Dual-specific molecular stabilization composition

Abstract

The present invention provides a pharmaceutical composition comprising a bispecific antigen-binding molecule at a high concentration, the pharmaceutical composition comprising at least one buffering agent, at least one sugar, and at least one stabilizing agent selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and citric acid to stabilize the bispecific antigen-binding agent even at higher concentrations.

Description

[Technical Field]

[0001] The present invention relates to biotechnology aspects, particularly to the provision of stabilized compositions for bispecific molecules. [Background technology]

[0002] Advances in commercial-scale purification processes have enabled protein-based pharmaceuticals, such as recombinant proteins, to be obtained with high purity upon initial production. However, proteins are only marginally stable and are highly susceptible to chemical and physical degradation, even during upstream manufacturing. Chemical degradation refers to covalent modifications such as deamidation, oxidation, cleavage or formation of new disulfide bridges, hydrolysis, isomerization, or deglycosylation. Physical degradation includes protein unfolding, unwanted adsorption to surfaces, and aggregation. Addressing these physical and chemical instabilities is one of the most difficult challenges in the development of protein pharmaceuticals (Chi et al., Pharm Res, Vol. 20, No. 9, September 2003, pp. 1325-1336; Roberts, Trends Biotechnol. 2014 July; 32(7):372-80).

[0003] Therefore, despite advances in manufacturing, new protein-based pharmaceuticals require proper formulation to avoid effects on product quality such as protein aggregation, which has implications for manufacturing, storage, and administration.

[0004] Such novel protein-based pharmaceuticals include, for example, bispecific (monoclonal) antibodies. Bispecific antibodies are artificial proteins that can simultaneously bind to two different types of antigens. They are known in several structural forms and are currently being explored for applications in cancer immunotherapy and drug delivery (Fan, Gaowei; Wang, Zujian; Hao, Mingju; Li, Jinming (2015). "Bispecific antibodies and their applications." Journal of Hematology & Oncology. 8:130).

[0005] In general, bispecific molecules useful in immuno-oncology can be antigen-binding polypeptides such as antibodies, for example, IgG-like bispecific antibodies (i.e., full-length bispecific antibodies) or non-IgG-like bispecific antibodies that are not full-length antigen-binding molecules. Full-length bispecific antibodies typically retain the traditional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab regions bind to different antigens. Non-full-length bispecific antibodies completely lack the Fc region. These include chemically linked Fabs consisting of only the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFvs). Fusion proteins that mimic the variable domains of two antibodies also exist. An example of such a format is the BiTE® (bispecific T cell engager) molecule (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). "Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies". International Journal of Molecular Sciences. 18(1):48).

[0006] Exemplary bispecific molecules, such as BiTE® antigen-binding molecules, are recombinant protein constructs composed of two flexibly linked antibody-derived binding domains. One binding domain of a BiTE® antigen-binding molecule is specific for a selected tumor-associated surface antigen on a target cell, and the second binding domain is specific for CD3, a subunit of the T cell receptor complex on a T cell. These special designs make BiTE® antigen-binding molecules uniquely suited to transiently bind T cells to target cells while simultaneously potently activating the cytotoxic potential of T cells against their natural target cells. An important further development of the first-generation BiTE® antigen-binding molecules deployed in the clinic as AMG 103 and AMG 110 (see WO 99 / 54440 and WO 2005 / 040220) provided bispecific antigen-binding molecules that bind to a context-independent epitope at the N-terminus of the CD3ε chain (WO 2008 / 119567). BiTE® antigen-binding molecules that bind to this selected epitope not only do not exhibit cross-species specificity for human and marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), or squirrel monkey (Saimiri sciureus) CD3ε chains, but also do not nonspecifically activate T cells to the same extent as observed with previous-generation T-cell-engaging antibodies, because they recognize this specific epitope instead of the epitope of previously described CD3-binding molecules in bispecific T-cell-engaging molecules. This reduction in T cell activation was associated with less or reduced T cell redistribution in patients, which was judged to be at risk for side effects.

[0007] Currently, approximately 1-5 mg / mL (i.e., 0.1-0.5% (w / v)) is a typical concentration of bispecific antigen-binding molecules, such as BiTE® (bispecific T cell engager) molecules conjugated to a single-chain Fc (scFc BiTE® molecule), in liquid (reconstituted) pharmaceutical compositions to avoid aggregation, such as HMW (high molecular weight) species formation. Such liquid compositions are typically lyophilized for storage and reconstituted for patient administration, typically by injection. However, for example, for subcutaneous administration, higher concentrations of bispecific antigen-binding molecules would be desirable to meet the required dosage within a limited volume. Furthermore, higher bispecific antigen-binding molecule concentrations mean, for example, reduced buffer volumes, fewer resources, and less energy consumption for development and manufacturing. Therefore, to obtain stable, highly concentrated formulations of bispecific antigen-binding molecules, it is necessary to provide a means to reduce the risk of aggregation. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 99 / 54440 Brochure [Patent Document 2] International Publication No. 2005 / 040220 Brochure [Patent Document 3] International Publication No. 2008 / 119567 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Chi et al.,Pharm Res,Vol.20,No.9,Sept 2003,pp.1325-1336,Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Non-patent document 2] Fan,Gaowei;Wang,Zujian;Hao,Mingju;Li,Jinming(2015).“Bispecific antibodies and their applications”.Journal of Hematology & Oncology.8:130 [Non-patent document 3] Yang, Fa; Wen, Weihong; Qin, Weijun (2016). “Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies”. International Journal of Molecular Sciences. 18(1):48 Summary of the Invention [Means for solving the problem]

[0010] Surprisingly, a liquid pharmaceutical composition comprising a stabilizer selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and citric acid, wherein the stabilizer is preferably present at a concentration in the range of 0.005% to 0.25% (w / v), preferably 0.01 to 0.2% (w / v), ensures improved quality of the bispecific antigen-binding molecule product at higher concentrations of about 8 to 35 mg / ml, and thus (i) allows for higher dosages within a limited volume (resulting in drug applications requiring stable drug at high concentrations, such as subcutaneous administration to patients in need thereof), and (ii) facilitates resource-efficient production and storage of frozen and reconstituted liquid pharmaceutical compositions comprising bispecific antigen-binding molecules, such as scFc BiTE® molecules.

[0011] Therefore, in a first aspect, in the context of the present invention, (a) a bispecific antigen-binding molecule comprising at least three domains, wherein: the first domain binds to a target cell surface antigen, the target cell surface antigen being a tumor antigen; The second domain binds to an extracellular epitope of the human and Macaca CD3 chain; and the third domain comprises two polypeptide monomers each comprising a hinge, a CH2 domain, and a CH3 domain, the two polypeptide monomers being fused to each other via a peptide linker, the third domain comprising, in amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3; The concentration of the bispecific antigen-binding molecule is 8 to 35 mg / ml; (b) at least one buffer; (c) at least one sugar; and (d) at least one stabilizer selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and citric acid, wherein the stabilizer is present at a concentration ranging from 0.005% to 0.25% (w / v), preferably from 0.01 to 0.2% (w / v); The pH of the pharmaceutical composition is in the range of 4.0 to 6.0. It is envisioned that a liquid pharmaceutical composition comprising:

[0012] According to the above aspect, it is also envisaged that the bispecific antigen-binding molecule is a single-chain molecule.

[0013] According to the above aspect, it is also envisioned that the multispecific antigen-binding molecule has an extended half-life.

[0014] According to said aspect, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the bispecific antigen-binding molecule is eliminated by an N314X substitution, wherein X is any amino acid except Q.

[0015] According to the above aspect, it is also envisaged that each of the polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-24, or has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24.

[0016] According to said embodiment, it is also envisaged that the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

[0017] According to the above aspect, it is also envisioned that the tumor antigen is selected from the group consisting of CDH19, CDH3, MSLN, DLL3, FLT3, EGFRvIII, BCMA, PSMA, CD33, CD19, CD20, CLDN18.2, MUC17, EpCAM, CD70, and CLDN6.

[0018] According to said embodiment, it is also envisaged that the second domain is an extracellular epitope of the human and / or macaque CD3 epsilon chain.

[0019] According to the above aspect, (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain; It is also expected that...

[0020] According to the above aspects, the antibody construct comprises, in order from amino to carboxyl: (a) First domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 189; (c) second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) the first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) a second polypeptide monomer of the third domain It is also assumed that the

[0021] According to said embodiment, the first binding domain of the construct comprises: (a) CDR-H1 as set forth in SEQ ID NO: 4, CDR-H2 as set forth in SEQ ID NO: 5, CDR-H3 as set forth in SEQ ID NO: 6, CDR-L1 as set forth in SEQ ID NO: 1, CDR-L2 as set forth in SEQ ID NO: 2 and CDR-L3 as set forth in SEQ ID NO: 3; (b) CDR-H1 as set forth in SEQ ID NO: 29, CDR-H2 as set forth in SEQ ID NO: 30, CDR-H3 as set forth in SEQ ID NO: 31, CDR-L1 as set forth in SEQ ID NO: 34, CDR-L2 as set forth in SEQ ID NO: 35 and CDR-L3 as set forth in SEQ ID NO: 36; (c) CDR-H1 as set forth in SEQ ID NO: 42, CDR-H2 as set forth in SEQ ID NO: 43, CDR-H3 as set forth in SEQ ID NO: 44, CDR-L1 as set forth in SEQ ID NO: 45, CDR-L2 as set forth in SEQ ID NO: 46 and CDR-L3 as set forth in SEQ ID NO: 47; (d) CDR-H1 as set forth in SEQ ID NO: 53, CDR-H2 as set forth in SEQ ID NO: 54, CDR-H3 as set forth in SEQ ID NO: 55, CDR-L1 as set forth in SEQ ID NO: 56, CDR-L2 as set forth in SEQ ID NO: 57 and CDR-L3 as set forth in SEQ ID NO: 58; (e) CDR-H1 as set forth in SEQ ID NO: 65, CDR-H2 as set forth in SEQ ID NO: 66, CDR-H3 as set forth in SEQ ID NO: 67, CDR-L1 as set forth in SEQ ID NO: 68, CDR-L2 as set forth in SEQ ID NO: 69, and CDR-L3 as set forth in SEQ ID NO: 70; (f) CDR-H1 as set forth in SEQ ID NO: 83, CDR-H2 as set forth in SEQ ID NO: 84, CDR-H3 as set forth in SEQ ID NO: 85, CDR-L1 as set forth in SEQ ID NO: 86, CDR-L2 as set forth in SEQ ID NO: 87, and CDR-L3 as set forth in SEQ ID NO: 88; (g) CDR-H1 as set forth in SEQ ID NO: 94, CDR-H2 as set forth in SEQ ID NO: 95, CDR-H3 as set forth in SEQ ID NO: 96, CDR-L1 as set forth in SEQ ID NO: 97, CDR-L2 as set forth in SEQ ID NO: 98 and CDR-L3 as set forth in SEQ ID NO: 99; (h) CDR-H1 as set forth in SEQ ID NO: 105, CDR-H2 as set forth in SEQ ID NO: 106, CDR-H3 as set forth in SEQ ID NO: 107, CDR-L1 as set forth in SEQ ID NO: 109, CDR-L2 as set forth in SEQ ID NO: 110, and CDR-L3 as set forth in SEQ ID NO: 111; (i) CDR-H1 as set forth in SEQ ID NO: 115, CDR-H2 as set forth in SEQ ID NO: 116, CDR-H3 as set forth in SEQ ID NO: 117, CDR-L1 as set forth in SEQ ID NO: 118, CDR-L2 as set forth in SEQ ID NO: 119, and CDR-L3 as set forth in SEQ ID NO: 120; (j) CDR-H1 as set forth in SEQ ID NO: 126, CDR-H2 as set forth in SEQ ID NO: 127, CDR-H3 as set forth in SEQ ID NO: 128, CDR-L1 as set forth in SEQ ID NO: 129, CDR-L2 as set forth in SEQ ID NO: 130, and CDR-L3 as set forth in SEQ ID NO: 131; (k) CDR-H1 as set forth in SEQ ID NO: 137, CDR-H2 as set forth in SEQ ID NO: 138, CDR-H3 as set forth in SEQ ID NO: 139, CDR-L1 as set forth in SEQ ID NO: 140, CDR-L2 as set forth in SEQ ID NO: 141 and CDR-L3 as set forth in SEQ ID NO: 142; (l) CDR-H1 as set forth in SEQ ID NO: 152, CDR-H2 as set forth in SEQ ID NO: 153, CDR-H3 as set forth in SEQ ID NO: 154, CDR-L1 as set forth in SEQ ID NO: 155, CDR-L2 as set forth in SEQ ID NO: 156, and CDR-L3 as set forth in SEQ ID NO: 157; (m) CDR-H1 as set forth in SEQ ID NO: 167, CDR-H2 as set forth in SEQ ID NO: 168, CDR-H3 as set forth in SEQ ID NO: 169, CDR-L1 as set forth in SEQ ID NO: 170, CDR-L2 as set forth in SEQ ID NO: 171 and CDR-L3 as set forth in SEQ ID NO: 172; (n) CDR-H1 as set forth in SEQ ID NO: 203, CDR-H2 as set forth in SEQ ID NO: 204, CDR-H3 as set forth in SEQ ID NO: 205, CDR-L1 as set forth in SEQ ID NO: 206, CDR-L2 as set forth in SEQ ID NO: 207 and CDR-L3 as set forth in SEQ ID NO: 208; (o) CDR-H1 as set forth in SEQ ID NO: 214, CDR-H2 as set forth in SEQ ID NO: 215, CDR-H3 as set forth in SEQ ID NO: 216, CDR-L1 as set forth in SEQ ID NO: 217, CDR-L2 as set forth in SEQ ID NO: 218, and CDR-L3 as set forth in SEQ ID NO: 219; (p) CDR-H1 as set forth in SEQ ID NO: 226, CDR-H2 as set forth in SEQ ID NO: 227, CDR-H3 as set forth in SEQ ID NO: 228, CDR-L1 as set forth in SEQ ID NO: 229, CDR-L2 as set forth in SEQ ID NO: 230, and CDR-L3 as set forth in SEQ ID NO: 231; (q) CDR-H1 as set forth in SEQ ID NO: 238, CDR-H2 as set forth in SEQ ID NO: 239, CDR-H3 as set forth in SEQ ID NO: 240, CDR-L1 as set forth in SEQ ID NO: 241, CDR-L2 as set forth in SEQ ID NO: 242, and CDR-L3 as set forth in SEQ ID NO: 243; and (r) CDR-H1 as set forth in SEQ ID NO: 248, CDR-H2 as set forth in SEQ ID NO: 249, CDR-H3 as set forth in SEQ ID NO: 250, CDR-L1 as set forth in SEQ ID NO: 251, CDR-L2 as set forth in SEQ ID NO: 252, and CDR-L3 as set forth in SEQ ID NO: 253. It is also contemplated that the antibody comprises a VH region comprising CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of:

[0022] According to the above-mentioned embodiment, the concentration of the bispecific antigen-binding molecule is also expected to be 10 to 35 or 15 to 31 mg / ml, more preferably 20 to 30 mg / ml or 25 to 30 mg / ml.

[0023] According to the above embodiment, it is also envisaged that the concentration of EDTA is in the range of 0.01% to 0.2% (w / V), preferably in the range of 0.01% to 0.16% (w / V), and more preferably 0.04% (w / V).

[0024] According to the above aspect, it is also envisaged that the at least one buffering agent is an acid selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate or a combination thereof, preferably glutamate.

[0025] According to said embodiment, it is also envisaged that the at least one buffering agent is present in a concentration range of 5 to 200 mM, more preferably in a concentration range of 10 to 50 mM, preferably at 15 mM.

[0026] According to the above aspect, it is also envisaged that the at least one sugar is selected from the group consisting of a monosaccharide, a disaccharide, a cyclic polysaccharide, a sugar alcohol, a linear branched dextran or a linear unbranched dextran.

[0027] According to said aspect, it is also envisaged that the disaccharide is selected from the group consisting of sucrose and trehalose and combinations thereof, preferably sucrose.

[0028] According to the above aspect, it is also envisioned that the sugar alcohol is selected from the group consisting of mannitol and sorbitol, and combinations thereof.

[0029] According to said embodiment, it is also envisaged that the at least one sugar is present in a concentration ranging from 1 to 15% (w / V), preferably from 8 to 12% (w / V), for example 8% (w / V).

[0030] According to the above aspect, it is also envisioned that the composition further comprises at least one surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, pluronic F68, Triton X-100, polyoxyethylene, PEG 3350, PEG 4000, and combinations thereof.

[0031] According to said embodiment, it is also envisaged that the composition comprises at least one surfactant in a concentration ranging from 0.004 to 0.5% (w / V), preferably in the range of 0.01 to 0.1% (w / V).

[0032] According to the above embodiment, it is also envisaged that the pH of the composition will be in the range of 4.0 to 5.3, preferably 4.2 to 5.2, and more preferably 4.3 to 4.6.

[0033] According to the above aspect, it is also envisaged that the pharmaceutical composition has an osmolality in the range of 150-500 mOsm.

[0034] According to the above aspect, it is also envisaged that the pharmaceutical composition further comprises an excipient selected from the group consisting of one or more polyols, preferably hydroxypropyl-β-cyclodextrin, and one or more amino acids, preferably phenylalanine (but preferably excluding arginine, proline, and tryptophan).

[0035] According to this aspect, it is also envisaged that the one or more excipients are present in a concentration range of 0.1-15% (w / V).

[0036] According to the above aspect, the composition comprises: (a) a bispecific antigen-binding molecule according to the present invention at a concentration ranging from 8 to 35 mg / ml, preferably from 15 to 30 mg / ml; (b) 15 mM glutamate or acetate; (c) 8% (w / V) sucrose or 8% (w / V) sucrose and 1% (w / V) hydroxypropyl-β-cyclodextrin; (d) optionally, 0.01% (w / V) polysorbate 80 Here, the pH of the liquid pharmaceutical composition is any value in the range of 4.0 to 5.2, preferably 4.2 to 4.6 or 4.3 to 4.6. It is also assumed that the

[0037] According to another aspect, it is also envisaged that a solid pharmaceutical composition may be obtained by lyophilization of a liquid pharmaceutical composition of the present invention.

[0038] According to another aspect, it is also envisaged that a liquid pharmaceutical composition may be obtained by reconstituting a solid pharmaceutical composition according to the invention with a pharmaceutically acceptable liquid.

[0039] According to another aspect, it is also envisaged that the pharmaceutical compositions of the present invention are for use in the treatment of a disease, preferably a proliferative disease such as cancer, preferably by intravenous or subcutaneous administration.

