Pharmaceutical preparations

Abstract

For example, to provide a stable aqueous formulation comprising a bispecific antibody construct, a buffer, sugars, and a surfactant, having a pH of 4.8 to 5.5. [Solution] In one embodiment, this specification describes a stable aqueous pharmaceutical formulation comprising the bispecific antibody construct, buffer, sugars and surfactant described herein, wherein the aqueous pharmaceutical formulation has a pH of 4.8 to 5.5. In some embodiments, the pH of the formulation is in the range of 4.5 to 5.3. In some embodiments, the pH of the formulation is 5.2.

Description

Technical Field

[0001] The present disclosure is included in the field of stable bispecific antibody construct formulations.

[0002] Incorporation by reference The following computer-readable nucleotide / amino acid sequence listing, which was filed simultaneously with this specification and identified as an ASCII (text) file named "54910_Seqlisting.txt" created on April 28, 2020 and having a size of 345,249 bytes, is incorporated herein by reference in its entirety.

Background Art

[0003] Protein-based pharmaceuticals are among the most rapidly growing therapeutics in (pre)-clinical development and as commercial products. Compared to small molecule chemical agents, protein pharmaceuticals have high specificity and activity at relatively low concentrations and typically provide treatment for major diseases such as various cancers, autoimmune diseases, and metabolic disorders (Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80, Wang, Int J Pharm. 1999 Aug 20;185(2):129-88). Advances in commercial-scale purification processes have made it possible to obtain protein-based pharmaceuticals, such as recombinant proteins, with high purity during initial production. However, proteins are only barely stable and are highly susceptible to chemical and physical degradation. Chemical degradation refers to modifications involving covalent bonds, such as deamide, oxidation, cleavage, clipping / fragmentation, formation of new disulfide crosslinks, hydrolysis, isomerization, or deglycosylation. Physical degradation includes protein unfolding, undesirable surface adsorption, and aggregation. Addressing these physical and chemical instabilities is one of the most challenging issues in the development of protein-based pharmaceuticals (Chi et al., Pharm Res, Vol.20, No.9, Sept 2003, pp.1325-1336, Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Non-Patent Document 2] Wang,Int J Pharm.1999 Aug 20;185(2):129-88) [Non-Patent Document 3] Chi et al.,Pharm Res,Vol.20,No.9,Sept 2003,pp.1325-1336 [Non-Patent Document 4] Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Overview of the Initiative] [Means for solving the problem]

[0005] Clipping (fragmentation) of protein-based pharmaceuticals can occur during the manufacturing process and storage due to chemical and enzymatic causes. Clipping is undesirable in the case of therapeutic polypeptides because it represents the degradation of the parent polypeptide, which can lead to a potential reduction in potency. Maintaining the stability and activity of proteins in biological and biotechnological applications presents a serious challenge. Optimized pharmaceutical compositions that enhance the stability of therapeutic proteins and prevent loss of function and adverse immunogenic reactions by reducing aggregation, denaturation, or degradation during formulation, filling, transport, storage, and administration are needed in the art.

[0006] Protein-based pharmaceuticals containing bispecific (and / or multispecific) antibodies that simultaneously bind to two (or more) different antigens, such as bispecific T-cell engaging (BiTE®) antibody constructs, tend to exhibit protein instability. This extends to their antibody constructs, including single-chain Fc forms (referred to as scFc), hetero-Fc forms (hetFc or heterodimer Fc, also referred to as hFc), and half-life extended forms (HLE forms) containing fusions of human serum albumin (also referred to as HSA or hALB).

[0007] Protein instability, and especially clipping (fragmentation), is an increasingly significant challenge in the biotechnology industry. All bispecific antibody constructs are susceptible to clipping in liquid formulations. Clipping generates low molecular weight (LMW) species of bispecific antibody constructs, resulting in inactive polypeptides. In liquid formulations with lower (i.e., more acidic) pH, clipping of bispecific antibody constructs tends to increase, while aggregation tends to decrease. In liquid formulations with higher (i.e., more basic) pH, aggregation of bispecific antibody constructs tends to increase, while clipping tends to decrease.

[0008] Unexpectedly, this disclosure identifies an optimized pH range for liquid bispecific antibody constructs that stabilize liquid formulations under various storage conditions. For example, the examples provided herein identify a specific pH range (i.e., pH 4.8–5.5) in which both the aggregation of bispecific antibody constructs (i.e., the amount of low molecular weight species) and the bispecific antibody constructs (i.e., the amount of molecular weight species) in liquid formulations decreases when stored, for example, at 2–8°C or 40°C. As shown in the examples described herein, the levels of clipping and aggregation of bispecific antibody constructs in liquid formulations with pH ranges of 4.8–5.5 were compared to the levels of clipping and aggregation in liquid formulations with pH outside the 4.8–5.5 range (i.e., pH 4.2 or pH 7.4). Therefore, liquid pharmaceutical formulations containing a bispecific antibody construct and having a pH range of approximately 4.8 to 5.5 (e.g., pH 5.2) are more stable and demonstrate improved structural integrity compared to liquid formulations containing a bispecific antibody construct at pH outside this range (e.g., pH 4.2 or pH 7.4) under the same storage conditions.

