Pharmaceutical preparations

Abstract

To provide a stable, high-concentration bispecific antibody construct formulation having less than 2% high molecular weight species of the bispecific antibody construct in the formulation under storage conditions at various time points. [Solution] This disclosure provides a stable formulation containing a high concentration (i.e., greater than 10 mg / mL) of a bispecific antibody construct (e.g., a BiTE® molecule), wherein less than approximately 3% of the bispecific antibody construct in the formulation is present as a high molecular weight (HMW) species. Formulations containing high concentrations of antibody are generally difficult to stabilize under storage conditions, although this is desirable. Surprisingly, formulations according to this disclosure containing a bispecific antibody construct at a higher concentration than expected have been found to be stable without the need for preservatives or stabilizers.

Description

Technical Field

[0001] The present disclosure is in the field of highly stable formulations of bispecific antibody constructs at high concentrations.

[0002] Incorporation by Reference The following ASCII (text) file, which was created on April 28, 2021, named "54911_Seqlisting.txt", sized 345,249 bytes, and identified as a computer-readable nucleotide / amino acid sequence listing, is incorporated by reference in its entirety and is filed simultaneously with this specification.

Background Art

[0003] Advances in purification processes at the commercial scale now enable protein-based pharmaceuticals, such as recombinant proteins, to be obtained with high purity at the first production. However, proteins are only marginally stable and are highly prone to chemical and physical degradation. Chemical degradation refers to modifications involving covalent bonds such as deamidation, oxidation, cleavage, clipping / fragmentation, 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 challenging issues in the development of protein pharmaceuticals (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325 - 1336, Roberts, Trends Biotechnol. 2014 Jul;32(7):372 - 80).

[0004] For example, bispecific T cell engagers (BiTE®), including half-life extension forms of molecules such as Fc, and other half-life extension antibody constructs should be protected from protein aggregation and / or other degradation phenomena. Protein aggregation of BiTE® molecules is problematic because it can weaken the biological activity of therapeutic proteins. Furthermore, aggregation of BiTE® molecules can reduce product yield due to the meticulous purification process required to remove aggregates from the final product. More recently, there has been growing concern and evidence that the presence of aggregated proteins (whether humanized or complete human proteins) can significantly increase the risk of patients developing an immune response to active protein monomers, resulting in the formation of neutralizing antibodies and drug resistance or other adverse side effects (Mahler J Pharm Sci. 2009 Sep;98(9):2909-34).

[0005] Aggregation is a major type of physical instability observed in high-concentration formulations. Aggregation initially involves the aggregation of naturally folded proteins into high molecular weight species (HMWs). Currently, protein aggregates are removed as impurities, primarily in the polishing process of downstream treatments. However, in the case of high levels of HMW species, significant HMW removal not only results in considerable yield loss but also makes it difficult to design robust downstream processes (Chi et al., Pharm Res, Vol.20, No.9, Sept 2003, pp.1325-1336). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chi et al.,Pharm Res,Vol.20,No.9,Sept 2003,pp.1325-1336 [Non-Patent Document 2] Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Overview of the Initiative] [Means for solving the problem]

[0006] This disclosure provides a stable formulation containing a high concentration (i.e., greater than 10 mg / mL) of a bispecific antibody construct (e.g., a BiTE® molecule), wherein approximately less than 3% of the bispecific antibody construct is present as a high molecular weight (HMW) species. Formulations containing high concentrations of antibody are generally difficult to stabilize under storage conditions, although this is desirable. Surprisingly, formulations according to this disclosure containing bispecific antibody constructs at higher concentrations than expected have been found to be stable without the need for preservatives or stabilizers.

[0007] In some embodiments, the formulation is a lyophilized formulation. In some embodiments, a bispecific antibody construct of 2.5% or less (e.g., 2.5%, 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, or 0.5%) is present as HMW species in the lyophilized formulation. In some embodiments, the amount of HMW species in the lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the formulation increases by approximately 0.1% to 0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of HMW species in the lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%).

[0008] In some embodiments, the bispecific antibody construct is present in amounts less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) as a low molecular weight (LMW) species in the lyophilized formulation. In some embodiments, the amount of LMW species in the lyophilized formulation increases by less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, after storage at 4°C for more than one month (e.g., one month, three months, or six months), the amount of LMW species in the formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%). In some embodiments, after storage at 40°C for more than one month (e.g., one week, two weeks, one month, or three months), the amount of LMW species increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of LMW species in the lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%).

[0009] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the lyophilized formulation is greater than 95% of the total protein content in the formulation.

[0010] In some embodiments, the lyophilized formulation is stable after storage at approximately 4°C for one month, and the amount of HMW species in the formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) during storage for at least one month. In some embodiments, the lyophilized formulation is stable after storage at approximately 4°C for three months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.2% (e.g., 0%, 0.1%, or 0.2%) during storage for at least three months. In some embodiments, the lyophilized formulation is stable after storage at approximately 4°C for at least six months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.4% (e.g., 0%, 0.1%, 0.2%, 0.3%, or 0.4%) during storage for at least six months.

[0011] In some embodiments, the lyophilized formulation, after storage at approximately 4°C for at least 1 month, 3 months, and 6 months, contains more than 95% of the total protein content during storage, representing a percentage of intact BiTE® molecules.

[0012] In some embodiments, the formulation is a liquid formulation. In some embodiments, less than 3% (e.g., 2.5%, or 2%, or 1.5%, or 1%, or 0.5%) of the bispecific antibody construct is present as HMW species in the liquid formulation. In some embodiments, the amount of HMW species in the liquid formulation increases by less than 3% (e.g., 3%, 2.5%, 2%, 1%, or 0.5%) after storage at 4°C for more than one month (e.g., one month, three months, six months, or one year). In some embodiments, after storage at 4°C for more than one month (e.g., one month, three months, six months, or one year), the amount of HMW species in the formulation increases by approximately 0.1% to 1% (e.g., 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of HMW species in the liquid formulation increases by less than 5% (e.g., 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, or 0.5%). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of HMW species in the liquid formulation increases by approximately 0.1% to 5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%).

[0013] In some embodiments, bispecific antibody constructs present in amounts less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5%) as low molecular weight (LMW) species in liquid formulations. In some embodiments, after storage at 4°C for more than one month (e.g., one, three, six, or twelve months), the amount of LMW species in the liquid formulation increases by less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%). In some embodiments, after storage at 4°C for more than one month (e.g., one, three, six, or twelve months), the amount of LMW species in the liquid formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%). In some embodiments, the amount of LMW species in the liquid formulation increases by less than 7% (e.g., 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of LMW species in the lyophilized formulation increases by approximately 0.1% to 7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 1.5%, or 2%, or 3%, or 5%, or 6%, or 7%).

[0014] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the liquid formulation is greater than 96% of the total protein content in the formulation.

[0015] In some embodiments, the liquid formulation is stable after storage at approximately 4°C for one month, and the amount of HMW species in the formulation increases by approximately 0.1% to 0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%). In some embodiments, the liquid formulation is stable after storage at approximately 4°C for three months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.3% (e.g., 0%, 0.1%, 0.2%, or 0.3%) during storage for at least three months. In some embodiments, the liquid formulation is stable after storage at approximately 4°C for six months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.6% (e.g., 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.6%) during storage for at least six months. In some embodiments, the liquid formulation is stable after 12 months of storage at approximately 4°C, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.2% (e.g., 0%, 0.1%, or 0.2%) during storage for at least 12 months.

[0016] In some embodiments, the lyophilized formulation is stable after storage at approximately 4°C for 1 month, 3 months, 6 months, and 12 months, with the percentage of intact BiTE® molecules exceeding 96% of the total protein content.

[0017] In some embodiments, the antibody-binding protein is a bispecific antibody construct comprising a first binding domain that binds to the surface antigen of the target cell and a second binding domain that binds to human CD3 on the surface of T cells.

[0018] In some embodiments, the bispecific antibody construct further comprises a third domain including a hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain in the order of amino to carboxyl. In some embodiments, each of the first and second binding domains of the bispecific antibody construct comprises a VH region and a VL region.

[0019] In some embodiments, the bispecific antibody construct is a single-chain antibody construct.

[0020] In some embodiments, the bispecific antibody construct binds to one target cell surface antigen, such as CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17.

[0021] In some embodiments, the first binding domain of the bispecific antibody construct includes one set of 6 CDRs shown in (a) SEQ ID NOs: 24-29, (b) SEQ ID NOs: 34-39, (c) SEQ ID NOs: 78-83, (d) SEQ ID NOs: 10-15, (e) SEQ ID NOs: 46-51, (f) SEQ ID NOs: 88-93, (g) SEQ ID NOs: 67-72, (h) SEQ ID NOs: 56-61, (i) SEQ ID NOs: 112-117, (j) SEQ ID NOs: 100-105, (k) SEQ ID NOs: 148-153, SEQ ID NOs: 157-162, SEQ ID NOs: 166-171, or SEQ ID NOs: 175-180, (l) SEQ ID NOs: 132-137, or (m) SEQ ID NOs: 123-128.