[0040] According to another aspect, it is also envisaged that the liquid pharmaceutical composition of the present invention is used to reduce the formation of high molecular weight species (HMWS) during storage, and that the amount of HMWS is kept below 5%, preferably below 3% or 2%, when the liquid pharmaceutical composition is stored at 4°C or below, preferably at -30°C or below. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows the percent high molecular weight (HMW) species determined by size exclusion chromatography (SEC) analysis as a function of protein concentration (4.5, 18.4, and 30.1 mg / ml of DLL3xCD3 bispecific antigen-binding molecule) and pH value (pH 4.4 (lower graph), pH 4.8 (middle graph), and pH 5.2 (upper graph)). The liquid pharmaceutical composition contains 15 mmol of glutamic acid, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 80, but does not contain any additional stabilizers such as EDTA. The percent HMW species significantly increases with increasing pH and protein concentration. [Figure 2] Figure 1 shows the percent high molecular weight (HMW) and low molecular weight (LMW) species after 4 weeks of storage at 40°C, as determined by size exclusion chromatography (SEC) analysis, depending on the pH value (individual values ​​range from pH 4.1 to 4.9). The liquid pharmaceutical composition contains 20 mg / ml of each bispecific antigen-binding molecule, 15 mmol of glutamic acid, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 80, but does not contain any additional stabilizers such as EDTA. Open symbols represent HMW data points, and closed symbols represent LMW data points. Squares represent BCMAxCD3 bispecific antigen-binding molecules, circles represent CD33xCD3 bispecific antigen-binding molecules, and triangles represent DLL3xCD3 bispecific antigen-binding molecules. The percent HMW species significantly increase with increasing pH, and the percent LMW species decrease with increasing pH. [Figure 3]The percent low molecular weight (LMW) species before storage (t=0, open circles) and after 4 weeks of storage at 40°C (filled circles) as determined by size exclusion chromatography (SEC) analysis depending on the pH value (individual values ​​ranging from pH 4.1 to 4.9) are shown. The liquid pharmaceutical compositions contained 5 mg / ml (dark symbols) or 20 mg / ml (light symbols) of each bispecific antigen-binding molecule. Each formulation contained 15 mmol of glutamic acid and 0.01% (w / v) polysorbate 80, and also (from left to right): 5% (w / v) sucrose, 5% + 1% (w / v) hydroxypropyl-β-cyclodextrin (HpbCD), sucrose, 5% (w / v) + 10 mM phenylalanine (Phe), and 0.3 M proline (Pro). "1" in the upper row shows the results for the DLL3xCD3 bispecific antigen-binding molecule, "2" in the middle row shows the results for the BCMAxCD3 bispecific antigen-binding molecule, and "3" in the lower row shows the results for the CD33xCD3 bispecific antigen-binding molecule. [Figure 4]Figure 4A shows the percent high molecular weight (HMW) species of BCMAxCD3 bispecific antigen-binding molecules from left to right in each set of four columns: (i, black) before storage at t = 0, (ii, medium grey) as determined by size exclusion chromatography (SEC) analysis depending on the pH value (for each set of excipients, from left to right, pH 4.3 ("4" in the x-axis caption), 4.6 ("46" in the x-axis caption), and 5.2 ("5" in the x-axis caption), respectively. ) after 2 weeks storage at -30°C, (iii, light grey) after 4 weeks storage at 4°C, and (iv, dark grey) after 4 weeks storage at 40°C. The liquid pharmaceutical compositions comprised 30 mg / ml BCMAxCD3 bispecific antigen-binding molecule, 15 mmol glutamic acid, 8% (w / v) sucrose and 0.01% (w / v) polysorbate 80, and for each triplet in the four columns from left to right were: (a) no additional stabilizer (control), (b), "CD") 1% (w / v) hydroxypropyl-β- Cyclodextrin (HpbCD), (c, "Phe") 50 mM phenylalanine, (d, "EDTA") 0.04% (w / v) EDTA, (e, "R") 50 mM arginine, (f, "Trp") 20 mM tryptophan, (g, "Pro") 50 mM proline, and (h, "BA") 0.45% (w / v) benzyl alcohol. Figure 4B shows the percent high molecular weight (HMW) of DLL3xCD3 bispecific antigen binding molecules from left to right in each set of four columns. ) indicates species: determined by size exclusion chromatography (SEC) analysis depending on pH value (from left to right, pH 4.3 ("4" in the x-axis caption), 4.6 ("46" in the x-axis caption), and 5.2 ("5" in the x-axis caption) for each set of excipients, respectively: (i, black) before storage at t=0, (ii, medium grey) after 2 weeks of storage at -30°C, (iii, light grey) after 4 weeks of storage at 4°C, and (iv, dark grey) after 4 weeks of storage at 40°C.The liquid pharmaceutical composition contained 30 mg / ml of DLL3xCD3 bispecific antigen-binding molecule, 15 mmol glutamic acid, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 80, with the following components for each triplet in the four columns, from left to right: (a) no additional stabilizer (control), (b, "CD") 1% (w / v) hydroxypropyl-β-cyclodextrin (HpbCD), (c, "Phe") 50 mM phenylalanine, (d, "EDTA") 0.04% (w / v) EDTA, (e, "R") 50 mM arginine, (f, "Trp") 20 mM tryptophan, (g, "Pro") 50 mM proline, and (h, "BA") 0.45% (w / v) benzyl alcohol. [Figure 5] Shown in each set of four columns from left to right are the percent high molecular weight (HMW) species of the MUC17xCD3 bispecific antigen-binding molecule as determined by size exclusion chromatography (SEC) analysis depending on the pH value (for each set of excipients, from left to right, pH 4.2 ("4" in the x-axis caption), 4.6 ("46" in the x-axis caption), and 5.0 ("5" in the x-axis caption), respectively: (i, black) before storage at t=0, (ii, medium grey) after 4 weeks of storage at -30°C, (iii, light grey) after 4 weeks of storage at 4°C, and (iv, dark grey) after 4 weeks of storage at 40°C. The liquid pharmaceutical composition contained 16 mg / ml of MUC17xCD3 bispecific antigen-binding molecule. Containing heteromeric antigen-binding molecule, 15 mmol glutamic acid, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 80, for each triplet in the four columns, from left to right, were: (a) no additional stabilizer (control), (b, "CD") 1% (w / v) hydroxypropyl-β-cyclodextrin (HpbCD), (c, "Phe") 50 mM phenylalanine, (d, "EDTAAp01") 0.01% (w / v) EDTA, (e, "R") 50 mM arginine HCl, (vi, "EDTAAp04") 0.04% (w / v) EDTA, (g, "Pro") 50 mM proline, and (h, "EDTAAp16") 0.16% (w / v) EDTA. DETAILED DESCRIPTION OF THE INVENTION

[0042] To facilitate convenient delivery of bispecific antigen-binding molecules, such as half-life extended (HLE) scFc BiTE® molecules, subcutaneous liquid formulations are highly desirable. However, highly concentrated formulations are often required to meet the required dosage within a limited volume. Unlike the typical concentration of mAbs (up to 70 mg / ml), bispecific antigen-binding molecules are typically lyophilized to a final reconstituted concentration of approximately 1-5 mg / ml (0.1-0.5% (w / v)). In addition, artificial bispecific antigen-binding molecules are more prone to aggregation than mAbs during product manufacturing, storage, and administration. It is difficult to formulate bispecific antigen-binding molecules into liquid pharmaceutical compositions at concentrations greater than approximately 5 mg (w / v) while minimizing undesired aggregation.

[0043] Thus, it was surprising that bispecific antigen-binding molecules such as HLE BiTE® molecules were stabilized at concentrations significantly greater than 5 mg / ml, up to approximately 35 mg / ml, i.e., the %HMW was found to be significantly lower in the optimized formulations compared to the formulation control under the same test conditions with respect to pH, incubation temperature, and duration. For example, for each of the DLL3xCD3, MUC17xCD3, and BCMAxCD3 bispecific antigen-binding molecules, a stabilizer with chelating properties, such as EDTA (ethylenediaminetetraacetic acid), significantly reduced the %HMW over a 4-week period compared to the control formulation. By way of illustration, at time t=0, the BCMAxCD3 bispecific antigen-binding molecule at 27 mg / ml and pH 5.2 exhibited approximately 2% HMW with EDTA as a stabilizer, compared to approximately 12% in pharmaceutical compositions without EDTA, ETPA, or citric acid. Other tested excipients, such as hydroxypropyl-β-cyclodextrin (HpbCD) and the amino acid phenylalanine (Phe), also showed some reduction in %HMW under certain conditions, particularly at pHs closer to 4 rather than 5, but lower than stabilizers with chelating properties. On the other hand, Arg-HCl promoted aggregation by increasing %HMW. Other tested excipients, such as Trp and Pro, did not appear to affect %HMW compared to the control buffer (potentially) without stabilizers.

[0044] The present invention shows for the first time that providing an optimized formulation by only adding one selected stabilizer can reduce the %HMW of various bispecific antigen-binding molecules at such high concentrations in liquid format, allowing them to be used for additional administration purposes, and overcoming the concentration-related stability disadvantages of bispecific antigen-binding molecules compared to conventional mAbs.

[0045] In the context of the present invention, it has surprisingly been found that the addition of a stabilizer, preferably EDTA, DFPA, or citric acid, has resulted in stabilization of such molecules, e.g., BiTE molecules, at concentrations greater than 5 mg, i.e., 8 mg / ml or greater, e.g., 8, 9, 10, 11, 12, 13, or 14 mg / ml, and preferably even 15-35 mg / ml, i.e., 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / ml. This is surprising because chelating agents such as EDTA have previously been used to reduce oxidative stress in mAbs, in addition to their chelating capabilities. While oxidation as a chemical modification of a molecule of interest can contribute to the alteration of the molecule, protein aggregation is a complex phenomenon that can occur through various pathways with different mechanisms. When bispecific antigen-binding molecules of the present invention are not formulated in pharmaceutical compositions containing the stabilizers described herein, they exhibit an increase in %HMWS over time as determined by SE-UHPLC (see Table 1). However, no chemical modification in terms of deamidation (e.g., at N350 and N353) or oxidation (e.g., at methionine position M281) was observed in the bispecific antigen-binding molecules according to the present invention. Without wishing to be bound by theory, the underlying principle of stabilization lies in the interaction of stabilizers with chelating properties with the CD3-binding domain. As determined by differential scanning fluorimetry (DSF) measurements, a typically rapid screening method for identifying low-molecular-weight ligands that bind and stabilize purified proteins, the melting temperature (Tm) of the bispecific antigen-binding molecules of the present invention is typically increased in the presence of stabilizers such as EDTA, indicating increased conformational stability of the CD3-binding domain. Thus, molecules of the present invention such as SEQ ID NO: 104 and SEQ ID NO: 258, which have different CD3 binding domains, abbreviated as I2C versus I2E, respectively, in the sequence listing (Table 5), are both stabilized by stabilizers with chelating properties of the present invention, such as EDTA.

[0046] [Table 1]

[0047] In the context of the present invention, HMWS are primarily dimers that share a similar conformation of the monomers and can be considered as native dimers. HMWS as understood in the context of the present invention are highly reversible, i.e., for multiple bispecific antigen-binding molecules, the dimers dissociate into monomers after 24 hours of thawing at 25°C. As shown in exemplary Table 1, HMWS increased without a corresponding increase in chemical modifications, either Met oxidation or deamidation.

[0048] Advantageously, the pharmaceutical compositions of the invention are capable of stabilizing bispecific antigen-binding molecules such as HLE BiTE molecules at higher concentrations, even at higher pH values, which may be beneficial in certain administration settings where too low a pH of the pharmaceutical composition is not feasible or where the stability of certain bispecific antigen-binding molecules requires a pH slightly higher than about 4, e.g., 4.6 or 5.2.

[0049] The bispecific antigen-binding molecule concentration can range from 8 to 35 mg / ml, preferably from 15 to 30 mg / ml. Stabilizing agents such as EDTA can be present at a concentration ranging from approximately 0.005 to 0.25% (w / v), the upper limit considered safe for human administration according to applicable guidelines. EDTA can be present as a salt, such as disodium EDTA, calcium edetate sodium, and tetrasodium EDTA, preferably disodium EDTA.

[0050] Bispecific antigen-binding molecules according to the present invention, such as ScFc-BiTE® molecules, typically contain an Fc region that is similar in size, pI, and hydrophobicity to the corresponding region of an IgG antibody. General downstream processing of IgG antibodies from host cell proteins (HCPs), other bioreactor impurities and reagents, high molecular weight (HMW) species or aggregates, low molecular weight species, or clips is known in the art (Shukla et al. 2006). Such downstream processing has been adapted for the purification of bispecific antigen-binding molecules, such as scFc-BiTE®, and typically includes the following steps: cell culture harvest; Protein A chromatography; virus inactivation by filter; a second column chromatography polishing step or two, e.g., CEX; virus filtration; and UF / DF.

[0051] The term "antibody product" refers to a "secreted protein" or "secreted recombinant protein," meaning a protein (e.g., a recombinant protein) that originally contains at least one secretory signal sequence when translated in a mammalian cell and that is secreted, at least in part, into the extracellular space (e.g., liquid culture medium) via enzymatic cleavage of the secretory signal sequence within the mammalian cell. One of skill in the art will understand that a "secreted" protein need not completely dissociate from the cell to be considered a secreted protein.

[0052] The term bispecific antibody product encompasses bispecific antibodies such as full-length, e.g., IgG-based antibodies and fragments thereof, which are generally referred to herein as bispecific antigen-binding molecules.

[0053] The term "antigen-binding molecule" refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length or complete immunoglobulin molecule, and / or derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or a fragment thereof. Thus, an antigen-binding molecule can bind to its specific target or antigen. Furthermore, the binding domain of an antigen-binding molecule according to the present invention comprises the minimum structural requirements of an antibody that enable target binding. This minimum requirement can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. Alternative approaches to defining the minimum structural requirements of an antibody are the structure of a specific target, the definition of an antibody epitope within the protein domains (epitope clusters) of the target protein that each constitute an epitope region, or by reference to specific antibodies that compete with the defined antibody epitope. Antibodies on which the constructs according to the invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies.

[0054] The binding domain of an antigen-binding molecule of the present invention may, for example, comprise the CDRs of the above-referenced groups. Preferably, these CDRs are contained within the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it is not necessary to contain both. Fd fragments, for example, have two VH regions and often retain some of the antigen-binding function of an intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants, or binding domains include: (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) an F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody; (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fvs (scFvs), the latter of which is preferred (e.g., derived from an scFv library). Examples of embodiments of antigen-binding molecules according to the present invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, U.S. Patent Application Publication Nos. 2014 / 0308285, 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910, and WO 2015 / 048272.

[0055] The definition of "binding domain" or "domain binding to" also includes fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "r IgG" ("half antibody"). Antigen-binding molecules according to the present invention may also include modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies, or single variable domain antibodies, which comprise only one variable domain, which may be VHH, VH, or VL, that specifically binds to an antigen or epitope independent of other V regions or domains.

[0056] As used herein, the term "single-chain Fv," "single-chain antibody," or "scFv" refers to an antibody fragment of a single polypeptide chain that contains the variable regions from both the heavy and light chains but lacks the constant region. Typically, single-chain antibodies further contain a polypeptide linker between the VH and VL domains, which enables them to form the desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods for making single chain antibodies are known, for example, as described in U.S. Pat. Nos. 4,694,778 and 5,260,203; WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single chain antibodies may also be bispecific, multispecific, human, and / or humanized, and / or synthetic.

[0057] Furthermore, the definition of the term "antigen-binding molecule" includes monovalent, bivalent, and polyvalent / multivalent constructs, and therefore bispecific constructs that specifically bind to only two antigenic structures, as well as polyspecific / multispecific constructs that specifically bind to three or more antigenic structures, for example, three, four, or more, through different binding domains. Furthermore, the definition of the term "antigen-binding molecule" includes molecules consisting of only one peptide chain, as well as molecules consisting of multiple polypeptide chains, which chains may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the above-identified antibodies and variants or derivatives thereof are described, inter alia, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Duebel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0058] The term "polypeptide," as used herein, refers to a group of molecules typically consisting of more than 30 amino acids. Polypeptides may also form multimers, such as dimers, trimers, and higher oligomers, i.e., composed of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are therefore referred to as homodimers or heterodimers, homotrimers, heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its native form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally occurring modified peptides / polypeptides / proteins that have been modified by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. "Peptides," "polypeptides," or "proteins," as referred to herein, may also be chemically modified, such as by pegylation. Such modifications are known in the art and are described herein below.

[0059] As used herein, the term "bispecific" refers to an antigen-binding molecule that is "at least bispecific," i.e., it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (e.g., a surface antigen on a target cell) and the second binding domain binds to another antigen or target (e.g., CD3). Thus, an antigen-binding molecule according to the present invention comprises specificities for at least two different antigens or targets. For example, the first domain preferably does not bind to one or more extracellular epitopes of CD3ε as described herein. The term "target cell surface antigen" refers to an antigenic structure that is expressed by a cell and present on the cell surface so that it is accessible to an antigen-binding molecule as described herein. A TAA can be a protein (preferably the extracellular portion of a protein) or a carbohydrate structure (preferably the carbohydrate structure of a protein such as a glycoprotein). Preferably, the TAA is a tumor antigen. The term "bispecific antigen-binding molecule" of the present invention also encompasses multispecific antigen-binding molecules, such as trispecific antigen-binding molecules comprising three binding domains, or constructs with more than three specificities (e.g., four, five, ...).

[0060] When an antigen-binding molecule of the present invention is (at least) bispecific, it does not occur in nature and is significantly different from naturally occurring products. Thus, a "bispecific" antigen-binding molecule or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sites with different specificities. Bispecific antigen-binding molecules can be produced by various methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0061] The at least two binding domains and the variable domain (VH / VL) of the antigen-binding molecule of the present invention may or may not contain a peptide linker (spacer peptide). In the context of the present invention, the term "peptide linker" includes an amino acid sequence that connects the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of the antigen-binding molecule of the present invention. This peptide linker can also be used to fuse a third domain to another domain of the antigen-binding molecule of the present invention. An essential technical feature of such a peptide linker is that it does not contain polymerization activity. Suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, or WO 88 / 09344. Peptide linkers can also be used to link other domains, modules, or regions (such as half-life-extending domains) to the antigen-binding molecules of the present invention.

[0062] The antigen-binding molecules of the present invention are preferably "antigen-binding molecules produced in vitro." This term refers to antigen-binding molecules as defined above in which all or part of the variable region (e.g., at least one CDR) is produced by non-immune cell selection, such as in vitro phage display, protein chips, or any other method that allows testing of candidate sequences for antigen-binding ability. Thus, this term preferably excludes sequences produced solely by genome rearrangement in animal immune cells. A "recombinant antibody" is an antibody produced by using recombinant DNA technology or genetic engineering.

[0063] The term "monoclonal antibody" (mAb) or monoclonal antigen-binding molecule, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, and are therefore uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method.

[0064] For the preparation of monoclonal antibodies, any technique that results in antibodies produced by continuous cell line cultures may be used. For example, the monoclonal antibodies used may be made by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Additional examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0065] The hybridomas can then be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to the designated antigen. Any form of the relevant antigen can be used as the immunogen, including, for example, recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof. Surface plasmon resonance, as employed in the BIAcore system, can be used to increase the efficiency of phage antibody binding to epitopes on surface antigens of target cells (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0066] Another exemplary method for generating monoclonal antibodies includes screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0067] In addition to using display libraries, the relevant antigen can be used to immunize a non-human animal, such as a rodent (e.g., a mouse, hamster, rabbit, or rat). In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, mouse strains deficient in mouse antibody production can be engineered with large fragments of the human Ig (immunoglobulin) locus. Hybridoma technology may be used to generate and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0068] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, chimerization, etc. Examples of modified antigen-binding molecules include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody mutants with modified effector functions (see, e.g., U.S. Pat. No. 5,648,260, Kontermann and Duebel (2010) supra, and Little (2009) supra).

[0069] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for antigens during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively higher affinities. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antigen-binding molecules, and antibody fragments. Random mutations within CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. Additionally, chain shuffling can increase genetic diversity. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinities in the low nanomolar range.

