of several conventional antibodies. The simultaneous binding of multiple selected targets through a novel and unique mechanism can produce beneficial effects superior to those of conventional antibodies. For example, blinatumomab (CD3×CD19, Amgen), which targets CD3 and CD19, effectively binds T cells by recognizing the Fv on its CD3- to kill CD19-expressing tumor cells. It has demonstrated remarkable therapeutic efficacy superior to conventional antibodies in indications such as acute lymphoblastic leukemia (ALL). Blinatumomab was approved for sale by the US FDA for the treatment of ALL in 2014.

[0007] Bispecific antibodies can be generated by chemical crosslinking, hybridization of hybridomas or transfected tumors, or by disulfide exchange at the hinges of two different Fab's. The first method produces heterogeneous and difficult-to-define products. The second method requires extensive purification of the bispecific antibody obtained from the by-products of multiple hybrid antibodies, which may affect cell crosslinking activity. The disulfide exchange method is essentially applicable only to F(ab')2s, which makes monoclonal antibodies susceptible to enzymatic degradation. Furthermore, because Fab's have little affinity for each other, very high protein concentrations are required to form disulfide bonds between Fab's. The disulfide exchange method has been improved by using Ellman's reagent, which modifies one Fab' with the other before oxidation, thereby reducing the occurrence of homodimerization. However, even with these improvements, heterodimeric F(ab')2s are rarely produced in yields greater than 50%. However, current antibody drugs suffer from unfavorable safety issues, low response rates, and limited efficacy. These disadvantages may stem from non-target effects on normal tissues / cells due to the antibody epitope being derived from an autoantigen, the suppressive microenvironment of immune effector cells, and unexpected Fc-mediated effector functions. Therefore, there is still a need in this field for highly purified multispecific (e.g., trispecific) antibodies with good therapeutic efficacy. Summary of the Invention

[0008] In one aspect, the invention provides an engineered antibody comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain VL1 that binds to a first target, a second heavy chain variable domain VH2 that binds to a second target, and a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; (iii) a third polypeptide comprising, from N- to C-terminus, a hinge domain containing a cysteine ​​and the CH2-CH3 domains of an IgG. where: VL1 and VH1 combine to form a domain capable of binding to a first target, said first target being CD3.

[0009] VL2 and VH2 combine to form a domain capable of binding to a second target, said second target being CD19.

[0010] VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 each independently contain one or more substitutions that introduce charged amino acids, which are electrostatically unfavorable for homodimer formation.

[0011] The hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently linked by a disulfide bond.

[0012] In the above antibody, the amino acid sequence of VL1 is SEQ ID NO.: 1, the amino acid sequence of VH1 is SEQ ID NO.: 2, the amino acid sequence of VL2 is SEQ ID NO.: 3, and the amino acid sequence of VH2 is SEQ ID NO.: 4. The amino acid sequence of the first polypeptide is SEQ ID NO.: 5, the amino acid sequence of the second polypeptide is SEQ ID NO.: 6, and the amino acid sequence of the third polypeptide is SEQ ID NO.: 7.

[0013] In other aspects, the invention further provides engineered antibodies comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain VL1 that binds to a first target, a second heavy chain variable domain VH2 that binds to a second target, and a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain of an IgG that binds to a third target, as well as a hinge domain containing a cysteine ​​and a CH2-CH3 domain of an IgG, wherein VH3 and CH1 are connected by a linker; and (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 that binds to the third target, and a light chain fixing domain CL that comprises a cysteine, wherein VL3 and CL are connected by a linker.

[0014] Here, VL1 and VH1 combine to form a domain capable of binding to a first target.

[0015] Here, VL2 and VH2 combine to form a domain capable of binding to a second target.

[0016] Here, VL3 and VH3 combine to form a domain capable of binding to a third target.

[0017] Here, VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 each independently contain one or more substitutions that introduce charged amino acids, and the substitutions of the charged amino acids are electrostatically unfavorable for homodimer formation.

[0018] Here, CH1 and CL are covalently linked by a disulfide bond.

[0019] Here, the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently linked by a disulfide bond.

[0020] In one embodiment of the above antibody, the first target is CD3, the second target is CD19, and the third target is CD20. The amino acid sequence of VL1 is SEQ ID NO.:1, the amino acid sequence of VH1 is SEQ ID NO.:2, the amino acid sequence of VL2 is SEQ ID NO.:3, the amino acid sequence of VH2 is SEQ ID NO.:4, the amino acid sequence of VL3 is SEQ ID NO.:8, and the amino acid sequence of VH3 is SEQ ID NO.:9. The amino acid sequence of the first polypeptide is SEQ ID NO.:10, the amino acid sequence of the second polypeptide is SEQ ID NO.:11, the amino acid sequence of the third polypeptide is SEQ ID NO.:12, and the amino acid sequence of the fourth polypeptide is SEQ ID NO.:13.