[0022] In some embodiments, the first binding domain of the bispecific antibody construct includes a VH region containing an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NOs: 30, 40, 84, 16 or 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143, or 129.

[0023] In some embodiments, the first binding domain of the bispecific antibody construct comprises a VL region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144, or 130. In some embodiments, the first binding domain of the bispecific antibody construct comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144, or 130.

[0024] In some embodiments, the first binding domain is: (a) a VH region containing the amino acid sequence shown in SEQ ID NO: 30 and a VL region showing in SEQ ID NO: 31; (b) a VH region containing the amino acid sequence shown in SEQ ID NO: 40 and a VL region containing the amino acid sequence shown in SEQ ID NO: 41; (c) a VH region containing the amino acid sequence shown in SEQ ID NO: 84 and a VL region containing the amino acid sequence shown in SEQ ID NO: 85; (d) a VH region containing the amino acid sequence shown in SEQ ID NO: 16 or 17 and a VL region containing the amino acid sequence shown in SEQ ID NO: 18 or 19; (e) a VH region containing the amino acid sequence shown in SEQ ID NO: 52 and a VL region containing the amino acid sequence shown in SEQ ID NO: 53; (f) a VH region containing the amino acid sequence shown in SEQ ID NO: 94 and a VL region containing the amino acid sequence shown in SEQ ID NO: 95; (g) a VH region containing the amino acid sequence shown in SEQ ID NO: 73 and an amino acid sequence shown in SEQ ID NO: 74 (h) A VL region containing an acid sequence; (i) A VH region containing the amino acid sequence shown in SEQ ID NO: 62 and a VL region containing the amino acid sequence shown in SEQ ID NO: 63; (j) A VH region containing the amino acid sequence shown in SEQ ID NO: 118 and a VL region containing the amino acid sequence shown in SEQ ID NO: 119; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 154, 163, 172 or 181 and a VL region containing the amino acid sequence shown in SEQ ID NO: 155, 164, 173 or 182; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 106 and a VL region containing the amino acid sequence shown in SEQ ID NO: 107; (l) A VH region containing the amino acid sequence shown in SEQ ID NO: 138 or 143 and a VL region containing the amino acid sequence shown in SEQ ID NO: 139 or 144; or (m) A VH region containing the amino acid sequence shown in SEQ ID NO: 129 and a VL region containing the amino acid sequence shown in SEQ ID NO: 130.

[0025] In some embodiments, the second binding domain of the bispecific antibody construct contains a set of 6CDRs shown in SEQ ID NOs: 1-6.

[0026] In some embodiments, the second binding domain of the bispecific antibody construct comprises a VH region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the second binding domain of the bispecific antibody construct comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 7.

[0027] In some embodiments, the second binding domain of the bispecific antibody construct comprises a VL region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the second binding domain of the bispecific antibody construct comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 8.

[0028] In some embodiments, the second binding domain comprises (a) a VH region comprising the amino acid sequence shown in SEQ ID NO: 7 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0029] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to CD19 and comprises an anti-CD19 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 85 and an anti-CD19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 84, a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7, and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 86 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct comprises the amino acid sequence shown in SEQ ID NO: 87.

[0030] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to MSLN, comprising an anti-MSLN variable light chain domain comprising the amino acid sequence of SEQ ID NO: 41 and an anti-MSLN variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 40; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and a second binding domain comprising an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 42 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 43, 44, or 45.

[0031] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to DLL3, comprising an anti-DLL3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 74 and an anti-DLL3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 73; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 75 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 76 or 77.

[0032] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to FLT3, comprising an anti-FLT3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 63 and an anti-FLT3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 62; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 64 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 65 or 66.

[0033] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to EGFRvIII, comprising an anti-EGFRvIII variable light chain domain comprising the amino acid sequence of SEQ ID NO: 31 and an anti-EGFRvIII variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 30; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 32 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 33.

[0034] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to BCMA, comprising an anti-BCMA variable light chain domain containing the amino acid sequence of SEQ ID NO: 95 and an anti-BCMA variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 94; a second binding domain comprising an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 96 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 98 or SEQ ID NO: 97.

[0035] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to PSMA, comprising an anti-PSMA variable light chain domain comprising the amino acid sequence of SEQ ID NO: 119 or 107 and an anti-PSMA variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 118 or 106; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 120 or 108 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NOs: 121, 122, 109, 110, or 111.

[0036] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CD33, comprising an anti-CD33 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 18 or 19 and an anti-CD33 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 16 or 17; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 189 or 190 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 20, 21, 22, or 23.

[0037] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CDH19, comprising an anti-CDH19 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 53 and an anti-CDH19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 52; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 54 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 55.

[0038] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to MUC17, comprising an anti-MUC17 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 155, 164, 173, or 182 and an anti-MUC17 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 154, 163, 172, or 181; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 156, 165, 174, or 183.

[0039] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to cldn18.2, which includes an anti-cldn18.2 variable light chain domain containing the amino acid sequence of SEQ ID NO: 139 or 144 and an anti-cldn18.2 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 138 or 143; a second binding domain containing an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain containing the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain containing the amino acid sequence of SEQ ID NO: 140 or 145, and a second binding domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 141, 142, 146, or 147.

[0040] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CD70, comprising an anti-CD70 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 130 and an anti-CD70 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 129; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 131.

[0041] The pharmaceutical formulations of this disclosure include a buffering agent. In some embodiments, the buffering agent is an acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, or a combination thereof. In some embodiments, the buffering agent is present in the formulation at a concentration ranging from about 5 mM to about 200 mM (or about 10 mM to about 50 mM).

[0042] The pharmaceutical formulations of this disclosure contain sugars. In some embodiments, the sugars are monosaccharides or disaccharides. In some embodiments, the sugars are sugar alcohols (e.g., sorbitol). In some embodiments, the sugars are sucrose, trehalose, mannitol, sorbitol, or a combination thereof. In some embodiments, the sugars are present in the formulation at concentrations ranging from about 1 to about 15 (w / V)% (or about 9 to about 12 (w / V)% or about 5 to about 12 (w / V)% or about 7 to about 12 (w / V)%).

[0043] The pharmaceutical formulations of this disclosure include surfactants. In some embodiments, the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X-100, polyoxyethylene, PEG3350, PEG4000, or a combination thereof. In some embodiments, the surfactant is present in the formulation at a concentration ranging from 0.004 to about 0.5 (w / V)% (or about 0.001 to about 0.01 (w / V)%, or about 0.001 to about 0.5 (w / V)%, or about 0.004 to about 0.01 (w / V)%).

[0044] In some embodiments, the formulation has a molar osmotic concentration in the range of about 150 to about 500 mOsm. In some embodiments, the formulation has a molar osmotic concentration of less than 500 mOsm / L, 450 mOsm / L, 400 mOsm / L, or 350 mOsm / L. In some embodiments, the formulation is close to isotonic, for example, 250 to 350 mOsm / L.

[0045] In some embodiments, the pharmaceutical formulation comprises 10 mM glutamate, 9 (w / V)% sucrose, and 0.01 (w / V)% polysorbate 80, where the pH of the pharmaceutical formulation is 4.2. In some embodiments, the bispecific antibody construct is present in the formulation at concentrations ranging from about 10 mg / mL to about 100 mg / mL. In some embodiments, the bispecific antibody construct is present in the formulation at concentrations of 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL. In some embodiments, the bispecific antibody is present in the formulation in an amount ranging from about 1000 μg to about 200 mg.

[0046] In some embodiments, the pharmaceutical formulations of this disclosure are liquid formulations.

[0047] In another embodiment, a method for treating cancer in a subject requiring such treatment is described, the method comprising the step of administering a formulation of the Disclosure to the subject.

[0048] While various embodiments in this specification are presented using the word “including” in various contexts, it should be understood that relevant embodiments may also be described using the words “consisting of” or “essentially consisting of.” This disclosure intends that embodiments described as “including” a certain feature include embodiments “consisting of” that feature. Note that the terms “one (a)” or “one (an)” refer to one or more; for example, “one immunoglobulin molecule” is understood to represent one or more immunoglobulin molecules. Therefore, the terms “one (a)” (or “one (an)”), “one or more,” and “at least one” may be used interchangeably in this specification.

[0049] When specifying a range of values, it should also be understood that the described characteristics may be individual values ​​found within that range. For example, "pH of approximately pH 4 to approximately pH 6" could be any value between pH 4, 4.2, 4.6, 5.1, 5.5, etc., without limitation. Furthermore, "pH of approximately pH 4 to approximately pH 6" should not be interpreted as meaning that the pH of the target formulation will fluctuate in 2 pH increments within the pH 4 to pH 6 range during storage, but rather that a value within that range may be selected for the pH of the solution, and the pH will remain buffered around that pH.