[0070] A preferred type of amino acid substitution variant of an antigen-binding molecule involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development have improved biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify potential hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domain and, for example, a surface antigen on a human target cell. Such contact and adjacent residues are candidates for substitution using the techniques detailed herein. After generating such variants, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0071] The monoclonal antibodies and antigen-binding molecules of the present invention particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkey, ape, etc.) and human constant region sequences. Various techniques for producing chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. Sci USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Nos. 0171496; 0173494; and British Patent No. 2177096.

[0072] Antibodies, antigen-binding molecules, antibody fragments, or antibody variants may also be modified by specific deletion of human T-cell epitopes (a method called "deimmunization"), for example, by the methods disclosed in WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for MHC class II-binding peptides, which represent potential T-cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). To detect potential T-cell epitopes, a computer modeling approach called "peptide threading," as described in WO 98 / 52976 and WO 00 / 34317, can be applied; in addition, databases of human MHC class II-binding peptides can be searched for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, making them potential T cell epitopes. Potential T cell epitopes detected can be eliminated by substituting a small number of amino acid residues in the variable domains, or preferably by substituting a single amino acid. Conservative substitutions are typically made. In many, but not all, positions common to human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16(5):237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The VBASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, L.A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs.Consensus human framework regions, such as those described in US Pat. No. 6,300,064, can also be used.

[0073] A "humanized" antibody, antigen-binding molecule, variant, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) is an antibody or immunoglobulin of largely human sequence that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also called a CDR) of the recipient are replaced by residues from a hypervariable region of a non-human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies," as used herein, may also comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0074] Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, 5,859,205, and 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids may be obtained from hybridomas producing antibodies against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0075] Humanized antibodies may also be produced using transgenic animals, such as mice, that express human heavy and light chain genes but are incapable of expressing endogenous mouse immunoglobulin heavy and light chain genes. Winter (U.S. Pat. No. 5,225,539) describes an exemplary CDR-grafting method that can be used to prepare the humanized antibodies described herein. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace as many CDRs as necessary for the humanized antibody to bind to a given antigen.

[0076] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such modified immunoglobulin molecules can be produced by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982, and EP 239400).

[0077] The terms "human antibody," "human antigen-binding molecule," and "human binding domain" include antibodies, antibody-binding molecules, and binding domains having antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described in Kabat et al. (1991) supra. Human antibodies, antigen-binding molecules, or binding domains of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. Human antibodies, antigen-binding molecules, or binding domains may have at least one, two, three, four, five, or more positions replaced with amino acid residues not encoded by human germline immunoglobulin sequences. However, as used herein, the definitions of human antibody, antigen-binding molecule, and binding domain also contemplate "fully human antibodies" which include only non-artificially and / or genetically modified human antibody sequences, such as those obtainable using technologies or systems such as Xenomouse. Preferably, a "fully human antibody" does not include any amino acid residues not encoded by human germline immunoglobulin sequences.

[0078] In some embodiments, the antigen-binding molecules of the present invention are "isolated" or "substantially pure" antigen-binding molecules. "Isolated" or "substantially pure," when used to describe antigen-binding molecules disclosed herein, refers to an antigen-binding molecule that has been identified, separated, and / or recovered from components of its production environment. Preferably, the antigen-binding molecule is free or substantially free from association with all other components from its production environment. Contaminant components of its production environment, such as components resulting from recombinant transfected cells, are typically materials that interfere with diagnostic or therapeutic uses for the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antigen-binding molecule may, for example, constitute at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein may constitute 5% to 99.9% by weight of the total protein content, depending on the circumstances. The polypeptide may be produced at significantly higher concentrations by using an inducible promoter or a high-expression promoter to produce at increased concentration levels. This definition includes production of antigen-binding molecules in a wide variety of organisms and / or host cells known in the art. In a preferred embodiment, the antigen-binding molecule is purified to a sufficient extent (1) to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, isolated antigen-binding molecules are usually prepared by at least one purification step.

[0079] The term "binding domain" in the context of the present invention is considered to be a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target site on a target molecule (antigen), such as CD33 and CD3, respectively. The structure and function of the first binding domain (e.g., recognizing CD33), and preferably also the structure and / or function of the second binding domain (e.g., recognizing CD3), are based on the structure and / or function of an antibody, e.g., a full-length or complete immunoglobulin molecule, and / or are derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or a fragment thereof. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). The second binding domain also preferably comprises the minimal structural requirements of an antibody that enable target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). It is envisaged that the first binding domain and / or the second binding domain are produced or obtained by phage display or library screening methods, rather than by grafting CDR sequences from an existing (monoclonal) antibody into a scaffold.

[0080] According to the present invention, a binding domain is in the form of one or more polypeptides. Such polypeptides may comprise proteinaceous and non-proteinaceous portions (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise two or more amino acids linked together via covalent peptide bonds (resulting in a chain of amino acids).

[0081] The term "polypeptide," as used herein, refers to a group of molecules typically consisting of more than 30 amino acids. Polypeptides may also form multimers, such as dimers, trimers, and higher oligomers, i.e., composed of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are therefore referred to as homodimers or heterodimers, homotrimers, heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its native form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally occurring modified peptides / polypeptides / proteins that have been modified by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. "Peptides," "polypeptides," or "proteins," as referred to herein, may also be chemically modified, such as by pegylation. Such modifications are known in the art and are described herein below.

[0082] Preferably, the binding domain that binds to a surface antigen of a target cell and / or the binding domain that binds to CD3ε are human binding domains. Antibodies and antigen-binding molecules comprising at least one human binding domain avoid some of the problems associated with antibodies or antigen-binding molecules having non-human variable and / or constant regions, such as those derived from rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins can result in rapid clearance of the antibody or antigen-binding molecule or can cause an immune response against the antibody or antigen-binding molecule by patients. To avoid using rodent-derived antibodies or antigen-binding molecules, human or fully human antibodies / antigen-binding molecules can be generated by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0083] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0084] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B cell development. Furthermore, such a strategy would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), which would represent an important milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antigen-binding molecules are expected to minimize the immunogenicity and allergic reactions inherent in mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of administered antibodies / antigen-binding molecules. The use of fully human antibodies or antigen-binding molecules is expected to provide significant advantages in the treatment of chronic and recurrent human diseases that require repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0085] One approach toward this goal has been to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci, with the expectation that such mice would produce a broad repertoire of human antibodies without producing mouse antibodies. Large human Ig fragments would preserve the broad diversity of variable genes and the appropriate regulation of antibody production and expression. By utilizing the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoire recapitulated in these mouse strains should produce high-affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity could be readily generated and selected. This general strategy was demonstrated in connection with the generation of the first XenoMouse mouse strains (see Green et al., Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered with yeast artificial chromosomes (YACs) containing 245-kb and 190-kb germline-configured fragments of the human heavy chain and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. The human Ig-containing YACs proved compatible with the mouse system for both antibody rearrangement and expression and were able to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and to generate antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing multiple V genes, additional regulatory elements, and human Ig constant regions, could recapitulate a virtually complete repertoire characterized by the human humoral response to infection and immunization. Recently, extending the work of Green et al., the introduction of megabase-sized germline-configured YAC fragments of the human heavy chain and kappa light chain loci, respectively, introduced approximately 80% of the human antibody repertoire. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application Serial No. 08 / 759,620.

[0086] The generation of XenoMouse mice is further described in U.S. patent application Ser. Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837 ...4,582, 08 / 463,191, 08 / 462,837, 08 / 464,582, 08 / 464,582, 08 / 463,191, 08 / 462,837, 08 / 464,582, 08 / 464,582, 08 / 464,582, 08 / 464,582, 08 / 464,582, 08 / 464,582, 08 / 464,582, 08 / 464, This is further discussed and detailed in U.S. Patent Nos. 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752 and 08 / 759,620, as well as U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181 and 5,939,598, and Japanese Patent Nos. 3068180B2, 3068506B2 and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0087] An alternative approach, utilized by others including GenPharm International, Inc., is the "minilocus" approach, in which small pieces (individual genes) from the Ig locus are included to mimic an exogenous Ig locus, such that one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a γ constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al., and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, and 5,877,397, all to Lonberg and Kay, respectively. Nos. 5,874,299, and 6,255,458 to Krimpenfort and Berns, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharm and U.S. Patent Application Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International.It is also described in European Patent No. 0546073B1, International Publication Nos. 92 / 03918, 92 / 22645, 92 / 22647, 92 / 22670, 93 / 12227, 94 / 00569, 94 / 25585, 96 / 14436, 97 / 13852, and 98 / 24884, and U.S. Pat. No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0088] Kirin has also demonstrated the production of human antibodies from mice into which large or entire chromosomes have been introduced by microcell fusion. See European Patent Applications 773288 and 843961. Xenerex Biosciences is developing a technology for producing promising human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes, e.g., B cells and / or T cells. The mice can then be immunized with an antigen to generate an immune response against the antigen. See U.S. Patent Nos. 5,476,996, 5,698,767, and 5,958,765.

[0089] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. However, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly when antibodies are used chronically or in multiple doses. Therefore, it would be desirable to provide an antigen-binding molecule that contains a human binding domain for a surface antigen of a target cell and a human binding domain for CD3ε to eliminate the concerns and / or effects of HAMA or HACA responses.

[0090] The terms "(specifically) bind", "(specifically) recognize", "(specifically) be attracted to" and "(specifically) react" mean, according to the present invention, that a binding domain interacts or specifically interacts with a target molecule (antigen), here a surface antigen of a target cell and a given epitope or a given target site on CD3ε, respectively.

[0091] The term "epitope" refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and therefore the term epitope is also sometimes referred to herein as an "antigen structure" or "antigenic determinant." The binding domain is therefore an "antigen interaction site." Said binding / interaction is also understood to define "specific recognition."

[0092] An "epitope" can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. A "linear epitope" is one in which a primary sequence of amino acids is recognized. A linear epitope typically contains at least three or at least four, more usually at least five, or at least six, or at least seven, e.g., about eight to about ten, amino acids in a unique sequence.

[0093] In contrast to linear epitopes, a "conformational epitope" is an epitope in which the primary sequence of amino acids comprising the epitope is not the only element defining the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by a binding domain). Generally, a conformational epitope contains a larger number of amino acids than a linear epitope. In recognizing a conformational epitope, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is contained within the surface antigen protein of a target cell). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones forming the conformational epitope are juxtaposed, thereby enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0094] The epitope mapping method is described below: when a region (a contiguous stretch of amino acids) of a human target cell surface antigen protein is exchanged / substituted with a corresponding region of a non-human or non-primate target cell surface antigen (e.g., a mouse target cell surface antigen, but also chicken, rat, hamster, rabbit, etc.), a reduction in binding activity of the binding domain is expected, as long as the binding domain is not cross-reactive with the non-human or non-primate target cell surface antigen used. The reduction is preferably at least 10%, 20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100% compared to binding to the corresponding region in the human target cell surface antigen protein, assuming binding to the corresponding region in the human target cell surface antigen protein to be 100%. It is envisioned that the above-mentioned human target cell surface antigen / non-human target cell surface antigen chimeras will be expressed in CHO cells. It is also envisaged that the human target cell surface antigen / non-human target cell surface antigen chimera is fused to the transmembrane and / or cytoplasmic domain of a different membrane-associated protein, such as EpCAM.

[0095] In an alternative or additional method of epitope mapping, several truncated forms of the extracellular domain of a human target cell surface antigen can be generated to determine the specific region recognized by the binding domain. In these truncated forms, different extracellular target cell surface antigen domains / subdomains or regions are deleted stepwise, starting from the N-terminus. It is contemplated that the truncated target cell surface antigen can be expressed in CHO cells. It is also contemplated that the truncated target cell surface antigen may be fused to the transmembrane and / or cytoplasmic domain of a different membrane-bound protein, such as EpCAM. It is also contemplated that the truncated target cell surface antigen may include a signal peptide domain at their N-terminus, such as a signal peptide derived from the mouse IgG heavy chain signal peptide. It is also contemplated that the truncated target cell surface antigen may include a v5 domain at the N-terminus (following the signal peptide), which can confirm their correct expression on the cell surface. It is expected that reduced or lost binding will occur for truncated target cell surface antigens that no longer encompass the target cell surface antigen region recognized by the binding domain. The reduction in binding is preferably at least 10%, 20%, 30%, 40% or 50%; more preferably at least 60%, 70%, 80%, and most preferably 90%, 95% or even 100%, relative to binding to the entire surface antigen protein (or its extracellular region or domain) of a human target cell, taken as 100.

[0096] Another method for determining the contribution of specific residues of target cell surface antigens to recognition by antigen-binding molecules or binding domains is alanine scanning, in which each analyzed residue is substituted with alanine, for example, by site-directed mutagenesis (see, e.g., Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7). Alanine is used because it is not bulky, is chemically inert, and yet contains a methyl functional group that mimics the secondary structure criterion of many other amino acids. In many cases, bulky amino acids such as valine or leucine can be used if it is desirable to maintain the size of the residue to be mutated. Alanine scanning is a mature technique that has been used for a long time.

[0097] The interaction between a binding domain and an epitope or an epitope-containing region means that the binding domain exhibits measurable affinity for the epitope / epitope-containing region on a particular protein or antigen (herein, target cell surface antigen and CD3, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than target cell surface antigen or CD3. "Appreciable affinity" means a binding domain with a measurable affinity of about 10 -6 M(KD) or higher. Preferably, the binding affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9If M, the binding is considered specific. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reactivity of the binding domain to a target protein or antigen with the reactivity of the binding domain to proteins or antigens other than the target cell surface antigen or CD3. Preferably, the binding domains of the present invention essentially or substantially do not bind to proteins or antigens other than the target cell surface antigen or CD3 (i.e., the first binding domain cannot bind to proteins other than the target cell surface antigen, and the second binding domain cannot bind to proteins other than CD3). Superior affinity characteristics compared to other HLE formats are anticipated as a feature of the antigen-binding molecules of the present invention. Such superior affinity suggests a prolonged in vivo half-life. The longer the half-life of the antigen-binding molecules of the present invention, the shorter the administration period and frequency, which typically contributes to improved patient compliance. This is particularly important because the antigen-binding molecules of the present invention are particularly beneficial for highly debilitated or multi-disease cancer patients.

[0098] The terms "does not essentially / substantially bind" or "cannot bind" mean that the binding domain of the invention does not bind to proteins or antigens other than target cell surface antigens or CD3, i.e., when binding to target cell surface antigens or CD3, respectively, is taken as 100%, it does not show reactivity to proteins or antigens other than target cell surface antigens or CD3 of more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably 9%, 8%, 7%, 6% or 5%.

[0099] Specific binding is believed to be mediated by specific motifs within the amino acid sequences of the binding domain and the antigen. Thus, binding is achieved not only as a result of their primary, secondary, and / or tertiary structures, but also as a result of secondary modifications of said structures. The specific interaction of the antigen-interaction site with its specific antigen can result in simple binding of said site to the antigen. Furthermore, the specific interaction of the antigen-interaction site with its specific antigen can alternatively or additionally initiate a signal, for example, by inducing a conformational change in the antigen, oligomerization of the antigen, etc.

[0100] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is responsible for determining the specificity and binding affinity of a particular antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen-binding site.

[0101] The variability is not uniformly distributed throughout the variable domains of antibodies, but is concentrated in subdomains within each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs in three-dimensional space that form the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4) that largely adopt a β-sheet configuration and are connected by three hypervariable regions that form loops connecting, and in some cases, part of, the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRMs with the hypervariable regions of the other chain, contributing to the formation of the antigen-binding site (see Kabat et al., supra).

[0102] The term "CDR," and its plural "CDRs," refers to complementarity-determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). The CDRs contain most of the residues responsible for specific interactions between the antibody and antigen and thus contribute to the functional activity of the antibody molecule; i.e., the CDRs are the primary determinants of antigen specificity.

[0103] The precise definition of CDR boundaries and lengths follows various classification and numbering systems. Thus, CDRs may be referred to by Kabat, Chothia, contact, or any other boundary definition, including the numbering systems described herein. Despite differences in boundaries, each of these systems has some overlap in the portions that constitute the so-called "hypervariable regions" within the variable sequences. Thus, CDR definitions according to these systems may differ in length and in the boundaries relative to the adjacent framework regions. For example, Kabat (an approach based on sequence variability between species), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol., 1987, 196:901-917; and MacCallum et al., J. Mol. Biol., 1996, 262:732). Another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). As long as two residue identification techniques define overlapping but non-identical regions, they can be combined to define hybrid CDRs. However, the so-called Kabat system of numbering is preferred.

[0104] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main-chain conformation adopted by the antigen-binding (CDR) loop. Comparative structural studies have revealed that five of the six antigen-binding loops have only a limited repertoire of useful conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures despite the high degree of variability in the amino acid sequences across most of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol., 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop as well as within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0105] The term "canonical structure" can also include considerations regarding the linear sequence of an antibody, for example, as classified by Kabat (Kabat et al., supra). The Kabat numbering scheme is a widely adopted standard for numbering amino acid residues in antibody variable domains in a consistent manner, and, as noted elsewhere herein, is the preferred scheme for application in the present invention. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering system can be accounted for by the numbering system of Chothia et al. and / or revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence can be classified into canonical classes for which, among other things, appropriate chassis sequences can be identified (e.g., based on the desire to include various canonical structures in a library). The Kabat numbering of antibody amino acid sequences and structural considerations as explained by Chothia et al., supra, and their implications for interpreting canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0106] The CDR3 of the light chain and, in particular, the CDR3 of the heavy chain may be the most important determinant of antigen binding within the light and heavy chain variable regions. In some antigen-binding molecules, the heavy chain CDR3 is thought to constitute the main contact area between the antigen and the antibody. An in vitro selection scheme that varies only the CDR3 can be used to alter the binding properties of the antibody or to determine which residues contribute to antigen binding. Therefore, the CDR3 typically represents the greatest source of molecular diversity within the antibody binding site. For example, H3 can be as few as 2 amino acid residues or more than 26 amino acids.

[0107] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to the VH is usually referred to as CH1. The constant ("C") domains are not directly involved in antigen binding but exhibit various effector functions, such as antibody-dependent and cell-mediated cytotoxicity and complement activation. The Fc region of an antibody is contained within the heavy chain constant domain and can interact, for example, with Fc receptors located on the cell surface.

[0108] The sequences of antibody genes after construction and somatic mutation are highly diverse, and these diverse genes are 10 It is predicted that they encode different antibody molecules (Immunoglobulin Genes, 2 nd(ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. The sequences may be generated by in vivo rearrangement of V, D, and J segments of heavy chains and V and J segments of light chains. Alternatively, the sequences may be generated from cells responding, for example, to in vitro stimuli that cause rearrangement. Alternatively, some or all of the sequences may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, e.g., U.S. Pat. No. 5,565,332). A repertoire may include only one sequence or may include multiple sequences, including sequences in a genetically diverse collection.