[0021] In one embodiment of the above antibody, the first target is CD20, the second target is CD19, and the third target is CD3. The amino acid sequence of VL1 is SEQ ID NO.:8, the amino acid sequence of VH1 is SEQ ID NO.:9, the amino acid sequence of VL2 is SEQ ID NO.:3, the amino acid sequence of VH2 is SEQ ID NO.:4, the amino acid sequence of VL3 is SEQ ID NO.:1, and the amino acid sequence of VH3 is SEQ ID NO.:2. The amino acid sequence of the first polypeptide is SEQ ID NO.:14, the amino acid sequence of the second polypeptide is SEQ ID NO.:15, the amino acid sequence of the third polypeptide is SEQ ID NO.:16, and the amino acid sequence of the fourth polypeptide is SEQ ID NO.:17.

[0022] The novel features of the present invention are set forth with particularity in the appended claims. The features and advantages of the present invention will become more fully apparent from the following detailed description of the specific embodiments and drawings, which illustrate exemplary embodiments to which the principles of the present invention may be applied. The drawings of the present invention are as follows: [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a schematic diagram of the structure of a bispecific antibody according to one embodiment of the present invention. [Figure 2] FIG. 1 shows the killing effect of antibody A of the present invention on Raji cells. [Figure 3] FIG. 1 shows the tumor-suppressing effect of administering antibody A of the present invention. [Figure 4] FIG. 1 shows the effect of administration of antibody A of the present invention on the body weight of mice. [Figure 5] FIG. 1 is a schematic diagram of the structure of a trispecific antibody according to one embodiment of the present invention. [Figure 6] FIG. 1 shows the killing effect of antibody #1 and antibody #2 of the present invention on Raji cells. [Figure 7] FIG. 1 shows the killing effect of antibody #1 and antibody #2 of the present invention on K562 cells. [Figure 8] FIG. 1 shows the effects of antibody #1 and antibody #2 of the present invention on mouse body weight under different concentration conditions. [Figure 9] FIG. 1 shows the tumor-suppressing effects of antibody #1 and antibody #2 of the present invention under different concentration conditions. DETAILED DESCRIPTION OF THE INVENTION

[0024] Generally, the present invention provides bispecific and trispecific engineered antibodies with a disulfide bond located between the VH and VL, mutations occurring in amino acids selected based on their electrostatic properties, and knob-in-hole structures located in the Fc fragment.

[0025] In one aspect, the present invention provides a bispecific engineered antibody comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain VL1 that binds to a first target, a second heavy chain variable domain VH2 that binds to a second target, and a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; (iii) a third polypeptide comprising, from N- to C-terminus, a hinge domain containing a cysteine ​​and the CH2-CH3 domains of an IgG. where: VL1 and VH1 combine to form a domain capable of binding to a first target, said first target being CD3.

[0026] VL2 and VH2 combine to form a domain capable of binding to a second target, said second target being CD19.

[0027] VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 each independently contain one or more substitutions that introduce charged amino acids, which are electrostatically unfavorable for homodimer formation.

[0028] The hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently linked by a disulfide bond.

[0029] In some embodiments, the first polypeptide has the following structure from N- to C-terminus: VL2-linker-VH1.

[0030] In some embodiments, the second polypeptide has the following structure from N- to C-terminus: VL1-linker-VH2-hinge region-CH2-CH3.

[0031] In some embodiments, the third polypeptide has the following structure from N- to C-terminus: hinge region-CH2-CH3.

[0032] The linker of the first polypeptide, the linker of the second polypeptide, and the linker of the third polypeptide have the sequence RTVAA, GGGGS, GGSGGS, or GGSGGSGGS.

[0033] The hinge region in the second polypeptide and the hinge region in the third polypeptide comprise a hinge from IgG1, IgG2, IgG3, IgG4, or IgA.

[0034] In one exemplary embodiment of the invention, VL2 comprises amino acid substitutions of Cys for Gln100 and Lys for Gln38, and VH2 comprises amino acid substitutions of Asp for Gln39 and Cys for Gly44. One of the CH3 domains of the second polypeptide and the third polypeptide comprises a substitution of Trp for Thr366, and the other CH3 domain comprises a substitution of Ser, Ala, and Val for Thr366, Leu368, and Tyr407, respectively.

[0035] The bispecific antibodies of the present invention can simultaneously recognize two antigens and have mutations in amino acids selected based on the disulfide bond located between the VH and VL and their electrostatic properties, and in some embodiments, the CH2CH3 domain of the second polypeptide chain and the CH2CH3 domain of the third polypeptide chain form a knob-in-hole structure.