[0050] When the term "approximately" is used, it means adding or subtracting 5%, 10%, 15%, or more of the listed number from the listed number. The actual intended variation can be determined from the context.

[0051] In any of the scopes described herein, the endpoints of the scope are included within that scope. However, this description also intends the same scope with the smaller and / or larger endpoints excluded. Additional features and variations of the present invention will be apparent to those skilled in the art from the whole of this application, including the drawings and detailed description, but all such features are intended as aspects of the present invention. Similarly, the features of the present invention described herein can be rearranged to form additional embodiments that are also intended as aspects of the present invention, whether or not the combination of features is specifically described above as an aspect or embodiment of the present invention. Furthermore, only such limitations described herein as essential to the present invention should be considered as such; variations of the present invention lacking limitations not described herein as essential are also intended as aspects of the present invention.

[0052] All references listed herein are incorporated herein by reference in their entirety. The present invention provides, for example, the following items: (Item 1) A stable pharmaceutical formulation comprising at least 10 mg / mL of a bispecific antibody construct, a buffer, sugars, and a surfactant, and having a pH in the range of 4 to 6. (Item 2) The preparation described in item 1, which has been freeze-dried. (Item 3) A formulation described in item 1 or 2, which is stable for up to 3 months at 4°C. (Item 4) The aforementioned formulation is a formulation according to any one of items 1 to 3, which contains less than 2% high molecular weight species after 3 months at 4°C. (Item 5) The aforementioned formulation is a formulation according to any one of items 1 to 4, having a pH of 4 to 5. (Item 6) The aforementioned formulation is a formulation according to any one of items 1 to 4, having a pH of 4.2. (Item 7) The preparation described in any one of items 1 to 6, wherein the buffering agent is an acetate buffer, a glutamate buffer, a citrate buffer, a lactic acid buffer, a succinate buffer, a tartaric acid buffer, a fumarate buffer, a maleate buffer, a histidine buffer, or a phosphate buffer. (Item 8) The buffering agent is present in a concentration in the range of 5 to 200 mM, and is a formulation according to any one of items 1 to 7. (Item 9) The buffering agent is present in a concentration in the range of 10 to 50 mM, as described in item 8. (Item 10) The preparation described in any one of items 1 to 9, wherein the aforementioned sugars are monosaccharides or disaccharides. (Item 11) The preparation according to any one of items 1 to 10, wherein the aforementioned sugars are glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, or xylitol. (Item 12) The aforementioned sugars are present in a concentration ranging from 1 to 15 (w / v)%, as described in any one of items 1 to 11. (Item 13) The aforementioned sugars are present in a concentration ranging from 5 to 12 (w / v)%, as described in item 12. (Item 14) The aforementioned sugars are present in a concentration in the range of 7-12 (w / v)% in the preparation described in item 12. (Item 15) The surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, Triton X-100, polyoxyethylene, PEG3350, PEG4000, or a combination thereof, as described in any one of items 1 to 154. (Item 16) The surfactant is present in a concentration ranging from 0.001% to 0.5(w / v)%, and the formulation is one of the items 1 to 15. (Item 17) The formulation described in item 16, wherein the surfactant is present in a concentration in the range of 0.001(w / v)% to 0.01(w / v)%. (Item 18) The aforementioned bispecific antibody construct is present in a concentration in the range of 10 mg / mL to 20 mg / mL, and is a formulation according to any one of items 1 to 17. (Item 19) The aforementioned bispecific antibody construct is a formulation described in any one of items 1 to 18, present in an amount of 20 mg / mL. (Item 20) The preparation described in any one of items 1 to 19 comprises 10 mM glutamate, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80, and the pH of the preparation is 4.2. (Item 21) The preparation according to any one of items 1 to 20, wherein the bispecific antibody construct comprises a first binding domain that binds to a target cell surface antigen and a second binding domain that binds to human CD3 on the surface of T cells. (Item 22) The formulation according to item 21, wherein the bispecific antibody construct further comprises a third domain including a hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain in the order of amino to carboxyl. (Item 22) The formulation according to item 21, wherein each of the first and second binding domains includes a VH region and a VL region. (Item 23) The formulation according to item 21 or 22, wherein the bispecific antibody construct is a single-chain antibody construct. (Item 24) The formulation according to any one of items 21 to 23, wherein the target cell surface antigen is CDH19, MSLN, DLL3, FLT3, EGFR, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, MUC17, or CLDN18.2. (Item 25) The formulation according to any one of items 21 to 24, wherein the first binding domain of the bispecific antibody construct comprises one set of 6 CDRs shown in (a) SEQ ID NOs. 24-29, (b) SEQ ID NOs. 34-39, (c) SEQ ID NOs. 78-83, (d) SEQ ID NOs. 10-15, (e) SEQ ID NOs. 46-51, (f) SEQ ID NOs. 88-93, (g) SEQ ID NOs. 67-72, (h) SEQ ID NOs. 56-61, (i) SEQ ID NOs. 112-117, (j) SEQ ID NOs. 100-105, (k) SEQ ID NOs. 148-153, SEQ ID NOs. 157-162, or SEQ ID NOs. 166-171, or SEQ ID NOs. 175-180, (l) SEQ ID NOs. 132-137, or (m) SEQ ID NOs. 123-128. (Item 26) The formulation according to any one of items 21 to 25, wherein the second binding domain of the bispecific antibody construct comprises one set of 6CDRs shown in SEQ ID NOs: 1 to 6. (Item 27) A formulation according to any one of items 21 to 26, wherein the first binding domain is: (a) The VH region containing the amino acid sequence shown in SEQ ID NO: 30, and the VL region containing the amino acid sequence shown in SEQ ID NO: 31; (b) The VH region containing the amino acid sequence shown in SEQ ID NO: 40 and the VL region containing the amino acid sequence shown in SEQ ID NO: 41; (c) The VH region containing the amino acid sequence shown in SEQ ID NO: 84 and the VL region containing the amino acid sequence shown in SEQ ID NO: 85; (d) The VH region containing the amino acid sequence shown in SEQ ID NO: 16 or 17, and the VL region containing the amino acid sequence shown in SEQ ID NO: 18 or 19; (e) The VH region containing the amino acid sequence shown in SEQ ID NO: 52 and the VL region containing the amino acid sequence shown in SEQ ID NO: 53; (f) The VH region containing the amino acid sequence shown in SEQ ID NO: 94 and the VL region containing the amino acid sequence shown in SEQ ID NO: 95; (g) The VH region containing the amino acid sequence shown in SEQ ID NO: 73 and the VL region containing the amino acid sequence shown in SEQ ID NO: 74; (h) The VH region containing the amino acid sequence shown in SEQ ID NO: 62 and the VL region containing the amino acid sequence shown in SEQ ID NO: 63; (i) The VH region containing the amino acid sequence shown in SEQ ID NO: 118 and the VL region containing the amino acid sequence shown in SEQ ID NO: 119; (j) A VH region containing the amino acid sequence shown in SEQ ID NOs. 154, 163, 172, or 181, and a VL region containing the amino acid sequence shown in SEQ ID NOs. 155, 164, 173, or 182; (k) The VH region containing the amino acid sequence shown in SEQ ID NO: 106 and the VL region containing the amino acid sequence shown in SEQ ID NO: 107; (l) The VH region containing the amino acid sequence shown in SEQ ID NO: 138 or 143, and the VL region containing the amino acid sequence shown in SEQ ID NO: 139 or 144; or (m) The VH region containing the amino acid sequence shown in SEQ ID NO: 129 and the VL region containing the amino acid sequence shown in SEQ ID NO: 130; A preparation containing the above ingredients. (Item 28) The formulation according to any one of items 21 to 27, wherein the second binding domain includes a VH region containing the amino acid sequence shown in SEQ ID NO: 7 and a VL region containing the amino acid sequence shown in SEQ ID NO: 8. (Item 29) The aforementioned bispecific antibody construct is a formulation according to any one of items 21 to 28, comprising the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 55, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 131, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 156, SEQ ID NO: 165, SEQ ID NO: 174, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188. (Item 30) A method for treating cancer in a subject requiring such treatment, comprising the step of administering the preparation described in any one of items 1 to 29 to the subject. [Brief explanation of the drawing]

[0053] [Figure 1] This graph shows the percentage of high molecular weight (HMW) species of BiTE®-I, BiTE®-C, and BiTE®-G, respectively, at 20 mg / mL in a lyophilized formulation containing 10 mM glutamate, 9 (w / V)% sucrose, 0.01 (w / V)% polysorbate 80, and pH 4.2, as evaluated by size exclusion ultrahigh performance liquid chromatography (SE-UHPLC) under storage conditions at 4°C at time points t (0, 1 month, and 3 months). [Figure 2] This graph shows the percentage of high molecular weight (HMW) species of BiTE®-I, BiTE®-C, and BiTE®-G at 20 mg / mL in a liquid formulation containing 10 mM glutamate, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, and pH 4.2, as evaluated by size exclusion ultrahigh performance liquid chromatography (SE-UHPLC) at time points t (0, 1 month, and 3 months) under storage conditions at 4°C. [Modes for carrying out the invention]

[0054] While current therapeutic biotechnology products are of high quality, and recombinant human proteins and antibodies closely resemble endogenous human proteins, protein instability remains a significant concern. In this field, there is a critical need for increased stability and reduced aggregation in therapeutic proteins, and optimized pharmaceutical formulations can help achieve this.