[0109] The term "Fc portion" or "Fc monomer" in the context of the present invention refers to a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having the function of a CH3 domain of an immunoglobulin molecule. As the term "Fc monomer" clearly indicates, a polypeptide comprising these CH domains is a "polypeptide monomer." An Fc monomer may be a polypeptide comprising a fragment of an immunoglobulin constant region excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but retaining a functional portion of at least one CH2 domain and one CH3 domain, with the CH2 domain located amino-terminal to the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region, and a CH3 region, with the hinge region located amino-terminal to the CH2 domain. The hinge region of the present invention is expected to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by papain digestion of an immunoglobulin region (which, of course, results in a dimer of two Fc polypeptides). In another aspect of this definition, an Fc monomer can be a polypeptide region comprising a portion of the CH2 region and the CH3 region. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by pepsin digestion of an immunoglobulin molecule. In one embodiment, the polypeptide sequence of an Fc monomer is substantially similar to the Fc polypeptide sequences of the IgG1 Fc region, the IgG2 Fc region, the IgG3 Fc region, the IgG4 Fc region, the IgM Fc region, the IgA Fc region, the IgD Fc region, and the IgE Fc region (see, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because there is some variation among immunoglobulins, and simply for clarity, the Fc monomer refers to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM. As mentioned, the Fc monomer may also include a flexible hinge N-terminal to these domains.In the case of IgA and IgM, the Fc monomer may comprise a J chain. In the case of IgG, the Fc portion comprises immunoglobulin domains CH2 and CH3, and the hinge between the first two domains and CH2. Although the boundaries of the Fc portion may vary, an example of a human IgG heavy chain Fc portion comprising functional hinge, CH2, and CH3 domains can be defined, for example, according to Kabat, to include residues D231 (in the hinge domain—corresponding to D234 in Table 1 below) at the carboxyl terminus of the CH3 domain to P476, L476 (in the case of IgG4), respectively. Two Fc portions or Fc monomers fused to each other via a peptide linker define the third domain of the antigen-binding molecule of the present invention, which may also be defined as an scFc domain.

[0110] In one embodiment of the present invention, it is envisioned that the scFc domains disclosed herein (respectively, Fc monomers fused to each other) are comprised solely in the third domain of an antigen-binding molecule. Consistent with the present invention, IgG hinge regions can be identified by similarity using the Kabat numbering set forth in Table 1. Consistent with the above, it is envisioned that the hinge domains / regions of the present invention comprise amino acid residues corresponding to the stretch of IgG1 sequence from D234 to P243 according to the Kabat numbering. Similarly, it is envisioned that the hinge domains / regions of the present invention comprise or consist of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 182) (corresponding to the D234 to P243 section as shown in Table 1 below - variants of said sequence are also envisioned, provided that the hinge region still promotes dimerization). In a preferred embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of an antigen-binding molecule is eliminated by an N314X substitution (where X is any amino acid except Q). The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321). It is also envisioned that the third domain of the antigen-binding molecule of the present invention comprises or consists of, in amino to carboxyl order, DKTHTCPPCP (SEQ ID NO: 182) (i.e., hinge)-CH2-CH3-linker-DKTHTCPPCP (SEQ ID NO: 182) (i.e., hinge)-CH2-CH3. In a preferred embodiment, the peptide linker of the antigen-binding molecule is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 5 (e.g., 5, 6, 7, 8, etc., or more), with 6 being preferred ((Gly4Ser)6). The construct may further comprise the aforementioned substitution N314X, preferably N314G, and / or the additional substitutions V321C and R309C.In a preferred embodiment of the antigen-binding molecule defined herein above, it is envisaged that the second domain binds to an extracellular epitope of the human and / or Macaca CD3 epsilon chain.

[0111] [Table 2]

[0112] In further embodiments of the invention, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 183), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 184) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 185), and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 186). The IgG1 subtype hinge sequence may have the following sequence EPKSCDKTHTCPPCP (as shown in Table 1 and SEQ ID NO: 183). Thus, these core hinge regions are also envisaged in the context of the present invention.

[0113] The locations and sequences of the IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering set forth in Table 2.

[0114] [Table 3]

[0115] In one embodiment of the present invention, the amino acid residues highlighted in bold are deleted in the CH3 domain of the first or both Fc monomers.

[0116] The peptide linker by which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomer") are fused to one another preferably comprises at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferred that the linker comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, and most preferably exactly 30 amino acid residues. A preferred embodiment of such a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 5 (e.g., 6, 7, or 8). Preferably, the integer is 6 or 7, more preferably, the integer is 6.

[0117] When a linker is used to fuse a first domain with a second domain, or to fuse a first or second domain with a third domain, the linker is preferably of sufficient length and sequence to ensure that the first and second domains can retain their distinct binding specificities independently of each other. Regarding peptide linkers connecting at least two binding domains (or two variable domains) in an antigen-binding molecule of the present invention, these peptide linkers preferably contain only a few amino acid residues (e.g., 12 or fewer amino acid residues). Therefore, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Conceivable peptide linkers with fewer than 5 amino acids contain 4, 3, 2, or 1 amino acid, with Gly-rich linkers being preferred. A preferred embodiment of a peptide linker for fusing the first and second domains is shown in SEQ ID NO: 1. Preferred linker embodiments of the peptide linker for fusing the second and third domains are (Gly)4-linkers and G4-linkers, respectively.

[0118] A particularly preferred "single" amino acid in connection with one of the above "peptide linkers" is Gly. Thus, the peptide linker may consist of a single amino acid, Gly. In a preferred embodiment of the present invention, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 1 (e.g., 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1-12. Features of the peptide linkers that do not promote secondary structure are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Furthermore, peptide linkers that do not promote any secondary structure are preferred. The interconnection of the domains can be provided, for example, by genetic engineering as described in the Examples. Methods for preparing fused, operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are known in the art (e.g., WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0119] In a preferred embodiment of the present invention, the first and second domains form an antigen-binding molecule of a type selected from the group consisting of (scFv)2, scFv-single domain mAb, diabody, and oligomers of any of these types.

[0120] According to a particularly preferred embodiment, and as described in the accompanying Examples, the first and second domains of the antigen-binding molecules of the present invention are "bispecific single-chain antigen-binding molecules," more preferably bispecific "single-chain Fvs" (scFvs). Although the two domains of an Fv fragment, the VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, as previously described herein, which allows them to be produced as a single protein chain using recombinant techniques; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as complete or full-length antibodies. Thus, single-chain variable fragments (scFvs) are fusion proteins of the variable region of an immunoglobulin heavy chain (VH) and the variable region of an immunoglobulin light chain (VL), linked by a short linker peptide, typically about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can either connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0121] Bispecific single-chain antigen-binding molecules are known in the art and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loeffler, Blood, (2000), 95, 6, 2098-2103, Bruehl, Immunol., (2001), 166, 2420-2426, and Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single-chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778; Kontermann and Duebel (2010), supra; and Little (2009), supra) can be adapted to produce single-chain antigen-binding molecules that specifically recognize a target of choice.

[0122] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv having the format (scFv)2) can be designed by linking two scFv molecules (e.g., by a linker as described previously herein). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably referred to as bivalent (i.e., has two valencies for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably referred to as bispecific. This linking can be achieved by generating a single peptide chain with two VH and two VL regions, resulting in a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to generate scFv molecules with a linker peptide that is too short (e.g., about 5 amino acids) to allow the two variable regions to fold together, and then allow the scFvs to dimerize. This type of entity is known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0123] Consistent with the present invention, either the first domain, the second domain, or the first and second domains may comprise a single domain antibody, a variable domain of a single domain antibody, or at least the CDRs of a single domain antibody, respectively. Single domain antibodies comprise only one (monomeric) antibody variable domain that can selectively bind to a specific antigen independently of other V regions or domains. The first single domain antibodies were developed from heavy chain antibodies found in camels; these comprised V H It is called H fragment. Cartilaginous fish also have V fragment. NARHeavy chain antibodies (IgNARs) have the ability to generate single-domain antibodies, called fragments. An alternative approach is to split the dimeric variable domains of common immunoglobulins, for example, from humans or rodents, into monomers, thereby obtaining VH or VL as single-domain Abs. Currently, most research on single-domain antibodies is based on heavy chain variable regions, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies are called sdAbs, nanobodies, or single variable domain antibodies.

[0124] Therefore, (single domain mAb)2 is V H , V L , V H H and V NAR A monoclonal antigen-binding molecule is a monoclonal antigen-binding molecule composed of (at least) two single-domain monoclonal antibodies independently selected from the group comprising: (a) a single-domain antibody (a single-domain antibody) and (b) a single-domain antibody (a single-domain antibody). The linker is preferably in the form of a peptide linker. Similarly, an "scFv-single-domain mAb" is a monoclonal antigen-binding molecule composed of at least one single-domain antibody as described above and one scFv molecule as described above. Again, the linker is preferably in the form of a peptide linker.

[0125] The binding of an antigen-binding molecule to another given antigen-binding molecule can be measured by a competitive assay, such as competitive ELISA or cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Similar to an avidin-coated ELISA plate, each of these beads can be used as a substrate when reacting with biotinylated proteins, and assays can be performed on them. The antigen is coated onto the beads, and then pre-coated with the first antibody. A secondary antibody is added to confirm any further binding. Possible reading methods include flow cytometry.

[0126] T cells or T lymphocytes are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several subsets of T cells, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T cell receptors (TCRs) on their cell surface. The TCR is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules and is composed of two distinct protein chains. In 95% of T cells, the TCR is composed of an alpha (α) chain and a beta (β) chain. When the TCR binds to an antigen peptide and MHC (peptide / MHC complex), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, coreceptors, specialized adapter molecules, and activated or released transcription factors.

[0127] The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor-CD3 complex, generating activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are closely related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif, or ITAM, which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans. The most preferred epitope of CD3 epsilon is contained within amino acid residues 1–27 of the human CD3 epsilon extracellular domain. Antigen-binding molecules according to the present invention are typically and advantageously expected to exhibit less undesirable non-specific T cell activation in certain immunotherapies, which in turn reduces the risk of side effects.

[0128] Lysis of redirected target cells via recruitment of T cells by multispecific (at least bispecific) antigen-binding molecules involves the formation of a cytolytic synapse and the delivery of perforin and granzymes. The bound T cells are capable of continuous target cell lysis and are not subject to immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, e.g., WO 2007 / 042261).

[0129] The cytotoxicity mediated by the antigen-binding molecules of the present invention can be measured in various ways. Effector cells can be, for example, stimulated enriched (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin, or express or are transfected with the surface antigen of the macaque target cell bound by the first domain, the effector cells should also be of macaque origin, such as a macaque T cell line, e.g., 4119LnPx. The target cells should express the surface antigen of the target cell, e.g., (at least the extracellular domain of) the surface antigen of a human or macaque target cell. The target cells can be a cell line (e.g., CHO) stably or transiently transfected with the surface antigen of the target cell, e.g., the surface antigen of a human or macaque target cell. Alternatively, the target cells can be a cell line positively expressing the surface antigen of the natural target cell. Typically, EC 50 The value is expected to be lower for target cell lines that express high levels of target cell surface antigens on the cell surface. The effector to target cell (E:T) ratio is usually about 10:1, but this can vary. The cytotoxic activity of target cell surface antigen x CD3 bispecific antigen binding molecules is 51Cytotoxicity can be measured in a Cr release assay (incubation time of about 18 hours) or a cytotoxicity assay using FACS (incubation time of about 48 hours). The incubation time (cytotoxic response) of the assay can be varied. Other methods for measuring cytotoxicity are known to those skilled in the art and include MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques.

[0130] The cytotoxic activity mediated by the target cell surface antigen x CD3 bispecific antigen-binding molecule of the present invention is preferably measured in a cell-based cytotoxicity assay. 51 Cytotoxicity can be measured by EC 50 The EC value corresponds to the half-maximal effective concentration (the concentration of the antigen-binding molecule that induces a cytotoxic response halfway between the baseline and maximum). Preferably, the EC value is 0.05 for the target cell surface antigen x CD3 bispecific antigen-binding molecule. 50 The value is ≦5000 pM or ≦4000 pM, more preferably ≦3000 pM or ≦2000 pM, even more preferably ≦1000 pM or ≦500 pM, even more preferably ≦400 pM or ≦300 pM, even more preferably ≦200 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦20 pM or ≦10 pM, and most preferably ≦5 pM.

[0131] Given the above EC 50 Values ​​can be measured by various assays: stimulated / enriched CD8 + When T cells are used as effector cells, ECs are significantly higher than unstimulated PBMCs. 50 Those skilled in the art will recognize that values ​​can be expected to be lower. 50 Values ​​can be expected to be lower if the target cells express a large number of target cell surface antigens compared to rats with fewer target antigens. For example, stimulated / enriched human CD8 +When T cells are used as effector cells (and when cells transfected with the target cell surface antigen, such as CHO cells, or target cell surface antigen-positive human cell lines are used as target cells), the EC of the target cell surface antigen x CD3 bispecific antigen-binding molecule is 50 The value is preferably ≦1000 pM, more preferably ≦500 pM, even more preferably ≦250 pM, even more preferably ≦100 pM, even more preferably ≦50 pM, even more preferably ≦10 pM, and most preferably ≦5 pM. When human PBMCs are used as effector cells, the EC 50 The value is preferably ≦5000 pM or ≦4000 pM (particularly when the target cells are a target cell surface antigen-positive human cell line), more preferably ≦2000 pM (particularly when the target cells are cells transfected with the target cell surface antigen, such as CHO cells), more preferably ≦1000 pM or ≦500 pM, even more preferably ≦200 pM, even more preferably ≦150 pM, even more preferably ≦100 pM, and most preferably ≦50 pM. When a macaque T cell line, such as LnPx4119, is used as the effector cell and a cell line transfected with the macaque target cell surface antigen, such as CHO cells, is used as the target cell line, the EC of the target cell surface antigen × CD3 bispecific antigen-binding molecule is 50 The value is preferably ≦2000 pM or ≦1500 pM, more preferably ≦1000 pM or ≦500 pM, even more preferably ≦300 pM or ≦250 pM, even more preferably ≦100 pM, and most preferably ≦50 pM.

[0132] Preferably, the target cell surface antigen x CD3 bispecific antigen-binding molecules of the present invention do not induce / mediate lysis of target cell surface antigen-negative cells, such as CHO cells, or do not substantially induce / mediate lysis. The terms "do not induce lysis," "do not substantially induce lysis," "do not mediate lysis," or "do not substantially mediate lysis" mean that, when the lysis of a target cell surface antigen-positive human cell line is taken as 100%, the antigen-binding molecules of the present invention do not induce or mediate lysis of target cell surface antigen-negative cells in more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5%. This generally applies to concentrations of antigen-binding molecules up to 500 nM. Those skilled in the art will know how to measure cytolysis without further effort. Furthermore, specific instructions for measuring cytolysis are taught herein.

[0133] The difference in cytotoxic activity between the monomeric and dimeric isoforms of a particular target cell surface antigen x CD3 bispecific antigen-binding molecule is referred to as the "potency gap." This potency gap can be measured, for example, by comparing the EC 50 EC values ​​and dimeric forms 50 The potency gap of the target cell surface antigen x CD3 bispecific antigen-binding molecule of the present invention is preferably ≦5, more preferably ≦4, even more preferably ≦3, even more preferably ≦2, and most preferably ≦1.

[0134] The first and / or second (or any further) binding domains of the antigen-binding molecules of the present invention are preferably cross-species specific for members of the mammalian order Primates. Cross-species specific CD3 binding domains are described, for example, in WO 2008 / 119567. According to one embodiment, the first and / or second binding domains, in addition to binding to surface antigens on human target cells and human CD3, also bind to surface antigens / CD3 on target cells of primates, including, but not limited to, New World primates (such as common marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus)), Old World primates (such as baboons and macaques), gibbons, and non-human homininae.

[0135] In one embodiment of the antigen-binding molecule of the present invention, the first domain binds to a surface antigen of a human target cell and further binds to a surface antigen of a macaque target cell, such as a surface antigen of a cynomolgus monkey (Macaca fascicularis) target cell, more preferably a surface antigen of a macaque target cell expressed on the surface of a macaque cell. The affinity of the first binding domain for the surface antigen of a macaque target cell is preferably ≦15 nM, more preferably ≦10 nM, even more preferably ≦5 nM, even more preferably ≦1 nM, even more preferably ≦0.5 nM, even more preferably ≦0.1 nM, and most preferably ≦0.05 nM or even ≦0.01 nM.

[0136] Preferably, the binding affinity gap of the antigen-binding molecule according to the present invention for the surface antigen of macaque target cells to the surface antigen of human target cells [surface antigen of maca target cells:surface antigen of hu target cells] (determined, for example, by BiaCore or Scatchard analysis) is <100, preferably <20, more preferably <15, even more preferably <10, even more preferably <8, more preferably <6, and most preferably <2. The preferred range of the binding affinity gap of the antigen-binding molecule according to the present invention for the surface antigen of macaque target cells to the surface antigen of human target cells is 0.1 to 20, more preferably 0.2 to 10, even more preferably 0.3 to 6, even more preferably 0.5 to 3 or 0.5 to 2.5, and most preferably 0.5 to 2 or 0.6 to 2.

[0137] The second (binding) domain of the antigen-binding molecule of the present invention binds to human CD3 epsilon and / or Macaca CD3 epsilon. In a preferred embodiment, the second domain further binds to marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), or squirrel monkey (Saimiri sciureus) CD3 epsilon. Both marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) are New World primates belonging to the marmoset (Callitrichidae) family, while squirrel monkeys (Saimiri sciureus) are New World primates belonging to the capuchin (Cebidae) family.

[0138] In the antigen-binding molecules of the present invention, the second binding domain that binds to an extracellular epitope of human and / or Macaca CD3 is (a) CDR-L1 as set forth in SEQ ID NO: 27 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 28 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 29 of WO 2008 / 119567; (b) CDR-L1 as set forth in SEQ ID NO: 117 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 118 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 119 of WO 2008 / 119567; and (c) Preferably, the VL region comprises CDR-L1, CDR-L2 and CDR-L3 selected from CDR-L1 as set forth in SEQ ID NO: 153 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 154 of WO 2008 / 119567 and CDR-L3 as set forth in SEQ ID NO: 155 of WO 2008 / 119567.

[0139] In a further preferred embodiment of the antigen-binding molecule of the present invention, the second domain binding to an extracellular epitope of the human and / or Macaca CD3 epsilon chain comprises: (a) CDR-H1 as set forth in SEQ ID NO: 12 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 13 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 14 of WO 2008 / 119567; (b) CDR-H1 as set forth in SEQ ID NO: 30 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 31 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 32 of WO 2008 / 119567; (c) CDR-H1 as set forth in SEQ ID NO: 48 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 49 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 50 of WO 2008 / 119567; (d) CDR-H1 as set forth in SEQ ID NO: 66 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 67 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 68 of WO 2008 / 119567; (e) CDR-H1 as set forth in SEQ ID NO: 84 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 85 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 86 of WO 2008 / 119567; (f) CDR-H1 as set forth in SEQ ID NO: 102 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 103 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 104 of WO 2008 / 119567; (g) CDR-H1 as set forth in SEQ ID NO: 120 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 121 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 122 of WO 2008 / 119567; (h) CDR-H1 as set forth in SEQ ID NO: 138 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 139 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 140 of WO 2008 / 119567; (i) CDR-H1 as set forth in SEQ ID NO: 156 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 157 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 158 of WO 2008 / 119567; and (j) A VH region comprising CDR-H1, CDR-H2, and CDR-H3 selected from CDR-H1 set forth in SEQ ID NO: 174 of WO 2008 / 119567, CDR-H2 set forth in SEQ ID NO: 175 of WO 2008 / 119567, and CDR-H3 set forth in SEQ ID NO: 176 of WO 2008 / 119567.

[0140] In a preferred embodiment of the antigen-binding molecule of the present invention, the above three groups of VL CDRs are combined with the above ten groups of VH CDRs in the second binding domain to form a group (30) comprising CDR-L1 to 3 and CDR-H1 to 3, respectively.