[0036] In another aspect, the present invention provides a trispecific engineered antibody comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, from N-terminal to C-terminal, a first light chain variable domain VL1 that binds to a first target, a second heavy chain variable domain VH2 that binds to a second target, and a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and a CH1 domain of an IgG that binds to a third target, as well as a hinge domain containing a cysteine ​​and a CH2-CH3 domain of an IgG, wherein VH3 and CH1 are connected by a linker; and (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 that binds to said third target, and a light chain locking domain CL that comprises a cysteine, wherein VL3 and CL are connected by a linker.

[0037] Here, VL1 and VH1 combine to form a domain capable of binding to a first target.

[0038] Here, VL2 and VH2 combine to form a domain capable of binding to a second target.

[0039] Here, VL3 and VH3 combine to form a domain capable of binding to a third target.

[0040] wherein VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 independently contain one or more substitutions that introduce charged amino acids, and the substitutions of the charged amino acids are electrostatically unfavorable for homodimer formation.

[0041] Here, CH1 and CL are covalently linked by a disulfide bond.

[0042] Here, the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently linked by a disulfide bond.

[0043] In some embodiments, the first polypeptide has the following structure from N- to C-terminus: VL2-linker-VH1.

[0044] In some embodiments, the second polypeptide has the following structure from N- to C-terminus: VL1-linker-VH2-hinge region-CH2-CH3.

[0045] In some embodiments, the third polypeptide has the following structure from N- to C-terminus: VH3-linker-CH1-hinge region-CH2-CH3.

[0046] In some embodiments, the fourth polypeptide has the structure, from N-terminus to C-terminus, VL3-linker-CL.

[0047] The linker of the first polypeptide, the linker of the second polypeptide, the linker of the third polypeptide, and the linker of the fourth polypeptide have the sequence RTVAA, or GGGGS, or GGSGGS, or GGSGGSGGS.

[0048] The hinge region in the second polypeptide and the hinge region in the third polypeptide comprise a hinge from IgG1, IgG2, IgG3, IgG4, or IgA.

[0049] In one exemplary embodiment of the invention, VL2 comprises amino acid substitutions of Cys for Gln100 and Lys for Gln38, and VH2 comprises amino acid substitutions of Asp for Gln39 and Cys for Gly44. One of the CH3 domains of the second polypeptide and the third polypeptide comprises a substitution of Trp for Thr366, and the other CH3 domain comprises a substitution of Ser, Ala, and Val for Thr366, Leu368, and Tyr407, respectively.

[0050] The trispecific antibodies of the present invention can simultaneously recognize three antigens, have disulfide bonds located between VH and VL, and mutations in amino acids selected based on their electrostatic properties, and in some embodiments, the CH3 domain of the second polypeptide chain and the CH3 domain of the third polypeptide chain form a knob-in-hole structure.

[0051] Disulfide bonds

[0052] In some embodiments of the present invention, VL2 and VH2 are covalently linked by a disulfide bond. In some embodiments, the FRs of VL2 and VH2 are covalently linked by a disulfide bond. Cysteine ​​mutations are introduced into the VL-VH interface to form disulfide bonds between VL and VH, thereby covalently linking VL and VH, thereby improving antibody stability.

[0053] Position 100 of VL2 and position 44 of VH2 were substituted with cysteines, which form disulfide bonds linking VL2 and VH2 of the diabodies and triabodies.

[0054] Charged amino acid substitutions

[0055] In addition to the introduction of a disulfide bond between VL2 and VH2, the VL2 and VH2 of the diabodies and triabodies of the invention have one or more amino acid substitutions of different charge properties.

[0056] Early studies have shown that the introduction of disulfide bonds significantly improves antibody stability, but a certain proportion of the light and heavy chains are bound non-covalently, affecting the purity of the product. In the trispecific antibody of the present invention, taking into account the influence of electrostatic forces between the regions, one or more amino acid substitutions with different charge properties are introduced into VH2 and VL2, respectively, to minimize undesired non-covalent binding and further improve the stability and purity of the product.

[0057] In some embodiments of the present invention, VL2 is substituted with a negatively charged amino acid, and VH2 is substituted with a positively charged amino acid. In some embodiments of the present invention, VL2 is substituted with a positively charged amino acid, and VH2 is substituted with a negatively charged amino acid. The negatively charged amino acid is aspartic acid or glutamic acid, and the positively charged amino acid is lysine or arginine.

[0058] In an exemplary embodiment of the invention, Gln38 on VL2 is substituted with Lys and Gln39 on VH2 is substituted with Asp, thereby forming a charged amino acid substitution.