[0055] Typically, the antibody constructs described herein are stored and / or used in lyophilized formulations at concentrations of approximately 1 mg / mL (International Publication No. 2018 / 204907) or at most 8 mg / mL (International Publication No. 2018 / 141910). At higher concentrations, a tendency for aggregation is generally observed. The formulations described in International Publication No. 2018 / 204907 include preservatives (e.g., chlorobutanol, methylparaben, or benzyl alcohol) with antibody concentrations of 0.5 mg / mL to 20 mg / mL, which contribute to the stabilization of the bispecific antibody constructs. In some cases, stability is achieved by other means, such as lowering the pH of the formulation. For example, International Publication No. 2018 / 141910 discloses that a formulation containing a bispecific antibody construct (5 mg / mL) with a low pH (i.e., pH 4.0) is more stable than a formulation with the same antibody concentration at a more basic pH (e.g., pH 6 or higher). Therefore, a low pH contributed to the stability of the formulation. However, as described herein, high-concentration (e.g., 20 mg / mL) bispecific antibody construct formulations (both liquid and lyophilized) were unexpectedly stable (e.g., had a low percentage of high molecular weight (HMW) species) at both 4°C and 40°C at various time points without the need for the addition of preservatives or other stabilizers with a more acidic pH. The high-concentration formulations with stability disclosed herein are preferable to formulations known in the art because it is beneficial to eliminate non-essential components in the pharmaceutical formulation, such as other components acting as preservatives or stabilizers.

[0056] This disclosure provides a stable formulation comprising a high concentration of a bispecific antibody construct (e.g., a BiTE® molecule), wherein approximately less than 2% of the bispecific antibody construct is present as a high molecular weight (HMW) species in the formulation.

[0057] Within the scope of this disclosure, the terms “stability” or “stabilization” refer to the stability of the entire pharmaceutical formulation, and more particularly to the stability of the active ingredient itself (e.g., a bispecific single-chain antibody construct), specifically during formulation, filling, transport, storage, and administration. A “stable formulation” is a formulation in which the bispecific antibody construct within it essentially maintains its physical and / or chemical integrity as well as its biological activity after storage and during processes (e.g., freeze / thaw, mechanical mixing, and lyophilization). Protein stability can be measured by the formation of high molecular weight (HMW) species, loss of enzyme activity, generation of peptide fragments, and shifts in charge profiles.

[0058] As used herein, the term "aggregation" refers to direct intermolecular attractive forces, such as van der Waals forces or chemical bonds. In particular, aggregation is understood to be the accumulation and clumping of proteins. Aggregates may include amorphous aggregates and oligomers, and are typically called high molecular weight (HMW) species, i.e., molecules with a higher molecular weight than the non-aggregated product molecules.

[0059] As used herein, the term "(protein) aggregate" typically encompasses high molecular weight protein species such as "oligomers" or "multimers," rather than a specific, predetermined species (e.g., monomers). This term is used interchangeably with the terms "high molecular weight" species and "HMW" herein. Protein aggregates can typically differ in size (ranging from small (dimers) to large aggregates (subvisible particles or even visible particles) and in diameters ranging from nanometers to micrometers), morphology (nearly spherical to fibrous), protein structure (natural vs. unnatural / denatured), type of intermolecular bonding (covalent vs. non-covalent), reversibility, and solubility. Soluble aggregates occupy a size range of approximately 1–100 nm, while protein microparticles occupy ranges from microscopically invisible (approximately 0.1–100 nm) to visible (>100 nm). All of the aforementioned types of protein aggregates are typically encompassed by this term. Therefore, the term "(protein) aggregate" refers to any type of unnatural species in which two or more protein monomers are physically associated or chemically bonded.

[0060] As used herein, the term "low molecular weight (LMW) species" refers to a fragment of a bispecific antibody construct.

[0061] As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a subject requiring it. The terms “subject,” “individual,” “animal,” or “patient” are used interchangeably herein and refer to any subject, particularly mammalian subjects, for which administration of the pharmaceutical composition of the present invention is desirable. Examples of mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc., but humans are preferred. The pharmaceutical formulations of this disclosure are stable and pharmaceutically acceptable, that is, they can exert the desired therapeutic effect without causing any significant undesirable local or systemic effects in the subject to which the pharmaceutical formulation is administered. The pharmaceutically acceptable formulations of the present invention may be sterile. Specifically, the term “pharmaceutically acceptable” may mean that it is approved by a regulatory authority or other generally recognized pharmacopoeia for use in animals (more specifically humans), and is not limited to those approved by a regulatory authority.

[0062] In some embodiments, this disclosure describes a formulation comprising a bispecific antibody construct that binds to CD3 on human T cells in an amount of at least 10 mg / mL, a buffer, a sugar, and a surfactant, wherein the formulation has a pH in the range of 4 to 6. In some embodiments, the bispecific antibody construct co-engages CD3 and one of human CDH19, human MSLN, human DLL3, human FLT3, human EGFRvlll, human BCMA, human PSMA, human CD33, human CD19, human CD70, human CLDN18.2, or MUC17 in such a manner that malignant cells transiently link to T cells, thereby inducing T cell-mediated elimination of the bound malignant cells.

[0063] Various aspects of this formulation are described below. The use of section headings in this specification is solely for convenience of reading and is not intended to limit them in themselves. This entire specification is intended to be considered a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated.

[0064] Antigen-binding protein An "antigen-binding protein" is a protein containing a domain that binds to a specific target antigen (such as CD3 and / or CDH19, MSLN, DLL3, FLT3, EGFRvll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17). An antigen-binding protein includes a backbone or framework portion that allows the antigen-binding domain to adopt a three-dimensional structure that promotes the binding of the antigen-binding protein to the antigen. In exemplary embodiments, the antigen-binding protein is an antibody or immunoglobulin, or an antigen-binding antibody fragment.

[0065] The term "antibody" refers to an intact antigen-binding immunoglobulin. An "antibody" is a type of antigen-binding protein. An antibody may be an IgA, IgD, IgE, IgG, or IgM antibody containing any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, an intact antibody contains two full-length heavy chains and two full-length light chains. An antibody has one variable region and one constant region. In the IgG form, the one variable region is generally about 100 to 110 or more amino acids and contains three complementarity-determining regions (CDRs) that are primarily involved in antigen recognition and are substantially different among other antibodies that bind to different antigens. A single variable region typically contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within a framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, cited above). The constant region allows the antibody to recruit cells and molecules of the immune system.

[0066] In some embodiments, the antibody in the formulation is a bispecific antibody, i.e., an antibody that binds to two different targets (e.g., CD3 and a second different target).

[0067] As used herein, the term “bispecificity” refers to an antibody construct that binds to two different target antigens, i.e., it comprises a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (e.g., a surface antigen of a target cell) and the second binding domain binds to another antigen or target (e.g., CD3). Thus, the antibody constructs according to this disclosure include specificity to two different antigens or targets. The term “surface antigen of a target cell” refers to an antigenic structure expressed by a cell and present on its cell surface so that the antibody construct described herein can access it. It may be a protein, preferably the extracellular portion of a protein, or a carbohydrate structure, preferably a carbohydrate structure of a protein such as a glycoprotein. It is preferably a tumor antigen. The present invention also encompasses multispecificity antibody constructs, such as a tripspecificity antibody construct, the latter encompassing constructs having three binding domains or more than three (e.g., four, five, etc.) specificity.

[0068] The bispecific antibodies and / or antibody constructs understood herein include, but are not limited to, conventional bispecific immunoglobulins (e.g., BsIgG), IgG containing an added antigen-binding domain (e.g., the amino or carboxyl terminus of the light or heavy chain is linked to an additional antigen-binding domain such as a single-domain antibody or a paired antibody variable domain (e.g., Fv or scFv)), BsAb fragments (e.g., bispecific single-chain antibodies), bispecific fusion proteins (e.g., an antigen-binding domain fused to an effector portion), and BsAb conjugates. For example, see Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015), which describes various forms of bispecificity and is incorporated herein by reference. Examples of bispecific constructs include, but are not limited to, diabodies, single-chain diabodies, tandem scFvs, bispecific T cell engagers (BiTE®) (fusion proteins consisting of two single-chain variable fragments (scFvs) linked by a linker), and modified constructs including Fab2 bispecifics and full-length antibodies. For example, all of the following are explicitly incorporated herein: Chames & Baty, 2009, mAbs 1[6]:1-9; and Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-1136; Wu et al., 2007, Nature Biotechnology 25

[11] :1290-1297; Michaelson et al., 2009, mAbs 1[2]:128-141; International Publication No. 2009032782 and International Publication No. 2006020258; Zuo et al., 2000, Protein Engineering 13[5]:361-367; U.S. Patent Application Publication No. 20020103345; Shen et al., 2006, J Biol Chem See 281

[16] :10706-10714; Lu et al., 2005, J Biol Chem 280

[20] :19665-19672; and Kontermann, 2012 MAbs 4(2):182.