[0141] In the antigen-binding molecules of the present invention, the second domain that binds to CD3 preferably comprises a VL region selected from the group consisting of the VL regions set forth in SEQ ID NOs: 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179, or 183 of WO 2008 / 119567, or as set forth in SEQ ID NO: 200.

[0142] It is also preferred that the second domain that binds to CD3 comprises a VH region selected from the group consisting of the VH regions as set forth in SEQ ID NOs: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008 / 119567 or as set forth in SEQ ID NO: 201.

[0143] More preferably, the antigen-binding molecule of the present invention comprises the following: (a) a VL region shown in SEQ ID NO: 17 or 21 of WO 2008 / 119567 and a VH region shown in SEQ ID NO: 15 or 19 of WO 2008 / 119567; (b) a VL region set forth in SEQ ID NO: 35 or 39 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 33 or 37 of WO 2008 / 119567; (c) a VL region as set forth in SEQ ID NO: 53 or 57 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 51 or 55 of WO 2008 / 119567; (d) a VL region set forth in SEQ ID NO: 71 or 75 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 69 or 73 of WO 2008 / 119567; (e) a VL region as set forth in SEQ ID NO: 89 or 93 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 87 or 91 of WO 2008 / 119567; (f) a VL region set forth in SEQ ID NO: 107 or 111 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 105 or 109 of WO 2008 / 119567; (g) a VL region set forth in SEQ ID NO: 125 or 129 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 123 or 127 of WO 2008 / 119567; (h) a VL region set forth in SEQ ID NO: 143 or 147 of WO 2008 / 119567 and a VH region set forth in SEQ ID NO: 141 or 145 of WO 2008 / 119567; (i) a VL region shown in SEQ ID NO: 161 or 165 of WO 2008 / 119567 and a VH region shown in SEQ ID NO: 159 or 163 of WO 2008 / 119567; (j) the VL region set forth in SEQ ID NO: 179 or 183 of WO 2008 / 119567 and the VH region set forth in SEQ ID NO: 177 or 181 of WO 2008 / 119567; and (k) the VL region shown in SEQ ID NO: 60 of WO 2022 / 096716 and the VH region shown in SEQ ID NO: 59 of WO 2022 / 096716 and a second domain that binds to CD3, comprising a VL region and a VH region selected from the group consisting of:

[0144] A CD3-binding second domain comprising a VL region as set forth in SEQ ID NO: 200 and a VH region as set forth in SEQ ID NO: 201 is also preferred in the context of the antigen-binding molecule of the present invention.

[0145] According to a preferred embodiment of the antigen-binding molecule of the present invention, the first domain and / or the second domain has the following format: a pair of a VH domain and a VL domain in the format of a single-chain antibody (scFv). The VH and VL domains are arranged in the order of VH-VL or VL-VH. It is preferred that the VH domain is arranged at the N-terminus of the linker sequence, and the VL domain is arranged at the C-terminus of the linker sequence.

[0146] A preferred embodiment of the above-mentioned antigen-binding molecule of the present invention is characterized by a CD3-binding second domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187 of WO2008 / 119567 or as set forth in SEQ ID NO: 202 (herein designated as I2C); or a second domain consisting of, in N- to C-terminus order, (i) a VH region set forth in SEQ ID NO: 59 of WO2022 / 096716, (ii) SEQ ID NO: 189, and (iii) a VL region set forth in SEQ ID NO: 60 of WO2022 / 096716 (herein designated as I2E). As discussed herein, without wishing to be bound by theory, stabilizers according to the present invention typically interact with and stabilize said second, i.e., CD3-binding domain despite structural differences between examples of second domains, e.g., I2C and I2E.

[0147] Covalent modifications of antigen-binding molecules are also included within the scope of the present invention, and are generally, but not necessarily, performed post-translationally. For example, some types of covalent modifications of antigen-binding molecules are introduced into the molecule by reacting specific amino acid residues of the antigen-binding molecule with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0148] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0149] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0150] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires that the reaction be performed under alkaline conditions. Furthermore, these reagents can react with lysine groups and the arginine epsilon-amino group.

[0151] The specific modification of tyrosyl residues may be undertaken to introduce spectral labels into tyrosyl residues, particularly by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively, to prepare labeled proteins for use in radioimmunoassay. 125 I or 131 The chloramine T method described above, in which tyrosyl residues are iodinated with I, is preferred.

[0152] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0153] Derivatization with bifunctional agents is useful for crosslinking the antigen-binding molecules of the present invention to water-insoluble support matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include homobifunctional imidoesters, including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates such as those described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are utilized for protein immobilization.

[0154] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, or they are deamidated under mildly acidic conditions, and both forms of these residues are within the scope of this invention.

[0155] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0156] Another type of covalent modification of an antigen-binding molecule within the scope of the present invention involves altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues, as discussed below) and the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0157] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0158] Addition of glycosylation sites to an antigen-binding molecule is conveniently accomplished by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). This modification can also be carried out by adding or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). Briefly, it is preferred to alter the amino acid sequence of an antigen-binding molecule by changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases to generate codons that translate into the desired amino acids.

[0159] Another means of increasing the number of carbohydrate moieties on an antigen-binding molecule is by chemically or enzymatically coupling glycosides to the protein. These procedures are advantageous in that they do not require the production of proteins in host cells with glycosylation capabilities for N-linked and O-linked glycosylation. Depending on the linkage mode used, sugars can be added to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0160] Removal of carbohydrate moieties present on the starting antigen-binding molecule can be accomplished chemically or enzymatically. Chemical deglycosylation involves exposing the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using various endoglycosidases and exoglycosidases, as described in Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by using tunicamycin, a compound described in Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin prevents protein-N-glycosidic bonds from forming.

[0161] Other modifications of antigen-binding molecules are also contemplated herein.For example, another type of covalent modification of antigen-binding molecules includes linking antigen-binding molecules to various non-proteinaceous polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.In addition, as known in the art, amino acid substitutions can be made at various positions within antigen-binding molecules to facilitate the addition of polymers such as PEG.

[0162] In some embodiments, covalent modification of the antigen-binding molecules of the present invention includes the addition of one or more labels. The labeling group may be attached to the antigen-binding molecule via a spacer arm of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in practicing the present invention. The term "label" or "labeling group" refers to any detectable label. Generally, labels are divided into various classes depending on the assay for detecting them, examples of which are listed below: a) Radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 Isotopic labels, which can be radioactive isotopes or heavy isotopes, such as I) b) Magnetic labels (e.g., magnetic particles) c) redox-active moieties d) optical dyes (including but not limited to chromophores, fluorophores and fluorophores), such as fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), chemiluminescent groups and fluorophores, which can be either "small molecule" fluorophores or proteinaceous fluorophores; e) Enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) f) Biotinylation group g) a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.); These include, but are not limited to:

[0163] "Fluorescent label" means any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland.

[0164] Suitable proteinaceous fluorescent labels include GFP from species of Renilla, Ptilosarcus, or Aequorea (Chalfie et al., 1994, Science 263:802-805), green fluorescent protein including EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8 th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607), and Renilla (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).

[0165] The antigen-binding molecules of the present invention may also contain additional domains, for example, useful for isolating the molecule or associated with a tailored pharmacokinetic profile of the molecule. Domains useful for isolating antigen-binding molecules may be selected from peptide motifs or auxiliary moieties that can be captured by isolation methods, such as isolation columns. Non-limiting examples of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and their variants (e.g., Strep II tags), and His tags. All of the antigen-binding molecules disclosed herein characterized by identified CDRs may contain a His tag domain, commonly known as a repeat of consecutive His residues, preferably five, more preferably six (hexahistidine) His residues, in the amino acid sequence of the molecule. The His tag may be located, for example, at the N-terminus or C-terminus of the antigen-binding molecule, preferably at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 199) is linked to the C-terminus of the antigen-binding molecule according to the present invention via a peptide bond. In addition, the PLGA-PEG-PLGA conjugate system may be combined with a polyhistidine tag for sustained release and improved pharmacokinetic profile.

[0166] Amino acid sequence modifications of the antigen-binding molecules described herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecule. Amino acid sequence variants of antigen-binding molecules are prepared by introducing appropriate nucleotide changes into the nucleic acid of the antigen-binding molecule or by synthesizing peptides. All of the amino acid sequence modifications described below should result in antigen-binding molecules that still retain the desired biological activity of the unmodified parent molecule (binding to surface antigens and CD3 on target cells).

[0167] The term "amino acid" or "amino acid residue" typically refers to an amino acid having an art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified according to the presence of a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0168] Amino acid modifications include, for example, deletion from, and / or insertion into, and / or substitution of, residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletion, insertion, and substitution can be performed to arrive at the final construct, provided that the final construct has the desired properties. Amino acid changes can also alter post-translational processes of the antigen-binding molecule, such as changing the number or location of glycosylation sites.

[0169] For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted, or deleted in each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be inserted, substituted, or deleted in each FR. Preferably, insertion of amino acid sequences into antigen-binding molecules includes intrasequence insertion of single or multiple amino acid residues as well as amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues. Corresponding modifications may also be made within the third domain of the antigen-binding molecules of the present invention. Insertional variants of the antigen-binding molecules of the present invention include fusion of an enzyme to the N- or C-terminus of the antigen-binding molecule or to a polypeptide.

[0170] The most important sites for substitutional mutagenesis include, but are not limited to, the CDRs of the heavy and / or light chains, particularly the hypervariable regions, although modifications of the FRs in the heavy and / or light chains are also contemplated. Substitutions are preferably conservative substitutions as described herein. Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDRs, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework regions (FRs). For example, if the CDR sequence contains 6 amino acids, it is contemplated that 1, 2, or 3 of these amino acids may be substituted. Similarly, if the CDR sequence contains 15 amino acids, it is contemplated that 1, 2, 3, 4, 5, or 6 of these amino acids may be substituted.

[0171] A useful method for identifying specific residues or regions of an antigen-binding molecule that are preferred positions for mutagenesis is the so-called "alanine scanning mutagenesis" method, as described in Cunningham and Wells in Science, 244:1081-1085 (1989). In this method, a residue or group of target residues in the antigen-binding molecule (e.g., charged residues such as arg, asp, his, lys, and glu) that affect the interaction of the amino acid with the epitope is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine).

[0172] Next, further or other variants are introduced into, or in place of, the substitution site to narrow down the range of amino acid positions that are functionally sensitive to the substitution. Thus, while the site or region for introducing an amino acid sequence mutation is predetermined, the nature of the mutation itself need not be. For example, to analyze or optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be performed at the target codon or region to screen for the optimal combination of desired activity in the expressed antigen-binding molecule variant. Techniques for making substitution mutations at predetermined sites within DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Mutants are screened using assays for antigen-binding activity, such as target cell surface antigen or CD3 binding.

[0173] Generally, when amino acids are substituted in one or more or all of the CDRs of the heavy and / or light chains, the resulting "substituted" sequence is preferably at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the "original" CDR sequence. This means that the degree of identity with the "substituted" sequence depends on the length of the CDR. For example, a CDR having five amino acids and having at least one substituted amino acid is preferably 80% identical to the substituted sequence. Thus, the CDRs of an antigen-binding molecule may have various degrees of identity with their substituted sequences; for example, CDRL1 may have 80% identity, while CDRL3 may have 90% identity.

[0174] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 3 below) is contemplated, as long as the antigen-binding molecule retains the ability to bind to a surface antigen of a target cell via the first domain and to bind to CD3 or CD3 epsilon via the second domain, and / or its CDRs have identity to the substituted sequence (at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, particularly preferably 90% or 95% identical to the "original" CDR sequence).

[0175] Conservative substitutions are shown under the heading of "preferred substitutions" in Table 3. If such substitutions result in altered biological activity, then substantial changes, such as those referred to in Table 3 as "exemplary substitutions" or further described below with reference to classes of amino acids, may be introduced and the products screened for desired characteristics.

[0176] [Table 4]

[0177] Substantial alterations in the biological properties of the antigen-binding molecules of the present invention can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the substituted region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk. Naturally occurring residues are classified into the following groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophobic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0178] Non-conservative substitutions involve exchanging a member of one of these classes for another. To avoid aberrant crosslinking, any cysteine ​​residue not involved in maintaining the proper conformation of the antigen-binding molecule can be substituted, generally with serine, to improve the oxidative stability of the molecule. Conversely, adding cysteine ​​bond(s) to an antibody can improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

[0179] For amino acid sequences, sequence identity and / or similarity may be determined by standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the BestFit sequence program described in Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by visual inspection. Preferably, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0180] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360. This method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0181] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, and word threshold (T) = 1. The HSP S and HSP S2 parameters are dynamic values ​​that are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched, however the values ​​can be adjusted to increase sensitivity.

[0182] An additional useful algorithm is Gapped BLAST, as reported in Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9, the cost of a gap length k is 10+k for a two-hit method that results in an ungapped extension, Xu is set to 16, and Xg is set to 40 in the database search stage and 67 in the output stage of the algorithm. Gapped alignments are given scores corresponding to approximately 22 bits.

[0183] Generally, the amino acid homology, similarity, or identity between individual variant CDR or VH / VL sequences is at least 60% relative to the sequences set forth herein, and more typically, the homology or identity is at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and approximately 100%. Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequences of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of the antigen-binding molecule. A specific method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with the overlap span and overlap fraction set to 1 and 0.125, respectively.

[0184] Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding each variant CDR or VH / VL sequence and the nucleotide sequences set forth herein will be at least 60%, more typically the homology or identity will be at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and preferably increases to nearly 100%. Thus, a "variant CDR" or "variant VH / VL region" is one that has a particular homology, similarity, or identity to a parent CDR / VH / VL of the invention and shares biological function, including but not limited to, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0185] In one embodiment, the percentage of identity of the antigen-binding molecule according to the present invention to human germline gene products is ≥70% or ≥75%, more preferably ≥80% or ≥85%, even more preferably ≥90%, and most preferably ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, or even ≥96%. Identity to human antibody germline gene products is considered to be an important feature for reducing the risk of a therapeutic protein inducing an immune response against the drug in patients undergoing treatment. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36 (2005) 3-10) demonstrated that reducing the non-human portion of a drug-antigen-binding molecule reduces the risk of inducing anti-drug antibodies in patients undergoing treatment. Comparison of a large number of clinically evaluated antibody drugs and their respective immunogenicity data has shown that humanized antibody V regions tend to result in proteins that are less immunogenic (5.1% of patients on average) than antibodies with unmodified non-human V regions (23.59% of patients on average). Therefore, for V region-based protein therapeutics in the form of antigen-binding molecules, a high degree of identity to human sequences is desirable. To determine this germline identity, the V region of the VL can be aligned with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the percentage of identical amino acid residues can be calculated by dividing the number of identical amino acid residues by the total number of amino acid residues in the VL. A similar method can be used for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), with the exception that the VH CDR3 may be excluded due to its high diversity and the lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase sequence identity to human antibody germline genes.

[0186] In a further embodiment, the bispecific antigen-binding molecules of the present invention exhibit high monomer yields under standard research-scale conditions (e.g., a standard two-step purification process). Preferably, the monomer yield of the antigen-binding molecules according to the present invention is ≧0.25 mg per L of supernatant, more preferably ≧0.5 mg per L, even more preferably ≧1 mg per L, and most preferably ≧3 mg per L of supernatant.

[0187] Similarly, the yield of dimeric antigen-binding molecule isoforms, and therefore the percentage of monomers in the antigen-binding molecule (i.e., monomer / (monomer+dimer)), can be determined. The productivity of monomeric and dimeric antigen-binding molecules and the calculated percentage of monomers can be obtained, for example, by an SEC purification step of culture supernatant obtained from standardized research-scale production in roller bottles. In one embodiment, the percentage of monomers in the antigen-binding molecule is ≧80%, more preferably ≧85%, even more preferably ≧90%, and most preferably ≧95%.

[0188] In one embodiment, the antigen-binding molecule preferably has a plasma stability (ratio of EC50 in the presence of plasma to EC50 in the absence of plasma) of ≦5 or ≦4, more preferably ≦3.5 or ≦3, even more preferably ≦2.5 or ≦2, and most preferably ≦1.5 or ≦1. The plasma stability of the antigen-binding molecule is determined by incubating the construct in human plasma at 37° C. for 24 hours, followed by 51Validation can be performed by determining the EC50 in a chromium release cytotoxicity assay. Effector cells in the cytotoxicity assay can be stimulated enriched human CD8-positive T cells. Target cells can be, for example, CHO cells transfected with a surface antigen of a human target cell. The effector cell to target cell (E:T) ratio can be selected as 10:1. The human plasma pool used for this purpose is derived from blood collected from healthy donors using an EDTA-coated syringe. Cellular components are removed by centrifugation, and the upper plasma phase is collected and subsequently pooled. As a control, the antigen-binding molecule is diluted in RPMI-1640 medium immediately before the cytotoxicity assay. Plasma stability is calculated as the ratio of EC50 (after plasma incubation) to EC50 (control).

[0189] It is further preferred that the monomer-to-dimer conversion rate of the antigen-binding molecules of the present invention is low. The conversion rate can be measured under different conditions and analyzed by high-performance size exclusion chromatography. For example, the monomeric isoform of the antigen-binding molecule can be incubated in an incubator at 37°C for 7 days at a concentration of, for example, 100 μg / ml or 250 μg / ml. Under these conditions, the antigen-binding molecules of the present invention preferably exhibit a dimer percentage of ≦5%, more preferably ≦4%, even more preferably ≦3%, even more preferably ≦2.5%, even more preferably ≦2%, even more preferably ≦1.5%, and most preferably ≦1%, ≦0.5%, or even 0%.

[0190] Furthermore, it is preferred that the bispecific antigen-binding molecules of the present invention exhibit a very low dimer conversion rate after several freeze / thaw cycles. For example, antigen-binding molecule monomers are adjusted to a concentration of 250 μg / ml in a common formulation buffer and subjected to three freeze / thaw cycles (freezing at -80°C for 30 minutes, followed by thawing at room temperature for 30 minutes), followed by high-speed SEC to determine the percentage of the initial monomeric antigen-binding molecules converted to dimeric antigen-binding molecules. Preferably, the percentage of dimers in the bispecific antigen-binding molecules is, for example, ≦5% after three freeze / thaw cycles, more preferably ≦4%, even more preferably ≦3%, even more preferably ≦2.5%, even more preferably ≦2%, even more preferably ≦1.5%, and most preferably ≦1% or even ≦0.5%.

[0191] The bispecific antigen-binding molecules of the present invention preferably exhibit good thermal stability with an aggregation temperature of ≥ 45°C or ≥ 50°C, more preferably ≥ 52°C or ≥ 54°C, even more preferably ≥ 56°C or ≥ 57°C, and most preferably ≥ 58°C or ≥ 59°C. In terms of the aggregation temperature of an antibody, the thermal stability parameter can be determined as follows: an antibody solution at a concentration of 250 μg / ml is transferred to a single-use cuvette and placed in a dynamic light scattering (DLS) instrument. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min, while the measured radius is constantly being acquired. The increase in radius indicates protein melting and aggregation and is used to calculate the aggregation temperature of the antibody.

[0192] Alternatively, melting temperature curves can be measured by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of antigen-binding molecules. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. The energy uptake of samples containing antigen-binding molecules is recorded from 20°C to 90°C and compared to samples containing formulation buffer only. The antigen-binding molecules are adjusted to a final concentration of, for example, 250 μg / ml in SEC running buffer. To record each melting curve, the temperature of the entire sample is increased stepwise. At each temperature T, the energy uptake of the sample and formulation buffer standard is recorded. The difference in energy uptake Cp (kcal / mole / °C) between the sample and the standard is plotted against each temperature. The melting temperature is defined as the temperature at the first maximum energy uptake.