[0059] Knobs-in-hole structure

[0060] In the bispecific and trispecific antibodies of the invention, the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently linked by a disulfide bond. In some embodiments, the CH2CH3 domain of the second polypeptide chain and the CH2CH3 domain of the third polypeptide chain form a knob-in-hole structure.

[0061] Knobs-in-holes architecture is also referred to as a "protuberance-into-cavity" strategy used to engineer the interface between the second and third polypeptides of a heterologous oligomer.

[0062] Generally, the preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. A "protrusion" is constructed by substituting a small amino acid side chain in the interface of the second polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary "cavity" of the same or similar size as the protrusion is optionally constructed in the interface of the third polypeptide by substituting a larger amino acid side chain with a smaller amino acid (e.g., alanine or threonine). When a protrusion or cavity of appropriate positioning and dimensions exists in the interface of the second or third polypeptide, it is sufficient to simply design a corresponding cavity or protrusion, respectively, in the adjacent interface. See U.S. Patent No. 8,216,805, the disclosure of which is incorporated herein by reference.

[0063] In one exemplary embodiment, the CH3 domain of the second polypeptide comprises a substitution of Trp for Thr366, and the CH3 domain of the third polypeptide comprises a substitution of Ser, Ala, and Val for Thr366, Leu368, and Tyr407, respectively, thereby forming a knob-in-hole structure.

[0064] In another exemplary embodiment, the CH3 domain of the second polypeptide comprises Ser, Ala, and Val substituting for Thr366, Leu368, and Tyr407, respectively, and the CH3 domain of the third polypeptide comprises Trp substituting for Thr366, thereby forming a knob-in-hole structure.

[0065] Antibody preparation

[0066] All antibody formats are based on the heavy and light chains of IgG antibodies and can be prepared using methods known in the art, which generally involve constructing expression cassettes for heavy and light chain genes, co-transfecting the two genes into a suitable cell system to produce recombinant antibody, preparing stable, high-yielding cell clones, and fermenting the cells to produce the final antibody product in cGMP. Example

[0067] The present invention is further illustrated, but not limited, by the following examples. The following examples section illustrates the generation of engineered antibodies of the present invention.

[0068] Example 1. Construction of bispecific antibodies

[0069] This example illustrates the construction of a CD3×CD19 bispecific antibody. The amino acid sequences of VH and VL of CD3 and CD19 used in the construction of this bispecific antibody are shown in SEQ ID NO. 1 to SEQ ID NO. 4 (CD3 VL: SEQ ID NO. 1, CD3 VH: SEQ ID NO. 2, CD19 VL: SEQ ID NO. 3, CD19 VH: SEQ ID NO. 4).

[0070] Construction method: The codon sequence was optimized using OptimumGene. The target gene was first constructed in the pUC57 vector and then subcloned into the pTGE5 vector. DNA was prepared for transfection using Maxiprep. CHO3E7 cells were cultured and transfected at 0.3 × 10 6 Subculture at a cell density of 1.8-2.5 x 10 cells / ml. 6Transfection was performed when the cell density reached 100 cells / ml. First, 300 μl of DNA heavy and light chains were added to 50 ml of Freestyle CHO medium and gently vortexed. Next, 3 mg of PEI transfection reagent was added and gently vortexed for 3 minutes. The mixture was incubated at 37°C for 7 minutes and then added to 450 ml of cell suspension for a total volume of 500 ml. After 24 hours, 25 ml of TN1 (200 g / L stock solution) was added to the mixture. 1 ml of suspension was removed for detection on days 1, 3, and 5 posttransfection. A 50 μl sample was taken for cell counting, and the remaining sample was centrifuged at 3000 rpm for 5 minutes. The supernatant was then stored at -20°C. On day 6, the culture was harvested and centrifuged at 5500 rpm for 30 minutes. The supernatant was separated and filtered through a 0.22 μm filter for further protein purification. Chromatography column: 5 ml Monofinity A resin (GenScript, lot number L00433) chromatography column; equilibration buffer A: 20 mM PB, 150 mM NaCl, pH 7.2; wash buffer B: 50 mM citric acid, pH 3.5; neutralization buffer C: 1 M Tris-HCl, pH 9.0; flow rate: 2 ml / min; gradient: 100% gradient elution. After separation, 0.155 ml of neutralization buffer C was added to each 1 ml of component. The collected protein solution was continuously dialyzed against PBS (pH 7.2) at 4°C for 16 hours.