[0069] In some embodiments, the formulations described herein are bispecific antibody constructs comprising a first binding domain that binds to a target cell surface antigen, a second binding domain that binds to human CD3 on the surface of T cells, and a third domain optionally comprising a hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain in the order of amino to carboxyl. In some embodiments, each of the first and second binding domains comprises a VH region and a VL region.

[0070] As used herein, the term "binding domain" refers to a domain that (specifically) binds to, interacts with, or recognizes a predetermined target epitope or target site on a target molecule (antigen), such as CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17, and CD3, respectively. The structure and function of the first binding domain (which recognizes CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17), and preferably the structure and / or function of the second binding domain (which recognizes CD3), are based on the structure and / or function of the antibody, for example, the full-length or complete immunoglobulin molecule, and / or derived from the variable heavy chain (VH) domain and / or variable light chain (VL) domain of the 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 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The second binding domain preferably also includes the minimum structural requirements of the antibody that enable target binding. More preferably, the second binding domain includes at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The first and / or second binding domains are assumed to be constructed or obtained by phage display or library screening, rather than by transplanting CDR sequences derived from an existing (monoclonal) antibody into the backbone.

[0071] In some embodiments, the first binding domain that binds to the surface antigen of the target cell and / or the second binding domain that binds to CD3ε are human binding domains. Antibodies and antibody constructs containing at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have non-human variable regions and / or constant regions, such as those of rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins may result in rapid clearance of the antibody or antibody construct, or may trigger an immune response by the patient to the antibody or antibody construct. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody function into rodents so that the rodents produce fully human antibodies.

[0072] In some embodiments, the antigen-binding protein comprises a single-chain antibody construct. The scFv comprises a variable heavy chain, an scFv linker, and a variable light chain domain. Optionally, the C-terminus of the variable light chain is bound to the N-terminus of the scFv linker, and its C-terminus is bound to the N-terminus of the variable heavy chain (N-vh-linker-vl-C), but the configuration is reversible (N-vl-linker-vh-C). Alternatively, the C-terminus of the variable heavy chain is bound to the N-terminus of the scFv linker, and its C-terminus is bound to the N-terminus of the variable light chain (N-vl-linker-vh-C), but the configuration is reversible (N-vh-linker-vC). Therefore, both orientations of the scFv are specifically included in the description and explanation of the scFv.

[0073] At least two binding domains and variable domains (VH / VL) of the antibody constructs of this disclosure may or may not contain a peptide linker (spacer peptide). According to the present invention, the term “peptide linker” includes an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antibody construct of this disclosure. A peptide linker may also be used to fuse a third domain to other domains of the antibody construct of the present invention. An essential technical feature of such a peptide linker is that it does not contain any polymerization activity. Particularly preferred peptide linkers are those described in U.S. Patent No. 4,751,180 and No. 4,935,233 or International Publication No. 88 / 09344, whose disclosures are incorporated herein by reference as a whole. Peptide linkers can also be used to add other domains, modules, or regions (e.g., half-life extension domains) to the bispecific antibody constructs described herein.

[0074] In some embodiments, the third domain includes "Fc" or "Fc region" or "Fc domain," referring to a polypeptide containing the constant region of an antibody that excludes the first constant region immunoglobulin domain. Thus, the "Fc domain" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the mobile hinge N-terminus to these domains. With respect to IgA and IgM, Fc may include the J chain. With respect to IgG, the Fc domain includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). The bispecific antibody construct is preferably an IgG antibody (including, but not limited to, several subclasses, including IgG1, IgG2, IgG3, and IgG4). The boundaries of the Fc region may vary, but the human IgG heavy chain Fc region is typically defined as encompassing residues C226 or P230 to its carboxyl terminus, and is numbered according to the EU index as described in Kabat. In some embodiments, amino acid modifications are made to the Fc region to alter, for example, binding to one or more FcγR receptors or FcRn receptors.

[0075] In some embodiments, the formulations described herein include a bispecific antibody construct that binds to human CD3 and human CDH19, or human CD3 and human MSLN, or human CD3 and human DLL3, or human CD3 and human FLT3, or human CD3 and human EGFRvIII, or human CD3 and human BCMA, or human CD3 and PSMA, or human CD3 and human CD33, or human CD3 and human CD19, human CD3 and human CD70, or human CD3 and human MUC17, or human CD3 and human CLDN18.2.

[0076] In some embodiments, the first binding domain of the bispecific antibody construct includes one set of 6 CDRs shown in (a) SEQ ID NOs: 24-29, (b) SEQ ID NOs: 34-39, (c) SEQ ID NOs: 78-83, (d) SEQ ID NOs: 10-15, (e) SEQ ID NOs: 46-51, (f) SEQ ID NOs: 88-93, (g) SEQ ID NOs: 67-72, (h) SEQ ID NOs: 56-61, (i) SEQ ID NOs: 112-117, (j) SEQ ID NOs: 100-105, (k) SEQ ID NOs: 148-153, SEQ ID NOs: 157-162, or SEQ ID NOs: 166-171, or SEQ ID NOs: 175-180, (l) SEQ ID NOs: 132-137, or (m) SEQ ID NOs: 123-128.

[0077] In some embodiments, the first binding domain of the bispecific antibody construct includes a VH region containing an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in sequence numbers 30, 40, 84, 16, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143, or 129.

[0078] In some embodiments, the first binding domain of the bispecific antibody construct includes a VL region containing an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NOs: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144, or 130.

[0079] In some embodiments, the first binding domain is (a) a VH region containing the amino acid sequence shown in SEQ ID NO: 30 and a VL region showing SEQ ID NO: 31; (b) a VH region containing the amino acid sequence shown in SEQ ID NO: 40 and a VL region showing the amino acid sequence shown in SEQ ID NO: 41; (c) a VH region containing the amino acid sequence shown in SEQ ID NO: 84 and a VL region containing the amino acid sequence shown in SEQ ID NO: 85; (d) a VH region containing the amino acid sequence shown in SEQ ID NO: 16 or 17 and a VL region containing the amino acid sequence shown in SEQ ID NO: 18 or 19; (e) a VH region containing the amino acid sequence shown in SEQ ID NO: 52 and a VL region containing the amino acid sequence shown in SEQ ID NO: 53; (f) a VH region containing the amino acid sequence shown in SEQ ID NO: 94 and a VL region containing the amino acid sequence shown in SEQ ID NO: 95; (g) a VH region containing the amino acid sequence shown in SEQ ID NO: 73 and an amino acid sequence shown in SEQ ID NO: 74 (h) A VL region containing a column; (i) A VH region containing the amino acid sequence shown in SEQ ID NO: 62 and a VL region containing the amino acid sequence shown in SEQ ID NO: 63; (j) A VH region containing the amino acid sequence shown in SEQ ID NO: 118 and a VL region containing the amino acid sequence shown in SEQ ID NO: 119; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 154, 163, 172, or 181 and a VL region containing the amino acid sequence shown in SEQ ID NO: 155, 164, 173, or 182; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 106 and a VL region containing the amino acid sequence shown in SEQ ID NO: 107; (l) A VH region containing the amino acid sequence shown in SEQ ID NO: 138 or 143 and a VL region containing the amino acid sequence shown in SEQ ID NO: 139 or 144; or (m) A VH region containing the amino acid sequence shown in SEQ ID NO: 129 and a VL region containing the amino acid sequence shown in SEQ ID NO: 130.

[0080] In some embodiments, the second binding domain of the bispecific antibody construct contains a set of 6CDRs shown in SEQ ID NOs: 1-6.

[0081] In some embodiments, the second binding domain of the bispecific antibody construct includes a VH region containing an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 7.

[0082] In some embodiments, the second binding domain of the bispecific antibody construct includes a VL region containing an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 8.

[0083] In some embodiments, the second binding domain includes (a) a VH region containing the amino acid sequence shown in SEQ ID NO: 7 and a VL region containing the amino acid sequence shown in SEQ ID NO: 8.

[0084] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CD19, comprising an anti-CD19 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 85 and an anti-CD19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 84; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 86 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 87.

[0085] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to MSLN, comprising an anti-MSLN variable light chain domain containing the amino acid sequence of SEQ ID NO: 41 and an anti-MSLN variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 40; a second binding domain comprising an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 42 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 43, 44, or 45.

[0086] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to DLL3, comprising an anti-DLL3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 74 and an anti-DLL3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 73; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 75 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 76 or 77.