[0193] The target cell surface antigen x CD3 bispecific antigen-binding molecules of the present invention are also expected to have a turbidity (measured by OD340 after concentrating purified monomeric antigen-binding molecules to 2.5 mg / ml and incubating overnight) of ≦0.2, preferably ≦0.15, more preferably ≦0.12, even more preferably ≦0.1, and most preferably ≦0.08.

[0194] It is further envisaged that the bispecific antigen-binding molecules of the invention will exhibit therapeutic efficacy or anti-tumour activity, which can be assessed, for example, in the tests disclosed in the Examples below in advanced stage human tumour xenograft models.

[0195] Those skilled in the art know how to modify or adapt certain parameters of this test, such as the number of tumor cells injected, the injection site, the number of human T cells implanted, the amount of bispecific antigen-binding molecule administered, and the schedule, while still obtaining meaningful and reproducible results. Preferably, the tumor growth inhibition T / C [%] is ≦70 or ≦60, more preferably ≦50 or ≦40, even more preferably ≦30 or ≦20, and most preferably ≦10 or ≦5, or even ≦2.5.

[0196] In a preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding molecule is a single-chain antigen-binding molecule.

[0197] In a preferred embodiment of the antigen-binding molecule of the present invention, the third domain comprises, in order from amino to carboxyl, hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0198] Also, in one embodiment of the present invention, the CH2 domain of one or preferably each (both) polypeptide monomer of the third domain contains an intradomain cysteine ​​disulfide bridge. As known in the art, the term "cysteine ​​disulfide bridge" refers to a functional group having the general structure RSSR. This linkage, also called an S-S bond or disulfide bridge, is obtained by coupling of two thiol groups of cysteine ​​residues. With respect to the antigen-binding molecules of the present invention, it is particularly preferred that the cysteines that form the cysteine ​​disulfide bridge in the mature antigen-binding molecule are introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0199] In one embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of the glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid other than Q. The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0200] For example, the preferred features of the antigen-binding molecules of the present invention compared with bispecific hetero-Fc antigen-binding molecules known in the art ( FIG. 1 b) may be particularly related to the introduction of the above-mentioned modifications in the CH2 domain. Thus, with respect to the constructs of the present invention, it is preferred that the CH2 domain in the third domain of the antigen-binding molecules of the present invention comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321, and / or the glycosylation site at Kabat position 314 has been removed by N314X substitution, preferably N314G substitution, as described above.

[0201] In a further preferred embodiment of the present invention, the CH2 domain within the third domain of the antigen-binding molecule of the present invention contains intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 has been eliminated by an N314G substitution.

[0202] In one embodiment, the present invention provides an antigen-binding molecule comprising: (182) The first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain; Antigen-binding molecules are provided.

[0203] Thus, the first domain and the second domain may each be a binding domain comprising two antibody variable domains, such as a VH domain and a VL domain. Examples of such binding domains comprising two antibody variable domains as described hereinabove include, for example, the Fv fragment, scFv fragment, or Fab fragment described hereinabove. Alternatively, either or both of the binding domains may comprise only a single variable domain. Examples of such single domain binding domains as described hereinabove include, for example, nanobodies or single variable domain antibodies comprising only one variable domain, which may be a VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0204] In a preferred embodiment of the antigen-binding molecule of the present invention, the first and second domains are fused to the third domain via a peptide linker. Preferred peptide linkers are described hereinabove and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 187), or a polymer thereof, i.e., (Gly4Ser)x (where x is an integer equal to or greater than 1 (e.g., 2 or 3)). A particularly preferred linker for fusing the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0205] In a preferred embodiment, the antigen-binding molecule of the present invention has, in order from amino to carboxyl, (a) First domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 189; (c) second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) the first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) a second polypeptide monomer of the third domain It is also assumed that the

[0206] In one embodiment of the present invention, the surface antigen of the target cell bound by the first domain is a tumor antigen, an antigen specific to an immune disorder, or a viral antigen. As used herein, the term "tumor antigen" can be understood as those antigens presented on tumor cells. These antigens can be presented on the cell surface with an extracellular portion, and often have both transmembrane and cytoplasmic portions of the molecule. These antigens may be presented only by tumor cells and never by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More common antigens are antigens presented by both tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. Non-limiting examples of tumor antigens used herein are CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, BCMA, and PSMA.

[0207] Further target cell surface antigens specific for immunological disorders in the context of the present invention include, for example, TL1A and TNF-alpha. Preferably, said targets are addressed by the bispecific antigen-binding molecules of the present invention, which are preferably full-length antibodies. In a highly preferred embodiment, the antibodies of the present invention are heterodimeric IgG antibodies.

[0208] In a preferred embodiment of the antigen-binding molecule of the present invention, the tumor antigen is preferably selected from the group consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, MUC17, CLDN18.2, CDH3, CD70, BCMA, and PSMA.

[0209] In one embodiment of the present invention, the antigen-binding molecule has, in order from amino to carboxyl, (a) SEQ ID NOs: 7, 8, 17, 27, 28, 37, 38, 39, 40, 41, 48, 49, 50, 51, 52, 59, 60, 61, 62, 63, 64, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93, 100, 101, 102, 103, 104, 113, 114, 121, 122, 123, 124, 125, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 80, 81, 89, 90, 91, 92, 93, 100, 101, 102, 103, 10 a first domain having an amino acid sequence selected from the group consisting of 32, 133, 134, 135, 136, 143, 144, 145, 146, 147, 148, 149, 150, 151, 158, 159, 160, 161, 162, 163, 164, 165, 166, 173, 174, 175, 176, 177, 178, 179, 180, 181, 223, 235, and 246; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 189; (c) a second domain having an amino acid sequence selected from the group consisting of 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187 of WO 2008 / 119567 or SEQ ID NO: 202; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197, and 198; (e) a first polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24 of WO 2017 / 134140; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) a second polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17 to 24 of WO 2017 / 134140;

[0210] In one embodiment, the bispecific antigen-binding molecule of the present invention is characterized in that it has an amino acid sequence selected from the group consisting of the following, derived from a surface antigen of each target cell: (a) SEQ ID NOs: 39 to 41; CD33 (b) SEQ ID NOs: 48 to 52, respectively; EGFRvIII (c) each of SEQ ID NOs: 59 to 64; MSLN (d) SEQ ID NOs: 71 to 82; CDH19 (e) each of SEQ ID NOs: 100 to 104; DLL3 (f) SEQ ID NOs: 7, 8, 17, 113 and 114; CD19 (g) each of SEQ ID NOs: 89 to 93; FLT3 (h) each of SEQ ID NOs: 121 to 125; CDH3 (i) each of SEQ ID NOs: 132 to 136; BCMA (j) SEQ ID NOs: 143 to 151, 158 to 166, and 173 to 181, respectively; PSMA (k) SEQ ID NO: 213; MUC17 (l) SEQ ID NOs: 225 and 237, respectively; CLDN18.2 (m) SEQ ID NO: 248; CD70 and (n) SEQ ID NOs: 258 and 261; CLDN 6

[0211] The present invention further provides polynucleotides / nucleic acid molecules encoding the antigen-binding molecules of the present invention. Polynucleotides are biopolymers composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (e.g., cDNA) and RNA (e.g., mRNA) are examples of polynucleotides with different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. Nucleic acid molecules or polynucleotides may be double-stranded or single-stranded, linear or circular. The nucleic acid molecule or polynucleotide is preferably contained in a vector, and the vector is preferably contained in a host cell. The host cell is capable of expressing the antigen-binding molecule after, for example, being transformed or transfected with the vector or polynucleotide of the present invention. To this end, the polynucleotide or nucleic acid molecule is operably linked to a regulatory sequence.

[0212] The genetic code is a set of rules for translating information encoded in genetic material (nucleic acids) into proteins. Biological decoding in living cells is accomplished by ribosomes, which carry amino acids and use tRNA molecules, which read the mRNA three nucleotides at a time, to link amino acids in the order specified by the mRNA. This code defines how triplet nucleotide sequences, called codons, specify the next amino acid to be added during protein synthesis. With some exceptions, each triplet codon in a nucleic acid sequence specifies a single amino acid. Because the majority of genes are coded using the exact same code, this particular code is often referred to as the canonical or standard genetic code. While the genetic code determines the protein sequence of a given coding region, other genomic regions can influence when and where these proteins are produced.

[0213] The present invention further provides vectors comprising the polynucleotides / nucleic acid molecules of the present invention. A vector is a nucleic acid molecule used as a vehicle for introducing (foreign) genetic material into cells. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Modified vectors generally contain an origin of replication, a multiple cloning site, and a selection marker. The vector itself is generally a nucleotide sequence (generally a DNA sequence) composed of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may include additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express transgenes in target cells and generally contain regulatory sequences.

[0214] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0215] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. This linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

[0216] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves creating transient pores or "holes" in the cell membrane to allow uptake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell squeezing, or by mixing cationic lipids with materials to generate liposomes that fuse with the cell membrane and deposit the internal cargo.

[0217] The term "transformation" is used to describe the non-viral introduction of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells, including plant cells. Transformation is thus the genetic modification of bacteria or non-animal eukaryotic cells resulting from the direct uptake of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane and subsequent integration. Transformation can occur by artificial means. For transformation to occur, cells or bacteria must be in a competent state in which transformation can occur as a timed response to environmental conditions such as starvation and cell density.

[0218] The present invention further provides host cells transformed or transfected with the polynucleotide / nucleic acid molecule or vector of the present invention. As used herein, the term "host cell" or "recipient cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antigen-binding molecules of the present invention; and / or the antigen-binding molecules themselves. Introduction of each substance into a cell is accomplished by transformation, transfection, or the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Because certain modifications may occur in successive generations due to spontaneous, accidental, or deliberate mutation, or due to environmental influences, such progeny may not actually be completely identical (morphologically or in terms of genomic or total DNA complement) to the parent cell, but still be within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells.

[0219] The antigen-binding molecules of the present invention can be produced in bacteria. After expression, the antigen-binding molecules of the present invention can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, by affinity chromatography and / or size exclusion chromatography. Final purification can be performed, for example, similar to the process used to purify antibodies expressed in CHO cells.

[0220] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the antigen-binding molecules of the present invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 24178), K. galactosyltransferase ... 36906, K. thermotolerans, and K. marxianus; Yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, for example Schwanniomyces occidentalis occidentalis); and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0221] Suitable host cells for expressing the glycosylated antigen-binding molecules of the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many strains and variants of baculovirus and corresponding permissive insect host cells derived from hosts (e.g., Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori) have been identified. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein according to the invention, particularly for transfection of Spodoptera frugiperda cells.

[0222] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell culture are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen et al. (1992) Bio / Technology 10:790-794; Artsaenko et al. (1995) The Plant J 8:745-750; and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0223] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0224] In a further embodiment, the present invention provides a process for producing an antigen-binding molecule of the present invention, the process comprising culturing a host cell of the present invention under conditions allowing expression of the antigen-binding molecule of the present invention, and recovering the produced antigen-binding molecule from the culture.

[0225] As used herein, the term "culturing" refers to the maintenance, differentiation, growth, proliferation, and / or propagation of cells in vitro under suitable conditions in a culture medium. The term "expression" includes any step involved in producing an antigen-binding molecule of the present invention, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0226] When using recombinant techniques, antigen-binding molecules can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. When antigen-binding molecules are produced intracellularly, the first step is to remove particulate host cell debris or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of Escherichia coli (E. coli). Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. When antibodies are secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0227] Antigen-binding molecules of the present invention prepared from host cells can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Depending on the antibody recovered, other protein purification techniques, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing SDS-PAGE, and ammonium sulfate precipitation, are also available. When the antigen-binding molecule of the present invention contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification.

[0228] A preferred purification technique is affinity chromatography. The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0229] The present invention also provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention or antigen-binding molecules produced by the methods of the present invention. In the pharmaceutical compositions of the present invention, the homogeneity of the antigen-binding molecules is preferably ≥80%, more preferably ≥81%, ≥82%, ≥83%, ≥84%, or ≥85%, even more preferably ≥86%, ≥87%, ≥88%, ≥89%, or ≥90%, even more preferably ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%, and most preferably ≥96%, ≥97%, ≥98%, or ≥99%.

[0230] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a patient (preferably a human patient). Particularly preferred pharmaceutical compositions of the present invention comprise one or more antigen-binding molecules of the present invention, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Preferably, components of an acceptable composition are non-toxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0231] The composition of the present invention may contain a pharmaceutically acceptable carrier. Generally, as used herein, "pharmaceutically acceptable carrier" refers to any aqueous or non-aqueous solution, sterile solution, solvent, buffer solution, such as phosphate-buffered saline (PBS) solution, water, suspension, emulsion such as oil / water emulsion, various types of wetting agents, liposomes, dispersion media and coatings that are suitable for pharmaceutical administration, especially parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known conventional methods.

[0232] Certain embodiments provide pharmaceutical compositions comprising an antigen-binding molecule of the invention and one or more excipients, such as those exemplarily described in this section and elsewhere herein. Excipients can be used in the invention for a wide range of purposes, such as adjusting the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or processes of the invention to improve efficacy and / or stabilize such formulations, and for example, against deterioration and damage due to stresses that occur during manufacturing, transportation, storage, preparation before use, administration, and thereafter.

[0233] In certain embodiments, pharmaceutical compositions may contain formulating materials intended to modify, sustain, or protect, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulating materials may include, but are not limited to, the following: amino acids, such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine, charged amino acids, such as lysine, lysine acetate, arginine, glutamate, and / or histidine; Antibacterial and antifungal agents Antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium bisulfite; buffers, buffer systems and buffering agents used to maintain compositions at physiological pH or slightly lower; examples of buffers are borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids, succinate, phosphate, and histidine; for example, Tris buffer with a pH of about 7.0 to 8.5; non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; Aqueous carriers such as water, alcoholic / aqueous solutions, emulsions, or suspensions, e.g., saline and buffered media; · Biodegradable polymers such as polyester; Bulking agents such as mannitol or glycine; · Chelating agents such as ethylenediaminetetraacetic acid (EDTA); ·Isotonic and absorption retarding agents; Complexing agents, such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin · Bulking agents; Monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrins); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol; (low molecular weight) proteins, polypeptides or proteinaceous carriers, such as human or bovine serum albumin, gelatin or immunoglobulins, preferably of human origin; · coloring and flavoring agents; Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate · Diluents; ·emulsifier; · Hydrophilic polymers such as polyvinylpyrrolidone; · Salt-forming counterions such as sodium; Preservatives, such as antimicrobials, antioxidants, chelating agents, and inert gases; examples include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide; · Metal complexes such as Zn-protein complexes; Solvents and cosolvents (such as glycine, propylene glycol or polyethylene glycol); sugars and sugar alcohols, for example, trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myo-inositose, galactose, lactitol, ribitol, myo-inositol, galactitol, glycerol, cyclitols (for example, inositol), polyethylene glycol; and polyhydric sugar alcohols; · suspending agents; surfactants or wetting agents, such as pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyloxapol; surfactants may be detergents, preferably with a molecular weight of >1.2 KD, and / or polyethers, preferably with a molecular weight of >3 KD; non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80, and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000, and PEG 5000; ·Stability enhancers such as sucrose or sorbitol; isotonicity enhancing agents, for example alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol; Parenteral delivery vehicles, such as sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils; Intravenous delivery vehicles, such as fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0234] It is contemplated that it will be apparent to one skilled in the art that different components of a pharmaceutical composition (e.g., the components listed above) may have different effects, for example, amino acids may act as buffers, stabilizers and / or antioxidants, mannitol may act as a bulking agent and / or tonicity enhancer, sodium chloride may act as a delivery vehicle and / or tonicity enhancer, etc.

[0235] It is envisioned that the compositions of the present invention may contain, in addition to the polypeptides of the present invention defined herein, additional biologically active agents depending on the intended use of the composition. Such agents may be drugs known in the art, such as drugs acting on the gastrointestinal system, drugs acting as cytostatics, drugs preventing hyperuricemia, drugs inhibiting immune responses (e.g., corticosteroids), drugs regulating inflammatory responses, drugs acting on the circulatory system, and / or cytokines. It is also envisioned that the antigen-binding molecules of the present invention may be used in combination therapy, i.e., in combination with another anticancer drug.

[0236] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the antigen-binding molecules of the compositions of the invention may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having the desired degree of purity with optional formulations (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Furthermore, in certain embodiments, the antigen-binding molecules of the present invention may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0237] When parenteral administration is intended, the therapeutic composition used in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antigen-binding molecule of the present invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antigen-binding molecule of the present invention is formulated as a sterile isotonic solution and appropriately stored. In certain embodiments, the preparation may include formulating the desired molecule with an agent that can be delivered via depot injection and that can provide sustained or sustained release of the product, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid, which has the effect of enhancing duration in the circulation, may also be used. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen-binding molecule.

[0238] Additional pharmaceutical compositions, including formulations containing the antigen-binding molecules of the present invention in sustained- or controlled-delivery / release formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Application PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Sustained-release formulations may include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and EP 058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyrate (EP 133,988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Applications 036,676; 088,046 and 143,949, which are incorporated by reference.

[0239] Antigen-binding molecules may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or by interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16 th edition, Oslo, A. Ed. (1980).

[0240] Pharmaceutical compositions used for in vivo administration are generally provided as sterile preparations.Sterilization can be achieved by filtration through sterile filtration membranes.When the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution.Compositions for parenteral administration can be stored in lyophilized form or as a solution.Parenteral compositions are generally filled into a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0241] Another aspect of the present invention includes the self-buffering antigen-binding molecules of the formulations of the present invention, which can be used as pharmaceutical compositions, as described in International Patent Application Publication No. WO 06138181 A2 (PCT / US2006 / 022599). Various reviews are available regarding protein stabilization and formulation materials and methods useful in this regard, such as Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution" in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002); and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13:159-75 (2002), particularly with respect to protein pharmaceuticals and processes for veterinary and / or human medical use, and reference is made in particular to the sections regarding the same excipients and processes as in the self-buffering protein formulations of the present invention.

[0242] Salts may be used in certain embodiments of the present invention, for example, to adjust the ionic strength and / or tonicity of a formulation and / or to improve the solubility and / or physical stability of a protein or other component of a composition according to the present invention. As is well known, ions can stabilize proteins in their native state by binding to charged residues on the protein's surface and by shielding charged and polar groups in the protein, reducing the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the protein's denatured peptide bond (--CONH). Furthermore, ionic interactions between charged and polar groups in proteins can reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0243] Ionic species vary significantly in their effects on proteins. Several taxonomic rankings of ions and their effects on proteins have been developed and can be used in formulating pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes according to their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate ("salting out") proteins from solution. Chaotropes are commonly used to denature and / or solubilize ("salting in") proteins. In the Hofmeister series, the position of an ion is defined by its relative effect on "salting in" and "salting out."

[0244] Free amino acids can be used in the antigen-binding molecules of the present invention's formulations according to various embodiments as bulking agents, stabilizers, and antioxidants, as well as for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful for ensuring proper cake structure and properties during lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0245] Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as glycerol and propylene glycol, and, for purposes of this discussion, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the tonicity of formulations. Among polyols useful in select embodiments of the present invention, mannitol is commonly used to ensure cake structural stability in lyophilized formulations. Mannitol ensures cake structural stability. Mannitol is commonly used in conjunction with a lyoprotectant, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting tonicity and as stabilizers for protection against freeze-thaw stress during transportation or bulk preparation during the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not included among the preferred polyols used in the present invention. In addition, sugars that form such reactive species, such as sucrose, are also not included among the preferred polyols of the present invention, because they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a cryoprotectant, and in this regard can be used in the present invention.