[0071] By the above method, a CD3×CD19 bispecific antibody (Antibody A, the structural diagram of which is shown in FIG. 1) containing the following three polypeptide chains is constructed and obtained. First polypeptide (SEQ ID NO. 5): CD19 VL-linker-CD3 VH Second polypeptide (SEQ ID NO. 6): CD3 VL-linker-CD19 VH-hinge region-CH2CH3 Third polypeptide C (SEQ ID NO. 7): hinge region - CH2CH3

[0072] The resulting antibody A was purified by size exclusion (SEC) and had a purity of 95% or more.

[0073] Example 2. Affinity Test

[0074] The binding affinity of antibody A to human CD3ε antigen and human CD19 antigen was tested using the BIAcore method, and the k, kd, and KD values ​​were calculated. In this example, affinity was evaluated using a capture method. Human CD19 was used as a ligand and captured on a chip to which a histamine antibody was bound. Five different concentrations of candidate drugs were then sampled as analytes and the affinity was analyzed. CD3ε antigen and CD19 antigen were immobilized on the chip, and the affinity test for antibody A was performed using the BIAcore method, with the following results obtained:

[0075] [Table 1]

[0076] Example 3. Cell killing assay on Raji cells

[0077] Lymphocytes were used as effector cells to analyze the antibody-mediated killing of target cells (Raji cells). The procedure was as follows.

[0078] Preparation of effector cells: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from blood by density gradient centrifugation. CD4+ T cells and CD8+ T cells were further isolated from PBMCs using a Stemcell isolation kit. PBMCs, CD4+ T cells, and CD8+ T cells were resuspended in cell culture medium, respectively, to detect cell density and cell viability. Cell culture medium was used to adjust the cell density to 6 × 10 6 The cell suspension was adjusted to 10 viable cells / mL and then added at 50 µL per well to a flat-bottom 96-well plate. The effector cell to target cell ratio (E / T) was 20:1 for the experiment. Cell culture medium: RPMI 1640 with 10% HI-FBS and 1% penicillin-streptomycin.

[0079] Target cell preparation: Raji cells were cultured at a density of 2 × 105 The cell density is 3 × 10 cells / mL, and they are used for experiments after 4 days of subculture. An appropriate amount of cell suspension is transferred to a 50 ml centrifuge tube and centrifuged at 200 g for 5 minutes at room temperature. The cells are resuspended in cell culture medium to determine cell density and viability. The cell density is adjusted to 3 × 10 cells / mL with cell culture medium. 5 The concentration is adjusted to 10 viable cells / mL, and then 50 μL / well of the cell suspension is added to a flat-bottom 96-well plate already containing Raji cells.

[0080] Antibody preparation: Dilute antibody A and controls (blinatumomab, MGD011, and RG6026) to different concentrations in cell culture medium. Add 50 μL of cell culture medium or diluted solution to the indicated wells to obtain final concentrations of 0 pM, 1 pM, or 100 pM.

[0081] The flat-bottom 96-well plate containing the antibodies, target cells, and effector cells was placed in a 37°C, 5% CO2 incubator and sampled and detected at 4, 20, and 40 hours. The samples were centrifuged at 350g for 5 minutes, and the cells were resuspended and stained with PI. 10 μL of counting beads was added to each well, followed by analysis by flow cytometry. The analysis results showed that the EC50 value of Antibody A was 1.086 pM, that of blinatumomab was 5.476 pM, that of MGD011 was 1.721 pM, and that of RG6026 was 0.6701 pM. The killing activity of Antibody A on target cells is shown in Figure 2.

[0082] Example 3. In vivo drug efficacy evaluation

[0083] The in vivo antitumor efficacy of Antibody A is tested in the Jeko-1 / NCG Mixeno model. Initially (day 0), 5 x 10 cells suspended in 100 μL of 1:1 PBS / gel were 6 Jeko-1 cells were inoculated subcutaneously into the right dorsal region of the animals. Three days after inoculation (day 3), 1 × 10 cells were injected intraperitoneally into the animals. 7 0.1 ml of PBMCs was injected per tumor.3 When the tumor size reaches 100 μg / mL, antibody samples are administered. Four antibodies (bornatuzumab @ 0.5 mg / kg, antibody A @ 0.3 mg / kg, MGD011 @ 0.3 mg / kg, RG6026 @ 0.7 mg / kg) and one control group (pH 6.0 PBS) are tested in six animals per group. All samples are administered intravenously via the tail vein. All antibodies and vehicle are administered twice weekly for three consecutive weeks. Treatment efficacy is evaluated based on relative tumor inhibition (TGIRTV), and safety is assessed based on animal weight change and death.

[0084] Relative tumor growth inhibition rate TGIRTV (%): TGIRTV = 1-TRTV / CRTV (%). TRTV / CRTV (%) is the relative tumor growth rate, i.e., the ratio of the tumor volume of the treated group to the tumor volume of the control group that received PBS at a certain time point. TRTV and CRTV are the tumor volumes (TV) of the treated group and the control group, respectively, at a certain time point.