[0009] In one embodiment, this specification describes a stable aqueous pharmaceutical formulation comprising the bispecific antibody construct, buffer, sugars, and surfactant described herein, wherein the aqueous pharmaceutical formulation has a pH of 4.8 to 5.5. In some embodiments, the pH of the formulation is in the range of 4.5 to 5.3. In some embodiments, the pH of the formulation is 5.2.

[0010] In some embodiments, the formulation contains less than 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3% or less) of a bispecific antibody construct of low molecular weight (LMW) species after storage at approximately 40°C for one month.

[0011] In some embodiments, the formulation contains less than 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3% or less) of a bispecific antibody construct of low molecular weight (LMW) species after storage for one month at approximately 2–8°C.

[0012] In some embodiments, the formulation contains a bispecific antibody construct of high molecular weight (HMW) species at less than 5% (e.g., 4%, 3%, 2%, 1% or less) after storage at approximately 40°C for one month.

[0013] In some embodiments, the formulation contains a bispecific antibody construct of high molecular weight (HMW) species at less than 5% (e.g., 4%, 3%, 2%, 1% or less) after storage at approximately 2–8°C for one month.

[0014] In some embodiments, the relative titer of the bispecific antibody construct in the formulation is more than 30% (e.g., more than 35%, 40%, 50%, 55%, or 60%) after storage at approximately 40°C for one month.

[0015] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the formulation is greater than 95% of the total protein content in the formulation.

[0016] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to a target cell surface antigen, which is a second binding domain that binds to human CD3 on the surface of T cells. In some embodiments, the bispecific antibody construct further includes a third domain 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 of the bispecific antibody construct includes a VH region and a VL region.

[0017] In some embodiments, the bispecific antibody construct is a single-chain antibody construct.

[0018] In some embodiments, the bispecific antibody construct binds to target cell surface antigens such as CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17.

[0019] 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.

[0020] 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, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143, or 129.

[0021] 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.

[0022] In some embodiments, the first binding domain comprises: (a) a VH region comprising the amino acid sequence shown in SEQ ID NO: 30 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 31; (b) a VH region comprising the amino acid sequence shown in SEQ ID NO: 40 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 41; (c) a VH region comprising the amino acid sequence shown in SEQ ID NO: 84 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 85; (d) a VH region comprising the amino acid sequence shown in SEQ ID NO: 16 or 17 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 18 or 19; (e) a VH region comprising the amino acid sequence shown in SEQ ID NO: 52 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 53; (f) a VH region comprising the amino acid sequence shown in SEQ ID NO: 94 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 95; (g) a VH region comprising the amino acid sequence shown in SEQ ID NO: 73 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 74; (h) a VH region comprising the amino acid sequence shown in SEQ ID NO: 62 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 63; (i) a VH region comprising the amino acid sequence shown in SEQ ID NO: 118 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 119; (j) a VH region comprising the amino acid sequence shown in SEQ ID NO: 154, 163, 172 or 181 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 155, 164, 173 or 182; (k) a VH region comprising the amino acid sequence shown in SEQ ID NO: 106 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 107; (l) a VH region comprising the amino acid sequence shown in SEQ ID NO: 138 or 143 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 139 or 144; or (m) a VH region comprising the amino acid sequence shown in SEQ ID NO: 129 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 130.

[0023] In some embodiments, the second binding domain of the bispecific antibody construct comprises a set of 6 CDRs shown in SEQ ID NOs: 1-6.

[0024] 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 region comprising the amino acid sequence shown in SEQ ID NO: 7.

[0025] 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 region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to PSMA, comprising an anti-PSMA variable light chain domain containing the amino acid sequence of SEQ ID NO: 119 or 107 and an anti-PSMA variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 118 or 106; 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: 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] The pharmaceutical formulations of this disclosure include a buffering agent. In some embodiments, the buffering agent is selected from the group consisting of acetates, glutamates, citrates, succinates, tartrates, fumarates, maleates, histidines, phosphates, and 2-(N-morpholino)ethanesulfonates or combinations 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). In some embodiments, the buffering agent is present in the formulation at a concentration of 10 mM.

[0040] 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 7 to about 12 (w / v)%).

[0041] The pharmaceutical formulations of this disclosure include surfactants. In some embodiments, the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, 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))%).

[0042] 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.