[0246] Embodiments of the antigen-binding molecule formulations of the present invention further comprise a surfactant. Protein molecules can be susceptible to adsorption onto surfaces, as well as denaturation and resulting aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These adverse interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical agitation, such as that encountered during product transportation and handling. Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. Surfactants useful in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188. Surfactants are also commonly used to control protein conformational stability. The use of surfactants in this regard is protein-specific, as any given surfactant typically stabilizes some proteins and destabilizes others.

[0247] Polysorbates are prone to oxidative degradation and often contain sufficient peroxide content when supplied to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should be used with caution and, when used, at the lowest effective concentration. In this regard, polysorbates exemplify the general principle that excipients should be used at their lowest effective concentration.

[0248] Embodiments of the antigen-binding molecule formulations of the present invention further comprise one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, particularly useful antioxidants include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants used in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the shelf life of the product. In this regard, a preferred antioxidant according to the present invention is EDTA. Antioxidants can damage proteins. For example, reducing agents (e.g., glutathione) can, in particular, disrupt intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected, inter alia, to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation.

[0249] The formulations of the present invention may contain metal ions, which are protein cofactors and are required to form protein coordination complexes, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins. Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 , Mn +2 , and Zn +2 may destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, and factor VIII is +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2The aggregation can be destabilized by Al +3 It may be increased by ions.

[0250] Embodiments of the antigen-binding molecule formulations of the present invention further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations involving multiple withdrawals from the same container. Their primary function is to inhibit microbial growth and ensure product sterility over the drug product's shelf life or usage period. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use with small molecule parenteral drugs, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have only been formulated for single-use. However, the possibility of multi-dose formulations offers added benefits of patient convenience and increased marketability. Human growth hormone (hGH) is a good example, where the development of a preserved formulation led to the proposal and commercialization of a more convenient multi-use injection pen. At least four such pen devices containing preserved hGH formulations are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol. During the formulation and development of preserved dosage forms, several aspects need to be considered. The effective preservative concentration in the drug product needs to be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that confer antimicrobial efficacy without compromising protein stability.

[0251] As expected, developing a liquid formulation containing a preservative is more challenging than a lyophilized formulation. Lyophilized products can be lyophilized without the preservative and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing the associated stability risks. For liquid formulations, the preservative's effectiveness and stability must be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that preservative effectiveness should be demonstrated in the final formulation containing the active drug and all excipient components.

[0252] The antigen-binding molecules disclosed herein may also be formulated as liposomes. Liposomes are small vesicles composed of various types of lipids, phospholipids, and / or surfactants, useful for drug delivery to mammals. Liposome components are usually organized in a bilayer structure, similar to the lipid structure of biological membranes. Liposomes containing antigen-binding molecules can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 97 / 38731. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antigen-binding molecules of the present invention can be conjugated to liposomes as described by Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide exchange reaction. Optionally, a chemotherapeutic agent is contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19)1484 (1989).

[0253] After the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0254] The biological activity of the pharmaceutical compositions defined herein is also described, for example, in the Examples below, in WO 99 / 54440, or in the publication by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to the response to therapy with the pharmaceutical composition of the present invention, for example, using standardized NCI response criteria. The success or in vivo efficacy of therapy using the pharmaceutical composition of the present invention refers to the effectiveness of the composition for its intended purpose, i.e., its ability to cause its desired effect, i.e., the depletion of pathological cells (e.g., tumor cells). In vivo efficacy can be monitored by standard methods established for each disease entity, including, but not limited to, white blood cell count, differential, fluorescence-activated cell sorting, and bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used. Additionally, computed tomography, x-ray, and nuclear magnetic resonance imaging (e.g., response assessment based on the National Cancer Institute criteria [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999] Apr;17(4):1244]), positron emission tomography scanning, white blood cell count, differential, fluorescence activated cell sorting, bone marrow aspiration, lymph node biopsy / histology, and various lymphoma-specific clinical chemistry parameters (e.g., lactate dehydrogenase), as well as other established standard methods may be used.

[0255] Another major challenge in the development of drugs, such as the pharmaceutical compositions of the present invention, is the predictable modulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of a candidate drug can be constructed, i.e., a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given pathological condition. Pharmacokinetic parameters of a drug that affect the ability of a drug to treat a particular disease entity include, but are not limited to, half-life, volume of distribution, hepatic first-pass metabolism, and degree of serum binding. The efficacy of a given drug may be affected by each of the above parameters. A presumed feature of the antigen-binding molecules of the present invention, resulting from a particular FC mode, is that they include, for example, differences in pharmacokinetic behavior. The targeted antigen-binding molecules with extended half-life according to the present invention preferably exhibit a surprisingly increased residence time in vivo compared to "canonical" non-HLE versions of the antigen-binding molecules.

[0256] "Half-life" refers to the time it takes for 50% of an administered drug to be eliminated through biological processes, such as metabolism, excretion, etc. "Hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" refers to the degree of retention of a drug across various compartments of the body, such as intracellular and extracellular spaces, tissues and organs, and the distribution of the drug within these compartments. "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins, such as albumin, resulting in a reduction or elimination of the drug's biological activity.

[0257] Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rate, onset of action, and / or Cmax for a given amount of drug administered. "Bioavailability" refers to the amount of drug in the blood compartment. "Lag time" refers to the time delay between administration of a drug and its detection and measurability in the blood or plasma. "Tmax" is the time after which the maximum blood concentration of the drug is reached, and "Cmax" is the maximum blood concentration attained by a given drug. The time it takes for a drug to reach the blood or tissue concentration required for biological effect is affected by all parameters. As outlined above, pharmacokinetic parameters of bispecific antigen-binding molecules exhibiting cross-species specificity, which can be determined in preclinical animal studies in non-chimpanzee primates, are also described, for example, in a publication by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12).

[0258] In a preferred embodiment of the present invention, the pharmaceutical composition is stable at about -20°C for at least 4 weeks. As is evident from the accompanying examples, the quality of the antigen-binding molecules of the present invention compared to that of corresponding state-of-the-art antigen-binding molecules can be tested using different systems. These tests are understood to comply with the "ICH Harmonized Tripartite Guideline: Stability Testing of Biotechnological / Biological Products Q5C and Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products Q6B" and are selected to provide a stability-indicating profile that reliably detects changes in the identity, purity, and potency of the product. It is well recognized that the term "purity" is a relative term. Due to the influence of glycosylation, deamidation, or other heterogeneity, the absolute purity of a biotechnological / biological product typically must be assessed by multiple methods, and the resulting purity value is method-dependent. For stability testing purposes, purity tests should be tailored to the determination of degradation products.

[0259] The quality of pharmaceutical compositions containing antigen-binding molecules of the present invention may be evaluated, for example, by analyzing the content of soluble aggregates (HMWS by size exclusion) in solution. Stability at about -20°C for at least 4 weeks is preferably characterized by a content of less than about 5% HMWS, more preferably less than 2.5%, and even more preferably less than 1.5% HMWS.

[0260] Other examples of evaluating the stability of antigen-binding molecules of the present invention in the form of pharmaceutical compositions are shown in the accompanying Examples 4 to 12. In these examples, embodiments of the antigen-binding molecules of the present invention are tested against different stress conditions in different pharmaceutical formulations, and the results are compared with other bispecific T-cell-engaging antigen-binding molecules in half-life extension (HLE) formats known in the art. In general, antigen-binding molecules provided in a specific FC format according to the present invention are typically expected to be more stable against a wide range of stress conditions, such as temperature and light stress, compared with both antigen-binding molecules provided in different HLE formats and antigen-binding molecules without any HLE format (e.g., "canonical" antigen-binding molecules). The temperature stability may relate to both low temperatures (below room temperature, including freezing temperatures) and high temperatures (above room temperature, including temperatures up to or above body temperature). As those skilled in the art will recognize, such improved stability against stresses that are difficult to avoid in clinical practice will result in safer antigen-binding molecules because fewer degradation products will be generated in clinical practice. Consequently, the improved stability means improved safety.

[0261] One embodiment provides an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the method of the present invention for use in the prevention, treatment, or amelioration of a proliferative disease, a neoplastic disease, a viral disease, or an immune disorder.

[0262] The formulations described herein are useful as pharmaceutical compositions for treating, ameliorating, and / or preventing the pathological medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the formulations to the body, isolated tissues, or cells of a patient with a disease / disorder, symptoms of a disease / disorder, or predisposition to a disease / disorder, with the intent to cure, treat, relieve, alleviate, alter, correct, ameliorate, improve, or affect the disease, symptoms of a disease, or predisposition to a disease.

[0263] As used herein, the term "amelioration" refers to the improvement of the disease state of a patient having a tumor or cancer, or metastatic cancer, as defined herein below, by administering an antigen-binding molecule according to the present invention to a subject in need thereof. Such improvement can also be considered as a slowing or halting of the progression of the patient's tumor or cancer or metastatic cancer. As used herein, the term "prevention" refers to the avoidance of the onset or recurrence of a patient having a tumor or cancer, or metastatic cancer, as defined herein below, by administering an antigen-binding molecule according to the present invention to a subject in need thereof.

[0264] The term "disease" refers to any condition that would benefit from treatment with the antigen-binding molecules or pharmaceutical compositions described herein. This term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question.

[0265] A "neoplasm" is an abnormal growth of tissue, usually, although not necessarily, forming a mass. When forming a mass, it is commonly referred to as a "tumor." Neoplasms or tumors can be benign, potentially malignant (pre-cancerous), or malignant. Malignant neoplasms are commonly called cancers. Malignant neoplasms usually invade and destroy surrounding tissues and can form metastases, i.e., spread to other parts, tissues, or organs of the body. Thus, the term "metastatic cancer" includes not only metastasis of the primary tumor, but also metastasis to other tissues or organs. Lymphomas and leukemias are lymphatic neoplasms. For purposes of the present invention, these are also encompassed by the terms "tumor" or "cancer."

[0266] The term "viral disease" refers to a disease that occurs as a result of a viral infection in a subject.

[0267] As used herein, the term "immune disorder" refers to an immune disorder that meets the general definition of the term, such as an autoimmune disease, hypersensitivity disorder, or immunodeficiency.

[0268] In one embodiment, the present invention provides a method for treating or ameliorating a proliferative disease, a neoplastic disease, a viral disease, or an immune disorder, the method comprising the step of administering to a subject in need thereof an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the method of the present invention.

[0269] The term "subject in need" or "subject in need of treatment" includes subjects already with the disorder as well as subjects in which the disorder is to be prevented. A subject in need or a "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0270] The antigen-binding molecules of the present invention are generally designed for a specific route and method of administration, a specific dosage and frequency, and a specific treatment of a specific disease, within the range of bioavailability and duration. It is preferable to formulate the materials of the composition at a concentration that is acceptable for the administration site.

[0271] Thus, formulations and compositions may be designed in accordance with the present invention for delivery by any suitable route of administration, including, but not limited to, the following in the context of the present invention: · Topical route (e.g., on the skin, inhalation, nose, eyes, pinna / ear, vagina, mucous membranes); Enteral routes (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and Parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intra-articular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal).

[0272] The pharmaceutical compositions and antigen-binding molecules of the present invention are particularly useful for parenteral administration, for example, subcutaneous or intravenous delivery, by injection, such as a bolus injection, or by infusion, such as continuous infusion. The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0273] In particular, the present invention provides for uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted administration, i.e., continuous administration, can be achieved by a miniature pump system attached to the patient to regulate the inflow of a therapeutic agent into the patient's body. Pharmaceutical compositions containing antigen-binding molecules of the present invention can be administered using such pump systems. Such pump systems are generally known in the art and typically involve periodic replacement of a cartridge containing the therapeutic agent to be infused. When replacing the cartridge in such a pump system, a temporary interruption in the otherwise uninterrupted inflow of the therapeutic agent into the patient's body may result. In such cases, the administration step before cartridge replacement and the administration step after cartridge replacement are still considered within the meaning of the pharmaceutical means and methods of the present invention, which together constitute the "uninterrupted administration" of such a therapeutic agent.

[0274] Continuous or uninterrupted administration of the antigen-binding molecules of the present invention can be intravenous or subcutaneous administration using a fluid delivery device or miniature pump system, including a fluid pumping mechanism for pumping fluid from a reservoir and a drive mechanism for driving the pumping mechanism. A pump system for subcutaneous administration can include a needle or cannula that penetrates the patient's skin and delivers the suitable composition into the patient's body. The pump system can be fixed or attached directly to the patient's skin, whether venous, arterial, or vascular, thereby allowing direct contact between the pump system and the patient's skin. The pump system can be worn on the patient's skin for 24 hours to several days at most. The pump system can be compact, with a small-volume reservoir. As a non-limiting example, the reservoir volume of the suitable pharmaceutical composition to be administered can be 0.1 to 50 ml.

[0275] Continuous administration can also be transdermally administered by a patch that is attached to the skin and replaced at intervals.Those skilled in the art are aware of suitable patch systems for drug delivery for this purpose.It should be noted that transdermal administration is particularly suitable for uninterrupted administration, since it has the advantage that, for example, a new second patch can be attached to the skin surface immediately adjacent to the first used patch, and immediately before removing the first used patch, the replacement of the first used patch can be completed at the same time.There is no problem of interrupted flow or battery failure.

[0276] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with an appropriate liquid prior to administration, for example, bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation the protein was in before lyophilization.

[0277] The compositions of the present invention can be administered to subjects at a suitable dose, which can be determined, for example, by a dose-escalation study in which the antigen-binding molecules of the present invention exhibiting cross-species specificity described herein are administered to non-chimpanzee primates, such as macaques, at increasing doses. As described above, the antigen-binding molecules of the present invention exhibiting cross-species specificity described herein have the advantage that they can be used in the same form in preclinical studies in non-chimpanzee primates and can also be used as drugs in humans. The administration schedule will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage administered to a given patient depends on many factors, including the patient's size, body surface area, age, the individual compound administered, sex, time and route of administration, general health, and other drugs being administered simultaneously.

[0278] The term "effective dose" or "effective administration amount" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest a disease and its complications in a patient already suffering from the disease. The amount or dosage effective for this use depends on the condition (indication) being treated, the antigen-binding molecule being delivered, the nature and purpose of the treatment, the severity of the disease, previous treatments, the patient's medical history and response to the therapeutic agent, the route of administration, the body size (weight, body surface area, or organ size), and / or the condition of the patient (age and general health), as well as the general state of the patient's own immune system. The appropriate dosage can be administered to the patient in a single administration or multiple administrations and can be adjusted according to the judgment of the attending physician to obtain the optimal therapeutic effect.

[0279] Typical dosages can range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In certain embodiments, dosages can range from 1.0 μg / kg up to about 20 mg / kg, optionally 10 μg / kg up to about 10 mg / kg, or 100 μg / kg up to about 5 mg / kg.

[0280] A therapeutically effective amount of an antigen-binding molecule of the present invention preferably reduces the severity of disease symptoms, increases the frequency or duration of symptom-free periods, or prevents functional impairment or disability due to disease affliction. For the treatment of antigen-expressing tumors in target cells, a therapeutically effective amount of an antigen-binding molecule of the present invention, e.g., an anti-target cell antigen / anti-CD3 antigen-binding molecule, preferably inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of a compound to inhibit tumor growth can be assessed in an animal model that predicts efficacy.

[0281] The pharmaceutical composition can be administered in a single treatment, or optionally in combination with additional treatments, such as anti-cancer therapies, e.g., other proteinaceous and non-proteinaceous drugs. These drugs may be administered simultaneously with a composition comprising an antigen-binding molecule of the present invention as defined herein, or may be administered separately at a predetermined time interval and dose before or after administration of the antigen-binding molecule.

[0282] As used herein, the term "effective and non-toxic dose" refers to a tolerable dose of an antigen-binding molecule of the present invention that is high enough to cause depletion of pathological cells, tumor elimination, tumor regression, or disease stabilization without or essentially without causing significant toxic effects. Such an effective and non-toxic dose may be determined, for example, by a dose escalation study as described in the art, and should be below the dose that induces serious adverse side effects (dose-limiting toxicity, DLT).

[0283] As used herein, the term "toxicity" refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These side effects may refer to a lack of systemic drug tolerance and / or a lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0284] As used herein, the terms "safety," "in vivo safety," or "tolerability" are defined as the administration of a drug that does not induce severe adverse events immediately after administration (local tolerance) and during longer periods of drug administration. "Safety," "in vivo safety," or "tolerability" can be assessed, for example, periodically during treatment and follow-up. Measurements include clinical evaluations, such as organ findings and screening for laboratory abnormalities. Clinical evaluations can be performed, and deviations from normal findings can be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings can include criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmias, and coagulation, as set forth in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profiles, and investigations of other body fluids, such as serum, plasma, lymph, or spinal fluid. Thus, safety can be assessed by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI), other measurements by technical devices (i.e. electrocardiogram), vital signs, for example, by measuring laboratory parameters and recording adverse events. For example, in the uses and methods according to the present invention, adverse events in non-chimpanzee primates can be examined by histopathological and / or histochemical methods.

[0285] The above terms are also referred to, for example, in Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6 of July 16, 1997; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting.

[0286] Finally, the present invention provides kits comprising the antigen-binding molecule of the present invention or the antigen-binding molecule produced according to the process of the present invention, the pharmaceutical composition of the present invention, the polynucleotide of the present invention, the vector of the present invention, and / or the host cell of the present invention.

[0287] In the context of the present invention, the term "kit" refers to two or more components packaged together in a container, vessel, or other package, one of which corresponds to an antigen-binding molecule, pharmaceutical composition, vector, or host cell of the present invention. Thus, a kit can be described as a set of products and / or tools sufficient to achieve a specific purpose, which can be sold individually.

[0288] The kit may comprise one or more containers (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing an antigen-binding molecule or pharmaceutical composition of the present invention in a dosage amount suitable for administration (see above). The kit may further comprise instructions for use (e.g., in the form of a leaflet or instruction manual), means for administering the antigen-binding molecule of the present invention, such as a syringe, pump, or infuser, means for reconstituting the antigen-binding molecule of the present invention, and / or means for diluting the antigen-binding molecule of the present invention.

[0289] The present invention also provides a kit for single-dose administration unit.The kit of the present invention can also include a first container containing dried / lyophilized antigen-binding molecules and a second container containing aqueous formulations.In a specific embodiment of the present invention, a kit is provided that includes single-chamber and multi-chamber pre-filled syringes (for example, liquid syringes and lyophilized syringes).

[0290] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the method described herein.

[0291] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0292] The term "and / or", wherever used in this specification, includes the meanings "and", "or" and "all or any other combination of the elements connected by said term".

[0293] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range, although the term also includes specific numbers, for example, about 20 includes 20.

[0294] The terms "less than" or "greater than" are inclusive of a specific number. For example, less than 20 means less than or equal to. Similarly, greater than or greater than means greater than or equal to, or greater than or equal to, respectively.

[0295] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," should be understood to mean the inclusion of the specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including," or, as sometimes used herein, the term "having."

[0296] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0297] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with one of the other two terms.

[0298] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0299] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) cited throughout the text of this specification, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will take precedence over any such material.