[0085] The experiment was terminated 40 days after inoculation. As shown in Figure 3, tumor growth was significantly suppressed in all treatment (antibody) groups. Furthermore, as shown in Figure 4, no significant weight loss was observed in any of the treatment (antibody) groups, indicating that the therapeutic antibodies do not have significant toxic effects in the body.

[0086] Example 4. Construction of trispecific antibodies

[0087] This example illustrates the construction of a CD3xCD19xCD20 trispecific antibody. In the process of constructing the trispecific antibody in this example, the VH and VL sequences of CD3, CD19, and CD20 are exemplified in SEQ ID NOs. 1 to 4, 8, and 9 (CD3 VL: SEQ ID NO. 1, CD3 VH: SEQ ID NO. 2, CD19 VL: SEQ ID NO. 3, CD19 VH: SEQ ID NO. 4, CD20 VL: SEQ ID NO. 8, and CD20 VH: SEQ ID NO. 9).

[0088] According to the construction method described in Example 1, a CD3xCD19xCD20 trispecific antibody (Antibody #1) is constructed, which comprises the following four polypeptide chains: Polypeptide A (SEQ ID NO. 10): CD19 VL-linker-CD3 VH Polypeptide B (SEQ ID NO. 11): CD3 VL-linker-CD19 VH-hinge region-CH2CH3 Polypeptide C (SEQ ID NO. 12): CD20 VH-CH1-hinge region-CH2CH3 Polypeptide D (SEQ ID NO. 13): CD20 VL-CL

[0089] Using the same method as above, only the positions of CD3, CD19, and CD20 are exchanged to construct and obtain a CD3xCD19xCD20 trispecific antibody (antibody #2) containing the following four polypeptide chains: Polypeptide E (SEQ ID NO. 14): CD19 VL-linker-CD20 VH Polypeptide F (SEQ ID NO. 15): CD20 VL-linker-CD19 VH-hinge region-CH2CH3 Polypeptide G (SEQ ID NO. 16): CD3 VH-CH1-hinge region-CH2CH3 Polypeptide H (SEQ ID NO. 17): CD3 VL-CL

[0090] The obtained antibodies #1 and #2 were purified by size exclusion (SEC), and both had a purity of 90% or higher.

[0091] Example 5. Affinity Test

[0092] The binding affinity of antibody #1 to human CD3ε antigen and human CD19 antigen was tested using the BIAcore method, and the k, kd, and KD values ​​were calculated. In this example, affinity was evaluated using a capture method. Human CD19 was used as a ligand and captured on a chip to which a histamine antibody was bound. Five different concentrations of candidate drugs were then sampled as analytes and the affinity was analyzed. CD3ε antigen and CD19 antigen were immobilized on the chip, and the affinity test for antibody #1 was performed using the BIAcore method. The results are as follows:

[0093] [Table 2]

[0094] Example 6. Cell killing assay on Raji cells

[0095] Lymphocytes were used as effector cells to analyze the antibody-mediated killing of target cells (Raji cells). The procedure was as follows.

[0096] Preparation of effector cells: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from blood by density gradient centrifugation. CD4+ T cells and CD8+ T cells were further isolated from PBMCs using a Stemcell isolation kit. PBMCs, CD4+ T cells, and CD8+ T cells were resuspended in cell culture medium, respectively, to detect cell density and cell viability. Cell culture medium was used to adjust the cell density to 6 × 10 6 The cell suspension was adjusted to 10 viable cells / mL and then added at 50 µL per well to a flat-bottom 96-well plate. The effector cell to target cell ratio (E / T) was 20:1 for the experiment. Cell culture medium: RPMI 1640 with 10% HI-FBS and 1% penicillin-streptomycin.

[0097] Target cell preparation: Raji cells were cultured at a density of 2 × 10 5The cell density is 3 × 10 cells / mL, and they are used for experiments after 4 days of subculture. An appropriate amount of cell suspension is transferred to a 50 ml centrifuge tube and centrifuged at 200 g for 5 minutes at room temperature. The cells are resuspended in cell culture medium to determine cell density and viability. The cell density is adjusted to 3 × 10 cells / mL with cell culture medium. 5 The concentration is adjusted to 10 viable cells / mL, and then 50 μL / well of the cell suspension is added to a flat-bottom 96-well plate already containing Raji cells.

[0098] Antibody preparation: Dilute antibody #1, antibody #2, and the control bispecific antibody CD3xCD19 to different concentrations in cell culture medium. Add 50 μL of cell culture medium or diluted solution to the indicated wells to obtain final concentrations of 0 pM, 1 pM, or 100 pM.