[0043] In some embodiments, the pharmaceutical formulation contains 10 mM glutamate, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80, and the pH of the pharmaceutical formulation is 5.2. In some embodiments, the bispecific antibody construct is present in the formulation at a concentration ranging from about 0.1 mg / mL to about 20 mg / mL. In some embodiments, the heterodimer antibody is present in the formulation at a concentration of 1 mg / mL, 5 mg / mL, 10 mg / mL, or 20 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.

[0044] In some embodiments, the pharmaceutical formulations of this disclosure are liquid formulations.

[0045] In another aspect, this specification describes a method of treating cancer in a subject, which includes administering the formulation of the present disclosure to a subject in need of the subject.

[0046] 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 term “one” refers to one or more; for example, “one immunoglobulin molecule” is understood to represent one or more immunoglobulin molecules. Thus, the terms “one,” “one or more,” and “at least one” may be used interchangeably in this specification.

[0047] 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 approximately 4.8 to approximately 5.5" could be any value between 4.6, 5.2, 5.5, etc., without limitation. Furthermore, "pH approximately 4.8 to approximately 5.5" should not be interpreted as meaning that the pH of the formulation in question fluctuates by 2 pH units within the range of pH 4.8 to pH 5.5 during storage, but rather as meaning that a value within that range can be selected for the pH of the solution, and that the pH remains buffered around that pH.

[0048] 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.

[0049] 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.

[0050] All references listed herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0051] [Figure 1]This graph shows the percentage of low molecular weight (LMW) measured by reduced capillary electrophoresis of sodium dodecyl sulfate (rCE-SDS) for BiTE-I, BiTE-C, BiTE-D, and BiTE-G from accelerated stability tests in high pH formulations. [Figure 2] This graph shows the clipping rate of BiTE-G in pH 4.2 and pH 4.8 liquid formulations under accelerated and recommended storage conditions. [Figure 3-1] These graphs show the %LMW (evaluated by rCE) (Figures 3A and 3B) and %HMW (evaluated by SEC) (Figures 3C and 3D) of BiTE-D and BiTE-G formulations at pH 3.8, pH 4.8, pH 5.2, and pH 7.4. [Figure 3-2] These graphs show the %LMW (evaluated by rCE) (Figures 3A and 3B) and %HMW (evaluated by SEC) (Figures 3C and 3D) of BiTE-D and BiTE-G formulations at pH 3.8, pH 4.8, pH 5.2, and pH 7.4. [Figure 3-3] These graphs show the %LMW (evaluated by rCE) (Figures 3A and 3B) and %HMW (evaluated by SEC) (Figures 3C and 3D) of BiTE-D and BiTE-G formulations at pH 3.8, pH 4.8, pH 5.2, and pH 7.4. [Figure 3-4] These graphs show the %LMW (evaluated by rCE) (Figures 3A and 3B) and %HMW (evaluated by SEC) (Figures 3C and 3D) of BiTE-D and BiTE-G formulations at pH 3.8, pH 4.8, pH 5.2, and pH 7.4. [Modes for carrying out the invention]

[0052] While current therapeutic biotechnology products are of high quality and recombinant human proteins and antibodies are similar to endogenous human proteins, protein instability remains a significant concern. Processes for generating recombinant proteins (e.g., antibodies) can lead to loss of structural integrity due to fragmentation or clipping (Vlasak et al., MAbs, 3:253-263, 2011; Wu et al., FEBS Lett., 588:935-941, 2014). Increasing the stability and structural integrity of therapeutic proteins is crucial in this field, and optimized pharmaceutical formulations can help achieve this.

[0053] Certain protein-based pharmaceuticals, such as BiTE® antibody construct molecules, are not stable in liquid formulations over long periods, and are particularly unstable at accelerating temperatures, such as refrigeration temperatures above 4°C. The general concept underlying this disclosure is the observation that the structural integrity of bispecific antibody constructs in liquid pharmaceutical formulations described herein is improved at pH levels between 4.8 and 5.5 compared to liquid formulations with more acidic pH (i.e., less than 4.8) or more basic pH (i.e., greater than 5.5). As shown in the examples provided herein, liquid formulations with pH levels between 4.8 and 5.5 result in minimal clipping-free (and minimal aggregation-free) bispecific antibody constructs in the liquid formulation.

[0054] Previously, formulations containing a bispecific antibody construct would need to be lyophilized to exhibit the stability observed herein in liquid formulations having a pH of 4.8–5.5 (see, for example, International Publication Nos. 2018 / 114190 and 2018 / 204907). The lyophilized formulation described in International Publication No. 2018 / 204907 includes preservatives (e.g., chlorobutanol, methylparaben, or benzyl alcohol) that contributed to the stabilization of the bispecific antibody construct. In some cases, stability is also achieved by other additional means, such as lowering the pH of the formulation. For example, International Publication No. 2018 / 141910 discloses that a lyophilized formulation containing a bispecific antibody construct with a low pH (i.e., pH 4.0) was more stable than a lyophilized formulation with the same antibody concentration at a more basic pH (e.g., pH 6 or higher). In contrast, the optimized pH range identification described in the examples of this disclosure allows for the storage of pharmaceutical formulations containing bispecific antibody constructs as liquid formulations at temperatures above 4°C (up to 40°C) at various time points.