[0300] A better understanding of the present invention and its advantages will be obtained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0301] Example 1: Determining the effect of protein concentration and pH on BCMAxCD3, DLL3xCD3 and MUC17xCD3 BiTE® antigen-binding molecule stability Materials and Methods for All Experiments: Three half-life extension (HLE) BiTE® molecules according to the present invention, BCMAxCD3 (SEQ ID NO: 136), DLL3xCD3 (SEQ ID NO: 104), and MUC17xCD3 (SEQ ID NO: 213), were prepared at high concentrations (≥15 mg / mL) in 24 formulations at three different pH values: 4.4, 4.6, and 5.2, via a plate-based buffer exchange system, Freeslate Core Module 3 (CM3) liquid handler (Unchained Labs, Pleasanton, CA). Microbuffer exchange filtration was performed in a pressure chamber, and the liquid volume in each well was measured with a VolumeCheck reader (BioMicroLab, Concord, CA), as outlined in more detail in Paper 1. Formulation stock solutions used in all studies were prepared according to commonly known standards. After buffer exchange was completed, pH and concentration were measured to ensure they were within target values. After preparation, samples were aliquoted into 96-well plates, sealed, and stored at -30°C, 2-8°C, and 40°C for up to 4 weeks. Sample stability was then assessed for % HMW by size exclusion chromatography (SEC) on an ACQUITY UPLC system (Waters, Milford, MA). Samples were injected onto a Waters BEH200 column with a mobile phase containing 250 mM NaCl at pH 6.5. High molecular weight (HMW) and monomer or main peak (MP) peak areas were calculated as a percentage of the total peak area.

[0302] First, the effects of protein concentration and pH on the DLL3xCD3 bispecific antigen-binding molecule were determined. As can be seen from Figure 1 (results shown below the figure capture), when no additional stabilizer is present in the pharmaceutical composition, %HMW significantly increases with increasing concentration and pH. Specifically, at pH 4.4, %HMW increases from 0.33% to 7.97% as the protein concentration increases from 4.5 mg / mL to 30.1 mg / mL. At a given 30.1 mg / mL concentration, %HMW also increases with increasing pH, i.e., from pH 4.4 to 5.2, where %HMW increases from 3.17% to 17.26%. Thus, from pH 4.4 to 5.2, %HMW increases only from 0.33% to 2.17% at a protein concentration of 4.5 mg / mL, but increases more significantly at higher concentrations, increasing from 7.97% to 17.26% at 30.1 mg / mL. Therefore, higher protein concentration formulations require even greater stabilization at higher pH values.

[0303] Example 2: Determining the effect of protein concentration, pH and various excipients on the stability of BCMAxCD3, CD33xCD3 and DLL3xCD3 BiTE® antigen-binding molecules As shown in Figure 2, for each of the representative BCMAxCD3 bispecific antigen-binding molecule (squares; SEQ ID NO: 136), CD33xCD3 bispecific antigen-binding molecule (circles; SEQ ID NO: 41), and DLL3xCD3 bispecific antigen-binding molecule (triangles; SEQ ID NO: 104), increasing the pH decreased %LMW but conversely increased %HMW, consistent with the findings in Example 1. As shown in Figure 3, %LMW decreased with pH and was concentration-independent. %LMW decreased from greater than 8% to less than 5% across the pH range tested. The range of excipients tested, i.e., amino acids, cyclodextrin, and benzyl alcohol, did not have a significant effect on %LMW, at least at the higher bispecific antigen-binding molecule concentration of 20 mg / ml, as an exemplary value for high-concentration formulations according to the present invention.

[0304] Example 3: Determination of the stabilizing excipient EDTA versus pH and the effect of various excipients on BCMAxCD3 and DLL3xCD3 BiTE® antigen-binding molecules As shown in Figure 4, EDTA maintains low %HMW over 4 weeks in a liquid formulation at approximately 27 mg / ml. Thus, EDTA and related chelators are identified as excipients for significantly reducing %HMW in bispecific antigen-binding molecules. Specifically, the molecules of the present invention can have significantly higher %HMW at high concentrations, e.g., approximately 12%, in a composition comprising sucrose and glutamic acid at pH 5.2 without additional stabilizers (see G52Su in Figure 4A) for the BCMAxCD3 bispecific antigen-binding molecule (SEQ ID NO: 41) at a concentration of 27 mg / ml. Here, EDTA reduces %HMW under all test conditions compared to controls at time 0 or time 4 weeks (at -30°C, 4°C, or 40°C), e.g., only approximately 2% after 4 weeks at 4°C or -30°C for the BCMAxCD3 molecule at a concentration of 27 mg / mL at a pH ranging from 4.3 to 5.2. ArgHCl was identified as significantly increasing %HMW, for example, by more than 20% after 4 weeks at 4°C, indicating that the choice of EDTA was not arbitrary, as it was not foreseeable that Arg would act in a destabilizing manner, inducing further aggregation, whereas EDTA would not. HpbCD moderately reduced %HMW compared to the control, while Phe appeared to be less effective; Pro, Trp, and benzyl alcohol did not show a significant reducing effect on %HMW at a given polymer concentration of 27 mg. Comparable results were obtained with the DLL3xCD3 bispecific antigen-binding molecule (SEQ ID NO: 104).

[0305] Example 4: Effect of the concentration of the stabilizer EDTA on the stability of high-concentration formulations of MUC17xCD3 bispecific antigen-binding molecules As shown in Figure 5, the MUC17xCD3 molecule ("G4Su") (SEQ ID NO: 213) without additional stabilizers exhibits significantly higher %HMW at higher pHs, i.e., approximately 6-7% at pH 5 versus approximately 1-2% at pH 4.2. ArgHCl significantly increases %HMW in all conditions, i.e., greater than approximately 10%. HpbCD, Phe, and Pro are less effective than the stabilizer EDTA, resulting in similar levels of %HMW as the control. The stabilizer EDTA reduces %HMW at all tested concentrations, i.e., 0.01%, 0.04%, and 0.16% (w / v), compared to other excipients at 0 or 4 weeks. Notably, at the slightly higher pH of 4.6 or 5.0, i.e., in pharmaceutical compositions containing EDTA at any of the three resting concentrations, only 2-3% HMW was found after 4 weeks at 4°C or -30°C, whereas those without stabilizers exhibited HMW values ​​approaching or exceeding 5%.

[0306] [Table 5]

[0307] [Table 6]

[0308] [Table 7]

[0309] [Table 8]

[0310] [Table 9]

[0311] [Table 10]

[0312]

Table 11

[0313]

Table 12

[0314]

Table 13

[0315]

Table 14

[0316]

Table 15

[0317] Table 16

[0318]

Table 17

[0319]

Table 18

[0320]

Table 19

[0321] Table 20

[0322] Table 21

[0323] Table 22

[0324] Table 23

[0325] Table 24

[0326] Table 25

[0327] Table 26

[0328] Table 27

[0329] Table 28

[0330] Table 29

[0331]

Table 30

[0332] Table 31

[0333] Table 32

[0334]

Table 33

[0335] Table 34

[0336] Table 35

[0337] Table 36

[0338] Table 37

[0339] Table 38

[0340] Table 39

[0341] Table 40

[0342] Table 41

[0343] Table 42

[0344] Table 43

[0345] Table 44

Claims

1. (a) a bispecific antigen-binding molecule comprising at least three domains, wherein: the first domain binds to a target cell surface antigen, said target cell surface antigen being a tumor antigen; The second domain binds to an extracellular epitope of the human and macaque (Macaca) CD3 chain; and the third domain comprises two polypeptide monomers each comprising a hinge, a CH2 domain, and a CH3 domain, said two polypeptide monomers being fused to each other via a peptide linker, said third domain comprising, in amino to carboxyl order, hinge-CH2-CH3-linker-hinge-CH2-CH3; the concentration of the bispecific antigen-binding molecule is 8 to 35 mg / ml; (b) at least one buffering agent; (c) at least one sugar; and (d) at least one stabilizer selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and citric acid, wherein said stabilizer is present at a concentration ranging from 0.005% to 0.25% (w / v), preferably from 0.01 to 0.2% (w / v); The pH of the pharmaceutical composition is in the range of 4.0 to 6.

0.

1. A pharmaceutical composition, preferably liquid, comprising:

2. The pharmaceutical composition of claim 1 , wherein the bispecific antigen-binding molecule is a single-chain molecule.

3. The pharmaceutical composition of claim 1, wherein the bispecific antigen-binding molecule has an extended half-life.

4. 2. The pharmaceutical composition of claim 1, wherein the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the bispecific antigen-binding molecule is eliminated by an N314X substitution, wherein X is any amino acid except Q.

5. 2. The pharmaceutical composition of claim 1, wherein each of the polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-24, or has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24.

6. The pharmaceutical composition of claim 1 , wherein the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

7. 2. The pharmaceutical composition of claim 1, wherein the tumor antigen is selected from the group consisting of CDH19, CDH3, MSLN, DLL3, FLT3, EGFRvIII, BCMA, PSMA, CD33, CD19, CD20, CLDN18.2, MUC17, EpCAM, CD70, and CLDN6.

8. 2. The pharmaceutical composition of claim 1, wherein the second domain is an extracellular epitope of the human and / or macaque CD3 epsilon chain.

9. (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain. The pharmaceutical composition of claim 1.

10. The antibody construct is arranged in amino to carboxyl order as follows: (a) the first domain; (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-189; (c) the second domain; (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 187, 188, 189, 195, 196, 197 and 198; (e) a first polypeptide monomer of the third domain; (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 192, 193, and 194; and (g) a second polypeptide monomer of the third domain.

2. The pharmaceutical composition of claim 1, comprising:

11. wherein the first binding domain of the construct comprises: (a) CDR-H1 shown in SEQ ID NO: 4, CDR-H2 shown in SEQ ID NO: 5, CDR-H3 shown in SEQ ID NO: 6, CDR-L1 shown in SEQ ID NO: 1, CDR-L2 shown in SEQ ID NO: 2, and CDR-L3 shown in SEQ ID NO: 3; (b) CDR-H1 shown in SEQ ID NO: 29, CDR-H2 shown in SEQ ID NO: 30, CDR-H3 shown in SEQ ID NO: 31, CDR-L1 shown in SEQ ID NO: 34, CDR-L2 shown in SEQ ID NO: 35, and CDR-L3 shown in SEQ ID NO: 36; (c) CDR-H1 shown in SEQ ID NO: 42, CDR-H2 shown in SEQ ID NO: 43, CDR-H3 shown in SEQ ID NO: 44, CDR-L1 shown in SEQ ID NO: 45, CDR-L2 shown in SEQ ID NO: 46, and CDR-L3 shown in SEQ ID NO: 47; (d) CDR-H1 shown in SEQ ID NO: 53, CDR-H2 shown in SEQ ID NO: 54, CDR-H3 shown in SEQ ID NO: 55, CDR-L1 shown in SEQ ID NO: 56, CDR-L2 shown in SEQ ID NO: 57, and CDR-L3 shown in SEQ ID NO: 58; (e) CDR-H1 shown in SEQ ID NO: 65, CDR-H2 shown in SEQ ID NO: 66, CDR-H3 shown in SEQ ID NO: 67, CDR-L1 shown in SEQ ID NO: 68, CDR-L2 shown in SEQ ID NO: 69, and CDR-L3 shown in SEQ ID NO: 70; (f) CDR-H1 represented by SEQ ID NO: 83, CDR-H2 represented by SEQ ID NO: 84, CDR-H3 represented by SEQ ID NO: 85, CDR-L1 represented by SEQ ID NO: 86, CDR-L2 represented by SEQ ID NO: 87, and CDR-L3 represented by SEQ ID NO: 88; (g) CDR-H1 shown in SEQ ID NO: 94, CDR-H2 shown in SEQ ID NO: 95, CDR-H3 shown in SEQ ID NO: 96, CDR-L1 shown in SEQ ID NO: 97, CDR-L2 shown in SEQ ID NO: 98, and CDR-L3 shown in SEQ ID NO: 99; (h) CDR-H1 shown in SEQ ID NO: 105, CDR-H2 shown in SEQ ID NO: 106, CDR-H3 shown in SEQ ID NO: 107, CDR-L1 shown in SEQ ID NO: 109, CDR-L2 shown in SEQ ID NO: 110, and CDR-L3 shown in SEQ ID NO: 111; (i) CDR-H1 shown in SEQ ID NO: 115, CDR-H2 shown in SEQ ID NO: 116, CDR-H3 shown in SEQ ID NO: 117, CDR-L1 shown in SEQ ID NO: 118, CDR-L2 shown in SEQ ID NO: 119, and CDR-L3 shown in SEQ ID NO: 120; (j) CDR-H1 shown in SEQ ID NO: 126, CDR-H2 shown in SEQ ID NO: 127, CDR-H3 shown in SEQ ID NO: 128, CDR-L1 shown in SEQ ID NO: 129, CDR-L2 shown in SEQ ID NO: 130, and CDR-L3 shown in SEQ ID NO: 131; (k) CDR-H1 shown in SEQ ID NO: 137, CDR-H2 shown in SEQ ID NO: 138, CDR-H3 shown in SEQ ID NO: 139, CDR-L1 shown in SEQ ID NO: 140, CDR-L2 shown in SEQ ID NO: 141, and CDR-L3 shown in SEQ ID NO: 142; (l) CDR-H1 shown in SEQ ID NO: 152, CDR-H2 shown in SEQ ID NO: 153, CDR-H3 shown in SEQ ID NO: 154, CDR-L1 shown in SEQ ID NO: 155, CDR-L2 shown in SEQ ID NO: 156, and CDR-L3 shown in SEQ ID NO: 157; (m) CDR-H1 shown in SEQ ID NO: 167, CDR-H2 shown in SEQ ID NO: 168, CDR-H3 shown in SEQ ID NO: 169, CDR-L1 shown in SEQ ID NO: 170, CDR-L2 shown in SEQ ID NO: 171, and CDR-L3 shown in SEQ ID NO: 172; (n) CDR-H1 shown in SEQ ID NO: 203, CDR-H2 shown in SEQ ID NO: 204, CDR-H3 shown in SEQ ID NO: 205, CDR-L1 shown in SEQ ID NO: 206, CDR-L2 shown in SEQ ID NO: 207, and CDR-L3 shown in SEQ ID NO: 208; (O) CDR-H1 represented by SEQ ID NO: 214, CDR-H2 represented by SEQ ID NO: 215, CDR-H3 represented by SEQ ID NO: 216, CDR-L1 represented by SEQ ID NO: 217, CDR-L2 represented by SEQ ID NO: 218, and CDR-L3 represented by SEQ ID NO: 219; (p) CDR-H1 represented by SEQ ID NO: 226, CDR-H2 represented by SEQ ID NO: 227, CDR-H3 represented by SEQ ID NO: 228, CDR-L1 represented by SEQ ID NO: 229, CDR-L2 represented by SEQ ID NO: 230, and CDR-L3 represented by SEQ ID NO: 231; (q) CDR-H1 represented by SEQ ID NO: 238, CDR-H2 represented by SEQ ID NO: 239, CDR-H3 represented by SEQ ID NO: 240, CDR-L1 represented by SEQ ID NO: 241, CDR-L2 represented by SEQ ID NO: 242, and CDR-L3 represented by SEQ ID NO: 243; and (r) CDR-H1 shown in SEQ ID NO: 248, CDR-H2 shown in SEQ ID NO: 249, CDR-H3 shown in SEQ ID NO: 250, CDR-L1 shown in SEQ ID NO: 251, CDR-L2 shown in SEQ ID NO: 252, and CDR-L3 shown in SEQ ID NO: 253 2. The pharmaceutical composition of claim 1, comprising a VH region comprising CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of:

12. 2. The pharmaceutical composition of claim 1, wherein the concentration of the bispecific antigen-binding molecule is 10 to 35 or 15 to 31 mg / ml, more preferably 20 to 30 mg / ml or 25 to 30 mg / ml.

13. 2. The pharmaceutical composition of claim 1, wherein the concentration of EDTA is in the range of 0.01% to 0.2% (w / v), preferably in the range of 0.01% to 0.16% (w / v), more preferably 0.04% (w / v).

14. 2. The pharmaceutical composition of claim 1, wherein the at least one buffering agent is an acid selected from the group consisting of acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, or a combination thereof, preferably glutamate.

15. 15. The pharmaceutical composition according to claim 14, wherein said at least one buffering agent is present in a concentration range of 5 to 200 mM, more preferably in a concentration range of 10 to 50 mM, preferably at 15 mM.

16. 2. The pharmaceutical composition of claim 1, wherein the at least one saccharide is selected from the group consisting of a monosaccharide, a disaccharide, a cyclic polysaccharide, a sugar alcohol, a linear branched dextran, and a linear unbranched dextran.

17. 17. The pharmaceutical composition of claim 16, wherein the disaccharide is selected from the group consisting of sucrose and trehalose and combinations thereof, preferably sucrose.

18. 17. The pharmaceutical composition of claim 16, wherein the sugar alcohol is selected from the group consisting of mannitol and sorbitol, and combinations thereof.

19. 17. The pharmaceutical composition of claim 16, wherein the at least one sugar is present at a concentration in the range of 1 to 15% (w / v), preferably in a concentration range of 8 to 12% (w / v), such as 8% (w / v).

20. 10. The pharmaceutical composition of claim 1, further comprising at least one surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, pluronic F68, Triton X-100, polyoxyethylene, PEG 3350, PEG 4000, and combinations thereof.

21. 2. The pharmaceutical composition according to claim 1, comprising at least one surfactant in a concentration ranging from 0.004 to 0.5% (w / V), preferably in the range of 0.01 to 0.1% (w / v).

22. 2. The pharmaceutical composition of claim 1, wherein the pH of the composition is in the range of 4.0 to 5.3, preferably 4.2 to 5.2, more preferably 4.3 to 4.

6.

23. 10. The pharmaceutical composition of claim 1, having an osmolality in the range of 150 to 500 mOsm.

24. 10. The pharmaceutical composition of claim 1, further comprising excipients selected from the group consisting of one or more polyols, preferably hydroxypropyl-β-cyclodextrin, and one or more amino acids, preferably phenylalanine, but preferably not arginine, proline, and tryptophan.

25. 25. The pharmaceutical composition of claim 24, wherein the one or more excipients are present in a concentration range of 0.1 to 15% (w / v).

26. (a) a bispecific antigen-binding molecule according to any one of claims 1 to 25; (b) 15 mM glutamate or acetate; (c) 8% (w / V) sucrose or 8% (w / V) sucrose and 1% (w / V) hydroxypropyl-β-cyclodextrin; (d) optionally, 0.01% (w / V) polysorbate 80 2. The pharmaceutical composition of claim 1, wherein the pH of the liquid pharmaceutical composition is any value in the range of 4.0 to 5.2, preferably 4.2 to 4.6, preferably 4.3 to 4.

6.

27. A solid pharmaceutical composition obtainable by lyophilization of a liquid pharmaceutical composition according to any one of claims 1 to 26.

28. 28. A liquid pharmaceutical composition obtainable by reconstituting the solid pharmaceutical composition of claim 27 with a pharmaceutically acceptable liquid.

29. A composition according to any one of claims 1 to 28 for use in the treatment of a disease, preferably a proliferative disease.

30. 10. Use of the pharmaceutical composition of claim 1 for reducing the formation of high molecular weight species (HMWS) during storage, wherein the amount of HMWS is kept below 5%, preferably below 3% or 2%, when the liquid pharmaceutical composition is stored at 4°C or below, preferably at -30°C or below.

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