[0099] The flat-bottom 96-well plate containing the antibodies, target cells, and effector cells was placed in a 37°C, 5% CO2 incubator and sampled and detected at 4, 20, and 40 hours. The samples were centrifuged at 350g for 5 minutes, and the cells were resuspended and stained with PI. 10 μL of counting beads were added to each well, followed by analysis by flow cytometry. The analysis results showed that the EC50 value of antibody #2 was 971.8 pM and that of antibody #1 was 1.423 pM. The killing effects of antibody #1 and antibody #2 on target cells are shown in Figure 6.

[0100] Example 7. Cell killing assay on K562 cells

[0101] To observe the CD19-independent cell killing effect of CD3×CD19×CD20, CD19-CD20 double-negative cells K562 were stably transfected with CD20 as target cells, and lymphocytes were used as effector cells to analyze the antibody-mediated killing effect on target cells (K562 cells).

[0102] After centrifugation, K562-CD20 cells were resuspended in 1640 + 2% FBS medium, centrifuged at 1000 rpm for 5 minutes, counted, and seeded at 10,000 cells / 100 μl per well of a 96-well cell culture plate. PBMCs were collected, centrifuged at 1000 rpm for 5 minutes, resuspended in 1640 + 2% FBS, counted, and then added to each well of the cell culture plate at 100,000 cells / 100 μl. Antibodies were diluted in PBS, and 10-fold gradient dilutions were made in eight points. 10 μl of each dilution was added to each well of the cell culture plate, with each concentration diluted in duplicate. Incubate at 37°C in a 5% CO2 incubator for 4 hours. Remove the substrate from the detection kit and resuspend each bottle in 12 ml of buffer. The culture plate was centrifuged at 1500 rpm for 5 minutes, and 50 μL of the supernatant was transferred to a new culture plate. 50 μL of the newly prepared substrate was added to each well, and the mixture was incubated at room temperature for 10 minutes in the dark. OD490 was measured using an enzyme labeling assay. The analysis showed that the EC50 value of antibody #1 was 1.222 pM. The killing effects of antibody #1 and the control antibody on target cells are shown in Figure 7.

[0103] Example 8. In vivo drug efficacy evaluation

[0104] The in vivo antitumor efficacy of Antibody #1 and Antibody #2 is tested in the Jeko-1 / NCG Mixeno model. Initially (day 0), 5 x 10 cells suspended in 100 μL of 1:1 PBS / gel were 6 Jeko-1 cells were inoculated subcutaneously into the right dorsal region of the animals. Three days after inoculation (day 3), 1 × 10 cells were injected intraperitoneally into the animals. 7 0.1 ml of PBMCs was injected per tumor. 3Antibody #1 and Antibody #2 are administered when tumor growth reaches 100%. Four antibodies (CD3xCD19 @ 0.5 mg / kg, Antibody #1 @ 0.5 mg / kg, Antibody #1 @ 3 mg / kg, Antibody #2 @ 0.5 mg / kg) and one control group (pH 6.0 PBS) are tested in six animals per group. All samples are administered intravenously via the tail vein. All antibodies and vehicle are administered twice weekly for three consecutive weeks. Treatment efficacy is evaluated based on relative tumor inhibition (TGIRTV), and safety is assessed based on animal weight change and death.

[0105] Relative tumor growth inhibition rate TGIRTV (%): TGIRTV = 1-TRTV / CRTV (%). TRTV / CRTV (%) is the relative tumor growth rate, i.e., the ratio of the tumor volume of the treated group to the tumor volume of the control group that received PBS at a certain time point. TRTV and CRTV are the tumor volumes (TV) of the treated group and the control group, respectively, at a certain time point.

[0106] The experiment was terminated 28 days after inoculation. As shown in Figure 9, tumor growth was significantly suppressed in all treatment (antibody) groups. Furthermore, as shown in Figure 8, no significant weight loss was observed in any of the treatment (antibody) groups, indicating that the therapeutic antibodies did not have significant toxic effects in vivo.

[0107] While exemplary embodiments of the present invention have been described and shown herein, it will be apparent to those skilled in the art that these embodiments are merely exemplary. Those skilled in the art will be able to make various changes, modifications, and substitutions to these embodiments without departing from the invention. The scope of the present invention is defined by the appended claims, and methods and structures within the scope of these claims and their equivalents are also encompassed therein.