[0055] Within the scope of this disclosure, the terms “stability,” “stable,” or “stabilized” refer to the structural stability of the entire pharmaceutical formulation, and in particular to the structural stability of the active ingredient (e.g., the bispecific antibody construct) itself, specifically during formulation, filling, transport, storage, and administration. For example, the stability of the active ingredient in the context of the pharmaceutical formulations of this disclosure is determined by the amount of fragmentation and / or clipping of the active ingredient. As used herein, “stable aqueous formulation” refers to a liquid (i.e., non-freeze-dried) pharmaceutical formulation containing the bispecific antibody construct of this disclosure that has minimal or no fragmentation or clipping of the bispecific antibody construct when stored, for example, at 4°C for at least 4 weeks. Typical stable aqueous formulations have a pH of 4.8 to 5.5 (e.g., pH 5.2). As used herein, “formulation” and “composition” are synonymous.

[0056] As used herein, the terms “clipping” or “fragmentation” typically refer to the partial splitting of an expressed protein by “proteolysis.” Fragmentation in the CDR of bispecific antibody constructs of the pharmaceutical formulations disclosed herein is likely to have an effect on the bispecific antibody’s ability to bind to its target and, consequently, on its potency. In the literature, there are very few reports, if any, that describe altered affinity to a target for bispecific antibody constructs with fragmentation occurring within the CDR; this is likely because, unless the CDR-clipping species is purified and tested, low levels of clipping in the CDR eliminate any detectable changes by potency assays. Fragmentation within the hinge region may have greater implications for the function of the bispecific antibody construct: the resulting Fab fragments may evade any Fc-mediated effector function and have a reduced circulating half-life; Fc-Fab fragments may be completely ineffective if interaction with the target receptor requires both Fab arms. See Vlasak et al., mAbs, 3:3, 253-263, 2011.

[0057] As used herein, the term "low molecular weight (LMW) species" refers to a fragment of a bispecific antibody construct resulting from clipping.

[0058] 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 within this term. Therefore, the term "(protein) aggregate" refers to any non-natural species that consists of two or more protein monomers that are physically associated or chemically bonded.

[0059] As used herein, the term "aggregation" refers to direct intermolecular attractive forces, such as van der Waals forces or chemical bonds. In particular, the term "aggregation" is understood as a protein that has accumulated and formed clumps. Aggregates may include amorphous aggregates, oligomers, and are typically called high molecular weight (HMW) species, i.e., molecules with a higher molecular weight than the pure product molecule.

[0060] 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 0.1 to 20 mg / mL, a buffer, a sugar, and a surfactant, and having a pH in the range of 4.8 to 5.5 (e.g., pH 5.2). 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.

[0061] Various aspects of this formulation are described below. The use of section headings is solely for the convenience of reading and is not intended to limit them in themselves. The entire Spec. is intended to be considered a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated.

[0062] 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.

[0063] 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, one variable region generally consists of about 100-110 or more amino acids and contains three complementarity-determining regions (CDRs) that are primarily involved in antigen recognition and substantially vary among other antibodies that bind to different antigens. One variable region typically contains at least three heavy or light chain CDRs (Kabat et al.). (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), these are located within framework regions (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 antibodies to recruit cells and molecules of the immune system.

[0064] In some embodiments, the antibody in the formulation is a bispecific antibody, i.e., a single antibody that binds to two different targets (e.g., CD3 and a second different target).

[0065] As used herein, the term “bispecificity” refers to an antibody construct that binds to two different target antigens, namely, 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 single 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 term “bispecificity antibody construct” of the present invention also includes multispecificity antibody constructs, such as a trispecificity antibody construct comprising three binding domains, or constructs having more than three (e.g., four, five...) specificities.

[0066] 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), incorporated herein by reference, which describes various forms of bispecificity. 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.

[0067] 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.

[0068] 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 expected to be prepared or obtained by phage display or library screening, rather than by grafting CDR sequences derived from existing (monoclonal) antibodies onto a scaffold.

[0069] 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 having non-human variable and / or constant regions, such as those from 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.

[0070] 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.

[0071] 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.

[0072] 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 flexible hinge N-terminus to these domains. With respect to IgA and IgM, Fc may include a 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.

[0073] In some embodiments, the formulations described herein include bispecific antibodies that bind 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.

[0074] 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.

[0075] 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, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143, or 129.

[0076] 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.

[0077] 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.

[0078] In some embodiments, the second binding domain of the bispecific antibody construct contains a set of 6CDRs shown in SEQ ID NOs: 1-6.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to PSMA, comprising an anti-PSMA variable light chain domain containing the amino acid sequence of SEQ ID NO: 119 or 107 and an anti-PSMA variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 118 or 106; 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: 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.