[0087] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to FLT3, comprising an anti-FLT3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 63 and an anti-FLT3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 62; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 64 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 65 or 66.

[0088] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to EGFRvIII, comprising an anti-EGFRvIII variable light chain domain comprising the amino acid sequence of SEQ ID NO: 31 and an anti-EGFRvIII variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 30; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 32 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 33.

[0089] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to BCMA, comprising an anti-BCMA variable light chain domain containing the amino acid sequence of SEQ ID NO: 95 and an anti-BCMA variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 94; a second binding domain comprising an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 96 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 98 or SEQ ID NO: 97.

[0090] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to PSMA, comprising an anti-PSMA variable light chain domain comprising the amino acid sequence of SEQ ID NO: 119 or 107 and an anti-PSMA variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 118 or 106; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 120 or 108 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NOs: 121, 122, 109, 110, or 111.

[0091] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CD33, comprising an anti-CD33 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 18 or 19 and an anti-CD33 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 16 or 17; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 189 or 190 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 20, 21, 22, or 23.

[0092] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CDH19, comprising an anti-CDH19 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 53 and an anti-CDH19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 52; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain comprising the amino acid sequence of SEQ ID NO: 54 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 55.

[0093] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to MUC17, comprising an anti-MUC17 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 155, 164, 173, or 182 and an anti-MUC17 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 154, 163, 172, or 181; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 156, 165, 174, or 183.

[0094] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to cldn18.2, which includes an anti-cldn18.2 variable light chain domain containing the amino acid sequence of SEQ ID NO: 139 or 144 and an anti-cldn18.2 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 138 or 143; a second binding domain containing an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain containing the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain containing the amino acid sequence of SEQ ID NO: 140 or 145, and a second binding domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 141, 142, 146, or 147.

[0095] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to CD70, comprising an anti-CD70 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 130 and an anti-CD70 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 129; a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 131.

[0096] In some embodiments, the formulation contains an antigen-binding protein described herein (e.g., a bispecific antibody construct) in an amount ranging from about 10 mg to about 50 mg (or about 10 mg to about 20 mg, or about 20 mg to about 50 mg, or about 15 mg to about 20 mg, or about 20 mg to about 55 mg). In some embodiments, the formulation contains a bispecific antibody construct in an amount of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg.

[0097] In some embodiments, the formulation contains a bispecific antibody construct in an amount of about 10 mg / mL to about 50 mg / mL (or about 10 mg / mL to about 20 mg / mL or about 15 mg / mL to about 20 mg / mL). In some embodiments, the formulation contains a bispecific antibody construct at concentrations of about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL or about 50 mg / mL. In some embodiments, the formulation contains a bispecific antibody construct at a concentration of about 20 mg / mL.

[0098] cushioning agent The pharmaceutical formulation of the present invention optionally contains a buffering agent which may be an acetate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate, or a combination thereof.

[0099] Buffers are often used to control the pH of a formulation. In some embodiments, buffers are added at concentrations that maintain the pH of the formulation at approximately 4–6, approximately 4–5, or approximately 4.2. The effect of pH on the formulation can be characterized using one or more of several methods, such as accelerated stability testing and calorimetry screening tests (Remmele RLJr., et al., Biochemistry, 38(16):5241-7 (1999)).

[0100] The buffer system present in the formulation is selected to be physiologically compatible and to maintain the desired pH. The buffer may be present at concentrations of about 0.1 mM to about 1000 mM (1 M), or about 5 mM to about 200 mM, or about 5 mM to about 100 mM, or about 10 mM to about 50 mM. Preferred buffer concentrations include concentrations of about 200 mM or less. In some embodiments, the buffer in the formulation is present at concentrations of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM, or about 5 mM. In some embodiments, the concentration of the buffer is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffer is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM and 100 mM. In some embodiments, the concentration of the buffer is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM and 50 mM. In some embodiments, the concentration of the buffer is about 10 mM.

[0101] surfactant The pharmaceutical formulations described herein include surfactants. Typical surfactants include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X-100, polyoxyethylene, PEG3350, PEG4000, or combinations thereof.

[0102] The pharmaceutical formulations described herein include at least a surfactant, either individually or in mixtures of various proportions. In some embodiments, the formulations contain a surfactant at a concentration of about 0.001% to about 5(w / v)% (or about 0.001 to about 0.5(w / v)%, or about 0.004 to about 0.5(w / v)%, or about 0.001 to about 0.01(w / v)%, or about 0.004 to about 0.01(w / v)%). In some embodiments, the formulation contains a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5 (w / v)%. In some embodiments, the formulation contains a surfactant at a concentration of about 0.001 (w / v)% to about 0.5 (w / v)%. In some embodiments, the formulation contains a surfactant at a concentration of about 0.001 to about 0.01 (w / v)%. In some embodiments, the formulation contains a surfactant at a concentration of about 0.001 to about 0.01 (w / v)%. In some embodiments, the formulation contains a surfactant at concentrations of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4% to about 0.5 (w / v)%. In some embodiments, the formulation contains a surfactant incorporated at a concentration of about 0.001% to about 0.01 (w / v)%. In some embodiments, the surfactant is polysorbate 80, and the polysorbate 80 is present at a concentration of about 0.01 (w / v)%.

[0103] Sugars The pharmaceutical formulations described herein contain sugars. In some embodiments, the sugars are monosaccharides or disaccharides. In some embodiments, the sugars are glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, or xylitol, or a combination thereof.

[0104] In some embodiments, the pharmaceutical formulation contains sugars at concentrations of about 0.01% to about 40(w / v)%, or about 0.001% to about 20(w / v)%, or about 1% to about 15%, or about 5% to about 12%, or about 7% to about 12(w / v)%. In some embodiments, the pharmaceutical formulation contains at least sugars at concentrations of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, or at least 40(w / v)%. In some embodiments, the pharmaceutical formulation contains at least sugars at concentrations of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15(w / v)%. In some embodiments, the pharmaceutical formulation contains at least sugars at concentrations of about 1% to about 15(w / v)%. In yet another embodiment, the pharmaceutical formulation contains at least sugars at concentrations of about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12(w / v)%. In some embodiments, the pharmaceutical formulation contains at least sugars at concentrations of about 7% to about 12(w / v)%. In some embodiments, at least sugars are present in the formulation at a concentration of about 9 (w / v)%. In some embodiments, the sugars are sucrose and are present in the formulation in a range of about 9% to about 12 (w / v)%.

[0105] In preferred embodiments, the pharmaceutical formulation comprises 10 mM glutamate, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80, where the pH of the pharmaceutical formulation is 4.2. In some embodiments, the formulation is lyophilized.

[0106] stability The stability of bispecific antibody construct formulations can be quantified by several methods. In some embodiments, the stability of the antibody formulation is characterized by size exclusion high-performance liquid chromatography (SE-HPLC), size exclusion high-performance liquid chromatography (SE-UHPLC), cation exchange high-performance liquid chromatography (CE-HPLC), dynamic light scattering, analytical centrifugation (AUC), field flow fractionation (FFF), isoelectric focusing, and ion exchange chromatography (IEX). In some embodiments, the stability of the antibody formulation is characterized by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) and / or sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is characterized by partial dissociation measured by SDS-PAGE. In some embodiments, the stability of the formulation is evaluated by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). The rCE-SDS method separates heavy chain (HC), light chain (LC), nonglycosylated HC (NGHC), and other fine peak species and groups under reducing conditions.

[0107] In some embodiments, the stability of the formulation is characterized by the amount of high molecular weight (HMW) species in the bispecific antibody construct or the rate of increase of the amount of HMW species in the bispecific antibody construct under storage conditions at various time points. In some embodiments, the amount of HMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4°C or 40°C. In some embodiments, the rate of increase of HMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4°C or 40°C. In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0108] The stability of a bispecific antibody construct, and the ability of a formulation to maintain the stability of the bispecific antibody construct, can be evaluated over long periods (e.g., several weeks or months). In the context of formulations, a stable formulation is one in which the bispecific antibody construct within it essentially maintains its physical and / or chemical integrity, as well as its biological activity after storage and during processing, such as through freeze / thaw, mechanical mixing, and lyophilization. The stability of a bispecific antibody construct can be evaluated, for example, by the level and / or rate of high molecular weight (HMW) aggregate formation, shifts in the charge profile, and changes in particle size.

[0109] In some embodiments, the relative value of any particular species of the bispecific antibody construct, such as intact BiTE® molecules or major species, or high molecular weight (HMW) species (i.e., aggregates), or low molecular weight (LMW) species (i.e., fragments), as described herein, is expressed in relation to each numerical value of the total product. For example, in some embodiments, the formulation is a lyophilized formulation, and 2.5% or less of the bispecific antibody construct (e.g., 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) is present as HMW species in the lyophilized formulation. In some embodiments, the amount of HMW species in the lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the formulation increases by approximately 0.1% to 0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of HMW species in the lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0110] In some embodiments, the stability of the formulation is characterized by the amount of high molecular weight (LMW) species in the bispecific antibody construct or the rate of increase of the amount of LMW species in the bispecific antibody construct under storage conditions at various time points. In some embodiments, the amount of LMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4°C or 40°C. In some embodiments, the rate of increase of LMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4°C or 40°C. In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by capillary electrophoresis of sodium dodecyl sulfate (rCE-SDS). In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by size exclusion chromatography (SEC).