Claims

1. 1. An engineered antibody, comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 that binds to a first target and a second heavy chain variable domain VH2 that binds to a second target, as well as a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; and (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a hinge domain including a cysteine ​​and the CH2-CH3 domains of IgG; where: VL1 and VH1 combine to form a domain capable of binding to a first target, said first target being CD3; VL2 and VH2 combine to form a domain capable of binding to a second target, wherein the second target is CD19; VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 each independently contain one or more substitutions introducing charged amino acids, wherein the substitutions of the charged amino acids electrostatically disfavor homodimer formation; The engineered antibody, wherein the hinge domain of said second polypeptide chain and the hinge domain of said third polypeptide chain are covalently linked by a disulfide bond.

2. 2. The antibody of claim 1, wherein the amino acid sequence of VL1 is SEQ ID NO.: 1, the amino acid sequence of VH1 is SEQ ID NO.: 2, the amino acid sequence of VL2 is SEQ ID NO.: 3, and the amino acid sequence of VH2 is SEQ ID NO.:

4.

3. 2. The antibody of claim 1, wherein the amino acid sequence of the first polypeptide is SEQ ID NO.: 5, the amino acid sequence of the second polypeptide is SEQ ID NO.: 6, and the amino acid sequence of the third polypeptide is SEQ ID NO.:

7.

4. 1. An engineered antibody, comprising: (i) a first polypeptide comprising, in an N-terminal to C-terminal direction, a second light chain variable domain VL2 that binds a second target and a first heavy chain variable domain VH1 that binds a first target, wherein VL2 and VH1 are connected by a linker; (ii) a second polypeptide comprising, in an N-terminal to C-terminal direction, a first light chain variable domain VL1 that binds a first target, a second heavy chain variable domain VH2 that binds a second target, and a hinge domain containing a cysteine ​​and the CH2-CH3 domains of IgG, wherein VL1 and VH2 are connected by a linker; (iii) a third polypeptide comprising, in an N-terminal to C-terminal direction, a third heavy chain variable domain VH3 and an IgG CH1 domain that binds to a third target, as well as a hinge domain containing a cysteine ​​and an IgG CH2-CH3 domain, wherein VH3 and CH1 are connected by a linker; and (iv) a fourth polypeptide comprising, in an N-terminal to C-terminal direction, a third light chain variable domain VL3 that binds to the third target, and a light chain locking domain CL that comprises a cysteine, wherein VL3 and CL are linked by a linker; wherein VL1 and VH1 combine to form a domain capable of binding to a first target; wherein VL2 and VH2 combine to form a domain capable of binding to a second target; wherein VL3 and VH3 combine to form a domain capable of binding to a third target; wherein VL2 and VH2 are covalently linked by a disulfide bond, and VL2 and VH2 each independently contain one or more substitutions introducing charged amino acids, and the substitutions of the charged amino acids are electrostatically unfavorable for homodimer formation; wherein CH1 and CL are covalently linked by a disulfide bond; wherein the hinge domain of said second polypeptide chain and the hinge domain of said third polypeptide chain are covalently linked by a disulfide bond.

5. The antibody of claim 4, wherein the first target is CD3, the second target is CD19, and the third target is CD20.

6. The antibody of claim 4, wherein the first target is CD20, the second target is CD19, and the third target is CD3.

7. The antibody of claim 5, wherein the amino acid sequence of VL1 is SEQ ID NO.: 1, the amino acid sequence of VH1 is SEQ ID NO.: 2, the amino acid sequence of VL2 is SEQ ID NO.: 3, the amino acid sequence of VH2 is SEQ ID NO.: 4, the amino acid sequence of VL3 is SEQ ID NO.: 8, and the amino acid sequence of VH3 is SEQ ID NO.:

9.

8. The antibody of claim 6, wherein the amino acid sequence of VL1 is SEQ ID NO.: 8, the amino acid sequence of VH1 is SEQ ID NO.: 9, the amino acid sequence of VL2 is SEQ ID NO.: 3, the amino acid sequence of VH2 is SEQ ID NO.: 4, the amino acid sequence of VL3 is SEQ ID NO.: 1, and the amino acid sequence of VH3 is SEQ ID NO.:

2.

9. 6. The antibody of claim 5, wherein the amino acid sequence of the first polypeptide is SEQ ID NO.: 10, the amino acid sequence of the second polypeptide is SEQ ID NO.: 11, the amino acid sequence of the third polypeptide is SEQ ID NO.: 12, and the amino acid sequence of the fourth polypeptide is SEQ ID NO.:

13.

10. 7. The antibody of claim 6, wherein the amino acid sequence of the first polypeptide is SEQ ID NO.: 14, the amino acid sequence of the second polypeptide is SEQ ID NO.: 15, the amino acid sequence of the third polypeptide is SEQ ID NO.: 16, and the amino acid sequence of the fourth polypeptide is SEQ ID NO.: 17.

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