[0089] 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 NOs: 20, 21, 22, or 23.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] In some embodiments, the formulation contains a bispecific antibody construct at a concentration of about 1 mg / mL to about 20 mg / mL (or about 1 mg / mL to about 8 mg / mL or about 1 mg / mL to about 5 mg / mL). In some embodiments, the formulation contains a bispecific antibody construct at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, 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 or about 20 mg / mL.

[0096] In some embodiments, the formulation contains a bispecific antibody construct at a concentration ranging from about 0.1 to about 8 mg / mL (or about 0.5 to about 5 mg / mL or about 1 to about 5 mg / mL or about 3 to about 6 mg / mL). In some embodiments, the formulation contains a bispecific antibody construct at a concentration of about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL.

[0097] 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.

[0098] Buffers are often used to control the pH of a formulation. In some embodiments, buffers are added at a concentration that maintains the pH of the liquid formulation at a pH that reduces the rate of clipping and aggregation of the bispecific antibody construct in the formulation. In some embodiments, the pH of the liquid formulation is approximately pH 4.8 to pH 5.5, or approximately pH 4 to pH 5.5, or approximately pH 5.2. 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)).

[0099] The buffer system present in the formulation is selected to be physiologically compatible and to maintain the desired pH (e.g., 4.8–5.5, or 5.2).

[0100] The buffer may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. 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. Suitable buffer concentrations include concentrations of about 200 mM or less. In some embodiments, the buffer in the formulation may be 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] surfactants 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 one surfactant, either individually or in mixtures of various proportions. In some embodiments, the formulations include the surfactant at concentrations 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 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 0.1% to about 20(w / v)%, or about 1% to about 15(w / v)%, or about 5% to about 12(w / v)%, or about 7% to about 12(w / v)%. In some embodiments, the pharmaceutical formulation contains at least one sugar 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 one sugar at a concentration 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 one sugar at a concentration of about 1% to about 15(w / v)%. In yet another embodiment, the pharmaceutical formulation contains at least one sugar at a concentration 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 one sugar at a concentration of about 7% to about 12(w / v)%. In some embodiments, at least one sugar is present in the formulation at a concentration of about 9 (w / v)%. In some embodiments, the sugar is sucrose and is present in the formulation in a range of about 9% to about 12 (w / v)%.

[0105] In some embodiments, the pharmaceutical formulation comprises 10 mM glutamate, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80, wherein the pH of the pharmaceutical formulation is 5.2.

[0106] In some embodiments, the pharmaceutical formulation comprises 10 mM acetate, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80, wherein the pH of the pharmaceutical formulation is 5.2.

[0107] stability As described in the examples, the identification of the optimized pH range (pH 4.8 to pH 5.2) allows for the storage of the bispecific antibody construct as a liquid formulation at both 4°C and 40°C at various time points. The stability of the bispecific antibody construct formulation 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 ultra-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 partial dissociation measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) and / or sodium dodecyl sulfate polyacrylamide gel electrophoresis (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 chains (HC), light chains (LC), non-glycosylated HC (NGHC), and other fine peak species and groups under reducing conditions.

[0108] 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, 12 months, 18 months, or 2 years when stored at approximately 2–8°C (e.g., 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, 12 months, 18 months, or 2 years when stored at approximately 2–8°C (e.g., 4°C) or 40°C. In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by size exclusion chromatography (SEC). In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0109] 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, for example, 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, changes in charge profile, and changes in particle size.

[0110] In some embodiments, the relative value of any particular species of the bispecific antibody construct described herein, such as intact BiTE® molecules or main species, or high molecular weight (HMW) species (i.e., aggregates), or low molecular weight (LMW) species (i.e., fragments), is expressed in relation to each numerical value of the total product. For example, in some embodiments, bispecific antibody constructs present in amounts of 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) are present in the formulation as HMW species. In some embodiments, the amount of HMW species in the formulation increases by less than 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3% or less) over a period of more than one month (e.g., one month, three months, six months, twelve months, eighteen months, or two years) after storage at 2–8°C. In some embodiments, the amount of HMW species in the formulation increases by 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) over a period of more than one month (e.g., one week, two weeks, one month, three months, six months, or one year) after storage at 40°C. In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by size exclusion chromatography (SEC). In some embodiments, the HMW species of the bispecific antibody construct in the formulation are measured by SE-UHPLC.

[0111] In some embodiments, the stability of the formulation is characterized by the amount of low 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 month, 3 months, 6 months, 12 months, 18 months, or 2 years when stored at 2–8°C. In some embodiments, the amount of LMW species is determined during storage or after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year at 40°C. In some embodiments, the rate of increase of LMW species is determined after 1 month, 3 months, 6 months, 12 months, 18 months, or 2 years while stored at approximately 2–8°C. In some embodiments, the rate of increase of LMW species is determined during storage or after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year at 40°C. In some embodiments, the LMW species of the bispecific antibody construct in the formulation is determined by rCE-SDS.