[0111] In some embodiments, bispecific antibody constructs present in amounts less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) as low molecular weight (LMW) species in the lyophilized formulation. In some embodiments, the amount of LMW species in the lyophilized formulation increases by less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of LMW species in the formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) after storage at 4°C for more than one month (e.g., one, two, one, or three months). In some embodiments, the amount of LMW species in the lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) after storage at 40°C for more than one week (e.g., one, two, one, or three months). In some embodiments, the amount of LMW species in the lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by size exclusion chromatography (SEC). In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by reductive capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS).

[0112] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the lyophilized formulation is greater than 95% of the total protein content in the formulation.

[0113] In some embodiments, the lyophilized formulation is stable after storage at approximately 4°C for one month, and the amount of HMW species in the formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) during storage for at least one month. In some embodiments, the lyophilized formulation is stable after storage at 4°C for three months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.2% (e.g., 0%, 0.1%, or 0.2%) during storage for at least three months. In some embodiments, the lyophilized formulation is stable after storage at 4°C for at least six months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.4% (e.g., 0%, 0.1%, 0.2%, 0.3%, or 0.4%) during storage for at least six months. In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0114] In one embodiment, the lyophilized formulation is stable after storage at approximately 4°C for 1 month, 3 months, and 6 months, and the percentage of intact BiTE® molecules is more than 95% of the total protein content during storage.

[0115] In some embodiments, the formulation is a liquid formulation, and less than 3% (e.g., 2.5%, or 2%, or 1.5%, or 1%, or 0.5%) of a bispecific antibody construct is present as HMW species in the liquid formulation. In some embodiments, the amount of HMW species in the liquid formulation increases by less than 3% (e.g., 3%, 2.5%, 2%, 1%, or 0.5%) after storage at 4°C for more than one month (e.g., one month, three months, six months, or one year). In some embodiments, after storage at 4°C for more than one month (e.g., one month, three months, six months, or one year), the amount of HMW species in the liquid formulation increases by approximately 0.1% to 1% (e.g., 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%). In some embodiments, the amount of HMW species in the liquid formulation increases by 5% (e.g., 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, or 0.5%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). This increases by approximately 0.1% to 5% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%). In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0116] In some embodiments, bispecific antibody constructs present in amounts less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5%) as low molecular weight (LMW) species in the liquid formulation. In some embodiments, after storage at 4°C for more than one month (e.g., one, three, six, or twelve months), the amount of LMW species in the liquid formulation increases by less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%). In some embodiments, after storage at 4°C for more than one month (e.g., one month, three months, six months, or twelve months), the amount of LMW species in the liquid formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of LMW species in the liquid formulation increases by less than 7% (e.g., 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%). In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of LMW species in the lyophilized formulation increases by approximately 0.1% to 7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 1.5%, or 2%, or 3%, or 5%, or 0.6%, or 7%). In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by size exclusion chromatography (SEC). In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by rCE-SDS.

[0117] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the liquid formulation is greater than 96% of the total protein content in the formulation.

[0118] In some embodiments, the liquid formulation is stable after storage at approximately 4°C for one month, and the amount of HMW species in the formulation increases by approximately 0.1% to 0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%) during storage for at least one month. In some embodiments, the liquid formulation is stable after storage at approximately 4°C for three months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.3% (e.g., 0%, 0.1%, 0.2%, or 0.3%) during storage for at least three months. In some embodiments, the liquid formulation is stable after storage at approximately 4°C for six months, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.6% (e.g., 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.6%) during storage for at least six months. In some embodiments, the liquid formulation is stable after 12 months of storage at approximately 4°C, and the amount of HMW species in the formulation increases by approximately 0.0% to 0.2% (e.g., 0%, 0.1%, or 0.2%) during at least 12 months of storage. In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0119] In one embodiment, the lyophilized formulation is stable after storage at approximately 4°C for 1 month, 3 months, 6 months, and 12 months, with the percentage of intact BiTE® molecules being over 96% of the total protein content during storage.

[0120] The stability of the formulations described herein is further characterized by changes in the charge distribution, for example, in the amount of antibody charge change peaks. For example, in some embodiments, the amount of acidic peaks in the formulation (e.g., deamidation, which is a charge change with a relatively low isoelectric point (pI) in the formulation) increases by less than 2% (e.g., 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%) when stored at 4°C for at least one month (e.g., one month, three months, six months, or twelve months). In some embodiments, the amount of basic peaks in the formulation (e.g., charge changes with relatively high pI in the formulation) increases by less than 6% (e.g., 6%, 5%, 4%, 3%, 2%, or 1%) when stored at 4°C for at least one month (e.g., one month, three months, six months, or twelve months). In some embodiments, the formulation is a lyophilized formulation, and the amount of major peaks in the formulation decreases by less than 4% (e.g., 4%, 3.5%, 3%, 2.5%, 2%, or 1%) when stored at 4°C for at least one month. In some embodiments, the amount of major peaks in a lyophilized formulation decreases by less than 6% (e.g., 6%, 5%, 4%, 3.5%, 3%, 2.5%, or 2%) when stored at 4°C for at least three months. In some embodiments, the amount of the major peak in the lyophilized formulation decreases by less than 9% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored at 4°C for at least 6 months. In some embodiments, the amount of the major peak in the lyophilized formulation decreases by less than 9% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored at 4°C for at least 12 months.

[0121] In some embodiments, the amount of acidic peaks in the formulation increases by less than 30% (e.g., 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 4%, 4%, 3%, 2%, or 1%) when stored at 40°C for at least one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of basic peaks (e.g., charge changes with relatively high pI) in the formulation increases by less than 15% (e.g., 15%, 10%, 9%, 8%, 7%, 6%, 4%, 4%, 3%, 2%, or 1%) when stored at 40°C for at least one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the formulation is a lyophilized formulation, and the amount of main peak in the formulation decreases by less than 4% (e.g., 4%, 3.5%, 3%, 2.5%, 2%, 1% or less) when stored at 4°C for at least one month. In some embodiments, the amount of main peak in the lyophilized formulation decreases by less than 6% (e.g., 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored at 4°C for at least three months.

[0122] Therapeutic use of the preparation The formulations described herein are useful as pharmaceutical formulations in the treatment of cancer in subjects in need of treatment. The term “subjects in need” or “subjects in need of treatment” includes subjects who already have the disorder and subjects for whom prevention of the disorder is sought. “Subjects in need” or “patients” includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment. The term “treatment” includes both therapeutic treatment and prophylactic or preventive measures. Treatment includes the application or administration of formulations to the body, isolated tissues or cells of a patient having the disorder, symptoms of the disorder, or predisposition to the disorder, with the aim of curing, resolving, alleviating, mitigating, altering, correcting, improving, improving, or influencing the disorder, symptoms of the disorder, or predisposition to the disorder.

[0123] As used herein, the term "improvement" refers to any improvement in the disease state of a patient with a tumor, cancer, or metastatic cancer as described below, by administration of a formulation containing the antigen-binding protein described herein to a target who requires it. Such improvement may also be considered as slowing or halting the progression of the patient's tumor, cancer, or metastatic cancer. As used herein, the term "prevention" refers to the avoidance of the onset or recurrence of a patient with a tumor, cancer, or metastatic cancer as described below, by administration of a composition containing the antigen-binding protein (i.e., antibody construct) described herein to a target who requires it.

[0124] This disclosure provides a method for treating cancer, comprising the step of administering a therapeutically effective amount of a recombinant protein or pharmaceutical formulation described herein to a subject in need thereof. In a given embodiment, the subject is a human. In a given embodiment, the cancer is a solid tumor.

[0125] In some embodiments, cancer is brain tumor, bladder cancer, breast cancer, clear cell kidney cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, lip and oral cancer, liver cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, oral cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer (SCLC), head and neck squamous cell carcinoma (SCCHN), triple-negative breast cancer, or thyroid cancer.

[0126] In some embodiments, the cancer is an adrenocortical tumor, alveolar soft tissue sarcoma, carcinoma, chondrosarcoma, colorectal cancer, tendonoid tumor, fibrous round cell tumor, endocrine tumor, endoderm sinus tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, non-rhagic soft tissue sarcoma (NRSTS), osteosarcoma, paravertebral sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, periosteosarcoma, or Wilms' tumor.

[0127] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML).

[0128] In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0129] In fact, treatable cancers include, but are not limited to, alveolar soft part sarcoma, osteosarcoma, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nostrils, or middle ear, cancer of the oral cavity, cancer of the vulva, esophageal cancer, gastrointestinal carcinoid tumors, pharyngeal cancer, cancer of the nasopharynx, peritoneum, pleura, and mesentery, pharyngeal cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, ureteral cancer, and bladder cancer.