[0112] In some embodiments, bispecific antibody constructs present in the formulation as LMW species at concentrations of 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less). In some embodiments, the amount of LMW species in the formulation increases by less than 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3% or less) over a period of one month or more (e.g., one month, three months, six months, twelve months, eighteen months, or two years) after storage at 2–8°C. In some embodiments, the amount of LMW species in the formulation increases by less than 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) over a period of one month or more (e.g., one week, two weeks, one month, three months, six months, or one year) after storage at 40°C. In some embodiments, the LMW species of the bispecific antibody construct in the formulation is measured by rCE-SDS.

[0113] In some embodiments, the percentage of intact BiTE® molecules (i.e., the main peak species) in the formulation is greater than 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the total protein content in the formulation.

[0114] In one embodiment, the liquid formulation is stable after storage at approximately 2–8°C for one month, and the percentage of intact BiTE® molecules (i.e., the main peak species) is greater than 95% of the total protein content in the formulation. In another embodiment, the liquid formulation is stable after storage at approximately 40°C for one month, and the percentage of intact BiTE® molecules (i.e., the main peak species) is greater than 95% of the total protein content in the formulation.

[0115] 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).

[0116] 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%, 5%, 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%, 5%, 4%, 3%, 2%, or 1%) when stored at 4°C for at least one week (e.g., one week, two weeks, one month, or three months).

[0117] The stability of the liquid formulations described herein can be further evaluated by determining the level of relative titer of the bispecific antibody construct in the liquid formulation under storage conditions. The relative titer of the bispecific antibody construct can be determined by a cell-based bioassay that measures cell death by loss of luminescence in carcinoma cell lines. The biological activity of the test sample is determined (relative titer) by comparing the response of the test sample with the response of a reference standard. In some embodiments, as evaluated as described in Example 3, the relative titer of the bispecific antibody construct in the formulation is more than 30% (e.g., about 35%, 40%, 50%, 55%, 60%) after 1 month of storage at about 40°C.

[0118] 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 procedures 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.

[0119] 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 described herein (i.e., a bispecific antibody construct) to a target who requires it.

[0120] This disclosure provides a method for treating cancer, comprising 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.

[0121] 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.

[0122] 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.

[0123] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML).

[0124] 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).

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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 volumes. In non-limiting examples, the reservoir volume for the preferred pharmaceutical formulation to be administered may be 0.1 to 50 ml.

[0129] 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 comprising 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 labels 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.

[0130] 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.

[0131] In general, various storage and / or dosage forms are possible for the pharmaceutical compositions of the present invention, 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.

[0132] 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]

[0133] Materials and methods Stability samples were analyzed using various techniques to monitor the critical quality characteristics of the drug product. The stability assays described below were performed under accelerated conditions: SE-UHPLC (size exclusion ultra-high performance liquid chromatography) to monitor aggregation, rCE-SDS and multi-characterization (MAM) to monitor clipping, and visual inspection for visible particle analysis.

[0134] Size exclusion ultra-high performance liquid chromatography (SE-UHPLC) separates proteins based on their hydrodynamic volume differences. Molecules with higher hydrodynamic volumes elute faster than those with lower volumes. The sample is loaded onto an SE-UHPLC column (BEH200, 4.6 x 300 mm, (Waters Corporation, 186005226)), separated to uniform concentration, and the eluate is monitored by UV absorbance. Purity is determined by calculating the percentage of each separated component compared to the integrated total area. The SE-UHPLC settings are as follows: flow rate: 0.4 mL / min, run time: 12 min, UV detection: 280 nm, column temperature: ambient temperature, target protein loading: 6 μg, protein-compatible flow cell: 5 mm.

[0135] Reducing capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) separates proteins based on their differences in hydrodynamic size under reducing and denaturing conditions. 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, under which 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 component. 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.

[0136] Example 1 - Increased pH reduction percentage of LMW species as evaluated by CE-SDS The stability of BiTE®-I, BiTE®-C, BiTE®-D, and BiTE®-G in the following formulations was evaluated in accelerated stability tests at various pH levels (pH 4.2, 4.8, and 5.5). The formulations were stored at 40°C for one month, and the LMW species levels were then analyzed by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS).

[0137] The liquid formulation tested contained 1 mg / mL of BiTE molecules, 10 mM L-glutamic acid, 9 (w / v)% sucrose, and 0.01 (w / v)% polysorbate 80.

[0138] As shown in Figure 1 and Table 1 below, the liquid formulations at pH 4.8 and pH 5.2 showed a percentage reduction after being stored at 40°C for one month, compared to the pH 4.2 liquid formulation stored under the same storage conditions.

[0139] [Table 1]

[0140] Example 2 - Evaluation of LMW species using MAM After storage at 40°C for one month, the clipping rate (%) of BiTE-G in liquid formulations (described in Example 1) at pH 4.2, pH 4.8, and pH 7.4 was evaluated by MAM (Multi-Analysis of Physics). Briefly, the sample was captured and denatured on a 30 kDa MWCO spin filter, and then alkylated on the filter. Subsequently, the sample was buffered in a digestion buffer and then digested with trypsin. The digested sample was centrifuged, and the tryptic peptide was captured. Next, continuous on-filter digestion was performed using human neutrophil elastase, and the resulting peptide was collected and combined with the tryptic peptide. The digestion was quenched by adding 8 M guanidine to an acetate buffer (pH 4.8). The peptide was then analyzed using LC-MS with Agilent 1290 UPLC (Waters BEH C18 column) and Thermo Q Exactive Biopharma with data-dependent acquisition. The resulting data were searched using MassAnalyzer to determine sequence coverage and quantified using Skyline Workbooks. The observed chemical modifications were within commercially acceptable limits (data not shown).

[0141] Example 3 - The BiTE molecule (registered trademark) retains its titer at a pH greater than 4.2. The relative titer of BiTE®-G in liquid formulations at pH 4.2 and pH 4.8 (as described in Example 1) was also evaluated after two years of storage at 2–8°C. Briefly, the titer of BiTE®-G was measured by a cell-based bioassay that measures cell death due to loss of luminescence in carcinoma cell lines. The biological activity of the test sample was determined (relative titer) by comparing the response of the test sample with the response of a reference standard. This assay demonstrated that BiTE®-G in liquid formulations at both pH 4.2 and pH 4.8 maintained an acceptable level of titer after two years of storage at 2–8°C.

[0142] Example 4 - The clipping rate of the BiTE(registered trademark) molecule varies at various pH levels. The clipping rate of BiTE®-G in liquid formulations (described in Example 1) at pH 4.2 and pH 4.8 was evaluated by rCE-SDS. As shown in Figure 2 and Table 2 below, the liquid formulation at pH 4.8 demonstrated a 27% reduction in clipping level compared to the liquid formulation at pH 4.2 after storage at 40°C for one month. A further 29% reduction in clipping level was observed in the liquid formulation at pH 4.8 after storage at 2-8°C for two years, compared to the liquid formulation at pH 4.2.

[0143] [Table 2]

[0144] Example 5 - Evaluation of HMW species using SE-UHLPC The percentage of high molecular weight (HMW) species was evaluated by size exclusion ultra-high performance liquid chromatography (SE-UHLPC) in the liquid formulations described in Example 1 containing BiTE®-G at pH 4.2 and pH 4.8 after storage at either 8°C or -30°C for 1 month, 3 months, 6 months, 12 months, 18 months, and 2 years. This analysis demonstrated that the percentage of BiTE®-G observed in both the pH 4.2 and pH 4.8 formulations was less than 0.35% under storage conditions at 2–8°C at all time points tested (data not shown). This analysis demonstrated that the percentage of BiTE®-G observed in both the pH 4.2 and pH 4.8 formulations was less than 1.3% under storage conditions at -30°C at all time points tested (data not shown).

[0145] Example 6 - Evaluation of both LMW and low HMW varieties under various storage conditions The percentages of LMW and HMW species of BiTE®-G and BiTE®-D in various liquid formulations were evaluated for each formulation specified below. The percentage of LMW was determined by reductive capillary electrophoresis (rCE), and the percentage of HMW was determined by size exclusion chromatography (SEC). The liquid formulations tested were as follows: Formulation A: 1 mg / mL of BiTE® molecule, 10 mM L-glutamic acid, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, pH 3.8; Formulation B: 1 mg / mL of BiTE® molecule, 10 mM L-glutamic acid, 9 wt / volt% sucrose, 0.01 (w / v)% polysorbate 80, pH 4.8; Formulation C: 1 mg / mL of BiTE® molecule, 10 mM L-glutamic acid, 9 wt / volt% sucrose, 0.01 (w / v)% polysorbate 80, pH 5.2; Formulation D: 1 mg / mL of BiTE® molecule, 10 mM acetate, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, pH 5.2; Formulation E: 1 mg / mL of BiTE® molecule, 10 mM phosphate, 9 (w / v)% sucrose, 0.01 (w / v)% polysorbate 80, pH 7.4.