[0130] Route of administration Preferably, the pharmaceutical formulation is administered parenterally, for example, intravenously, subcutaneously, or intramuscularly. Parenteral administration can be achieved by injection, such as a bolus injection, or by infusion, such as a continuous infusion. Administration can be achieved via depot for long-term release. In some embodiments, the formulation is administered intravenously by continuous infusion after an initial bolus to maintain therapeutic circulating levels of the drug product. In some embodiments, the formulation is administered as a single dose. The pharmaceutical formulation may be administered using a medical device. Examples of medical devices for administering pharmaceutical preparations are described in U.S. Patent 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.

[0131] In some embodiments, the formulation is a lyophilized formulation that is reconstituted with sterile water or a suitable diluent for injection before administration.

[0132] This disclosure also intends to enable uninterrupted administration of the formulation. In a non-limiting example, uninterrupted or substantially uninterrupted, i.e., continuous administration can be achieved by a patient-worn miniature pump system for regulating the infusion of the therapeutic agent into the patient's body. The pharmaceutical formulation can be administered using such a pump system. Such pump systems are generally known in the art and typically rely on the periodic replacement of cartridges containing the therapeutic agent to be infused. When replacing cartridges in such a pump system, a temporary interruption may result in the infusion of the therapeutic agent into the patient's body, which is otherwise uninterrupted. Even in such cases, the administration stage before and after cartridge replacement will still be considered within the meaning of the pharmaceutical means and methods of the present invention, which together constitute “uninterrupted administration” of such therapeutic agent.

[0133] Continuous or uninterrupted administration of the formulation may be intravenous or subcutaneous via a fluid delivery device or a small pump system, which includes a fluid delivery mechanism for dispensing fluid from a reservoir and a drive mechanism for driving the delivery mechanism. A pump system for subcutaneous administration may include a needle or cannula for penetrating the patient's skin and delivering the preferred formulation into the patient's body. Direct contact between the pump system and the patient's skin is possible by fixing or attaching the pump system directly to the patient's skin, whether via a vein, artery, or blood vessel. This pump system can be attached to the patient's skin for 24 hours to several days. There may also be small pump systems with small reservoir capacities. In non-limiting examples, the reservoir volume for the preferred pharmaceutical formulation to be administered may be 0.1 to 50 ml.

[0134] kit In a further embodiment, a kit comprising one or more pharmaceutical compositions described herein, packaged in a manner that facilitates their use for administration to a subject. In one embodiment, such a kit comprises a compound or formulation described herein (e.g., a formulation containing an antibody described herein), packaged in a container such as a sealed bottle, container, single-use or multi-use vial, pre-filled syringe, or pre-filled injection device, with optionally affixed a label to the container, or contained in a package describing the use of the compound or formulation in practicing the method. In one embodiment, the formulation is packaged in unit dosage forms. The kit may further include a device suitable for administering the formulation according to a specific route of administration. Preferably, the kit contains a label describing the use of the antibody or formulation described herein.

[0135] The pharmaceutical formulations described herein can be formulated in various forms, such as solid, liquid, frozen, gaseous or lyophilized, and may be in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts.

[0136] In general, with respect to the pharmaceutical compositions of the present invention, various storage forms and / or dosage forms are possible, depending on the intended route of administration, delivery method and desired dose (see, for example, Remington's Pharmaceutical Sciences, 22nd edition, Oslo, A., Ed., (2012)). Those skilled in the art will recognize that the selection of such a particular dosage form may affect, for example, the physical state of the antibody, its stability, the in vivo release rate and the in vivo clearance rate.

[0137] For example, the main vehicle or carrier in a pharmaceutical formulation may be essentially aqueous or non-aqueous. Suitable vehicles or carriers may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, supplemented with other materials common in parenteral formulations. Neutral buffered saline or physiological saline mixed with serum albumin are further exemplary vehicles. [Examples]

[0138] Example 1 - Stability over time of liquid formulations and lyophilized formulations The stability of the following formulations was evaluated by size exclusion high-performance liquid chromatography (SE-HPLC) and cation exchange high-performance liquid chromatography (CE-HPLC): Liquid formulation: 20 mg / mL of BiTE® molecules, 10 mM L-glutamic acid, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, pH 4.2. Lyophilized formulation: 20 mg / mL of BiTE® molecules, 10 mM L-glutamic acid, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, pH 4.2.

[0139] Table 1 below provides data on the peaks (%) of high molecular weight (HMW) species, main peak (monomer), and low molecular weight (LMW) species of lyophilized and liquid formulations, evaluated by SE-HPLC at time points 0, 1 month, 3 months, 6 months, and 12 months after storage at 4°C.

[0140] [Table 1-1]

[0141] [Table 1-2]

[0142] Table 2 below provides data on the peak peaks of lyophilized and liquid formulations, including the high molecular weight (HMW) peak (%), main (monomer) peak, and low molecular weight (LMW) peak, as evaluated by SE-HPLC at time points 0, 1 week, 2 weeks, 1 month, and 3 months after storage at 40°C.

[0143] [Table 2]

[0144] High-performance liquid chromatography (CE-HPLC) was also performed to evaluate the charge change distribution of various BiTE® molecules in the test formulations at 4°C and 40°C at various time points. Table 3 below provides data on the main peak, acidic peak, and basic peak in percentage for lyophilized and liquid formulations evaluated by CE-HPLC after storage at 4°C at 0, 1 month, 3 months, 6 months, and 12 months.

[0145] [Table 3-1]

[0146] [Table 3-2]

[0147] Table 4 below provides data on the main peak, acidic peak, and basic peak in percentage for lyophilized and liquid formulations, evaluated by CE-HPLC at time points 0, 1 week, 2 weeks, 1 month, and 3 months after storage at 40°C.

[0148] [Table 4-1]

[0149] [Table 4-2]

[0150] Reducing capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) method: Protein species are bound to SDS, an anionic detergent, and electrodynamically injected into a bare fused silica capillary filled with SDS gel buffer. A voltage is applied throughout the capillary, and under it, the SDS-coated proteins are separated by their differences in migration in a hydrophilic polymer-based solution. The proteins are detected by a photodiode array (PDA) detector as they pass through a UV detection window. Purity is assessed by determining the precise peak area percentage of the leech components. The rCE-SDS method separates heavy chains (HC), light chains (LC), non-glycosylated HC (NGHC), and other fine peak species and groups under reducing conditions.

[0151] Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of various sizes enter or are excluded from these pores in the matrix. Small molecules diffuse into these pores, and their flow through the column is delayed according to their size, while larger molecules do not enter these pores and are eluted into the void volume of the column. As a result, molecules are separated based on their size as they pass through the column, and are eluted in order of increasing molecular weight (MW). The operating conditions and gel selection depend on the application and the desired solution.

[0152] Analysis of particles invisible to the naked eye is performed by the HIAC method. An electronic liquid particle counting system containing a photo-obscuring sensor equipped with a liquid sampler quantifies the number and size range of particles in a given test sample. When particles are in the liquid passage between the light source and the detector, they weaken or "blur" the beam of light falling on the detector. If the particle concentration is within the normal range of these sensors, these particles are detected one by one. The passage of each particle through the detection zone reduces the incident light on the photodetector, and the voltage output of the photodetector is momentarily reduced. The change in the voltage resistor as an electrical pulse is converted by the instrument to the number of particles present. This method is nonspecific and measures particles regardless of their origin.

[0153] Finally, the water content of the lyophilized formulations was determined by calorimetry using an oven. The principle of the Karl Fischer method is based on the water content in the sample determined by calorimetry. Water is released by heating the sample in the oven. Dry air or an inert gas such as nitrogen carried the evaporated water to the titrator. The amount of water present is determined by measuring the amount of coulombs (current / time) generated during the titration. When all the water is consumed by the titration, excess iodine is generated. The endpoint is indicated in terms of volume by applying a constant strength alternating current to two Pt electrodes. This results in a voltage difference between the Pt lines of the indicator electrodes, which decreases significantly in the presence of a minimal amount of free iodine. This voltage difference is used to determine the titration endpoint. The water content of lyophilized formulations containing BiTE®-I, BiTE®-C, and BiTE®-G was evaluated after storage at 2-8°C at 0, 1 month, 3 months, 6 months, and 1 year. As shown in Table 5 below, none of the lyophilized formulations tested had a water content greater than 1.70% throughout the entire testing period.

[0154] [Table 5]

[0155] conclusion In this embodiment, it is demonstrated that the high-concentration formulations described herein are stable after storage at approximately 4°C for 6 months. In these stable formulations, the amount of HMW species increased by approximately 0.0% to 0.4%, while the presence of preservatives or stabilizers was not required after storage for at least 6 months.

Claims

[Claim 1] The invention described herein.

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