[0146] The results are shown in Figures 3A-3D and Tables 3-6 below:

[0147] [Table 3]

[0148] [Table 4]

[0149] [Table 5]

[0150] [Table 6]

[0151] As shown in Figures 3A-3D, formulations B (pH 4.8) and C (pH 5.2) showed the lowest percentages for both LMW and HMW species after storage at both 4°C and 40°C for 4 weeks, compared to formulations A (pH 3.8) and E (pH 7.4). Formulation D (pH 5.2) also showed low percentages for both LMW and HMW, demonstrating that the choice of buffer in the formulation has no effect on the fragmentation of BiTE® molecules. The data provided in this embodiment demonstrate that, regardless of the buffer in the formulation, the identification of an optimized pH range (pH 4.8-pH 5.5) allows for the storage of pharmaceutical formulations containing bispecific antibody constructs as liquid formulations at both 4°C and 40°C at various time points. The present invention provides, for example, the following items: (Item 1) (a) Bispecific antibody construct, (b) sugars, (c) Surfactants, and (d) A stable aqueous pharmaceutical formulation containing a buffering agent, The aforementioned formulation is a stable aqueous pharmaceutical preparation with a pH of approximately 4.8 to 5.5. (Item 2) The formulation described in item 1, wherein the pH of the formulation is approximately 4.8 to approximately 5.3. (Item 3) The formulation according to item 1 or 2, wherein the pH of the formulation is approximately 5.2. (Item 4) The aforementioned sugars are monosaccharides or disaccharides, as described in any one of items 1 to 3. (Item 5) The preparation according to any one of items 1 to 4, wherein the aforementioned sugars are glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, or xylitol. (Item 6) The surfactant is a nonionic surfactant, manufactured according to any one of items 1 to 5. Agent. (Item 7) The formulation according to any one of items 1 to 6, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, or Triton X-100. (Item 8) The formulation according to item 7, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. (Item 9) The formulation according to item 7, wherein the surfactant is poloxamer 188 or poloxamer 407. (Item 10) The preparation described in any one of items 1 to 9, 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 11) The preparation is one of the preparations described in any one of items 1 to 10, comprising the bispecific antibody construct at a concentration of approximately 0.1 mg / ml to approximately 20 mg / ml. (Item 12) The formulation according to any one of items 1 to 12, 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 13) The formulation described in item 12, further comprising a third domain containing a hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain in the order of amino to carboxyl. (Item 14) The formulation according to item 13, wherein each of the first and second binding domains includes a VH region and a VL region. (Item 15) The formulation according to item 13 or 14, wherein the bispecific antibody construct is a single-chain antibody construct. (Item 16) The composition according to any one of items 12 to 15, wherein the target cell surface antigen is CDH19, MSLN, DLL3, FLT3, EGFR, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, MUC17, or CLDN18.2. (Item 17) The composition according to any one of items 12 to 16, 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 18) The second binding domain of the bispecific antibody construct is the composition according to any one of items 12 to 17, comprising one set of 6CDRs shown in SEQ ID NOs: 1 to 6. (Item 19) 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) 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 composition according to any one of items 12 to 18, comprising 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. (Item 20) The composition according to any one of items 12 to 19, wherein the second binding domain comprises 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 21) The aforementioned bispecific antibody construct is a formulation according to any one of items 12 to 20, 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 22) The formulation according to any one of items 1 to 21, wherein the formulation contains less than 10% of the low molecular weight (LMW) clipping product of the bispecific antibody construct after storage at approximately 40°C for one month. (Item 23) The formulation according to item 22, wherein the formulation contains less than 8% of the low molecular weight (LMW) clipping product of the bispecific antibody construct after storage at approximately 40°C for one month. (Item 24) The aforementioned formulation, after storage at approximately 40°C for one month, exhibits approximately 5% of the bispecific antibody construct. The formulation according to item 22 or 23, comprising less than % low molecular weight (LMW) clipping products. (Item 25) The formulation according to any one of items 22 to 24, wherein the formulation contains less than 3% of the low molecular weight (LMW) clipping product of the bispecific antibody construct after storage at approximately 40°C for one month. (Item 26) The preparation is the preparation according to any one of items 22 to 25, wherein, after storage at approximately 40°C for approximately one month, it contains less than 5% high molecular weight (HMW) aggregates of the bispecific antibody construct. (Item 27) The formulation according to any one of items 22 to 26, wherein the relative titer of the bispecific antibody construct is more than 30% after about one month at about 40°C. (Item 28) The formulation according to any one of items 22 to 26, wherein the relative titer of the bispecific antibody construct is more than 50% after about one month at about 40°C. (Item 29) The formulation according to any one of items 22 to 26, wherein the relative titer of the bispecific antibody construct is more than 60% after about one month at about 40°C. (Item 30) A method for treating cancer in a subject in need, comprising administering a preparation described in any one of items 1 to 29 to the subject. (Item 31) The method according to item 30, further comprising the step of diluting the formulation with a pharmaceutically acceptable liquid before administering it to the subject. (Item 32) The preparation is administered by intravenous administration, according to the method described in item 30 or 31.

Claims

[Claim 1] The invention described herein.

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