Pharmaceutical compositions comprising bispecific antibodies against CD3 and CD20, and uses thereof

Abstract

To provide pharmaceutical compositions and dosage unit forms of bispecific antibodies against CD3 and CD20, and uses thereof.SOLUTION: Novel compositions comprising CD3xCD20 bispecific antibodies are useful in therapeutic settings in which specific targeting and T cell-mediated killing of cells that express CD20 are desired. Formulations are useful both for IV administration and for subcutaneous administration.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to pharmaceutical compositions and unit dosage forms of bispecific antibodies against CD3 and CD20, and their uses. [Background technology]

[0002] 2. Background of the Invention CD3 has been known for many years and is therefore of interest in many areas. Specifically, antibodies directed against CD3 or the T cell receptor complex of which CD3 is a part The body is known.

[0003] A promising approach to improve targeted antibody therapy is to target cytotoxic cells to antigen-expressing By specifically delivering the drug to cancer cells, T cells can be used to efficiently kill tumor cells. This concept is described in Staerz, et al., 1985, Nature 314:628-631 (Non-Patent Document 1). However, early clinical trials have shown that bispecific antibodies have low efficacy and have serious side effects. However, the results were rather disappointing due to adverse effects (cytokine storm) and immunogenicity ( Muller and Kontermann, 2010, BioDrugs 24: 89-98 (Non-Patent Document 2)). bispecific antibody Advances in design and application of therapeutics have overcome some of the initial cytokine storm barriers and have made it easier to use. Enhanced clinical efficacy without dose-limiting toxicity (Garber, 2014, Nat. Rev. Drug Disorders v. 13: 799-801 (non-patent document 3); Lum and Thakur, 2011, BioDrugs 25: 365-379 (non-patent document 3); Ref. 4) The initial cytokine storm disruption described for catumaxomab The key to overcoming this barrier was (Berek et al. 2014, Int. J. Gynecol. Cancer 24(9): 1583 -1589 (Non-patent Document 5); Mau-Sorensen et al. 2015, Cancer Chemother. Pharmacol. 75: 1065-1073 (Non-Patent Document 6)), and the absence or silencing of the Fc domain.

[0004] The CD20 molecule (also called the human B-lymphocyte-restricted differentiation antigen or Bp35) is a cellular signaling molecule that mediates the differentiation of pre-B and mature B lymphocytes. It is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD present on B lymphocytes (Valenti ne et al. (1989) J. Biol. Chem. 264(19):11282-11287 (Non-Patent Document 7); and Einfiel d et al., (1988) EMBO J. 7(3):711-717 (Non-Patent Document 8). CD20 is expressed in peripheral blood or lymphocytes. It is found on the surface of more than 90% of B cells derived from host organs and is expressed early in pre-B cell development to induce plasma cell CD20 is present on normal B cells as well as malignant B cells. CD20 is expressed in more than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al. (1984) ) Blood 63(6):1424-1433 (Non-Patent Document 9)) is a marker for hematopoietic stem cells, pro-B cells, and normal plasma cells. It is not found in the cytoplasm of the leukocyte or in other normal tissues (Tedder et al. (1985) J. Immunol. 135(2 ):973-979 (Non-Patent Document 10).

[0005] Methods for treating cancer as well as autoimmune and immune diseases by targeting CD20 - Patent application For example, the chimeric CD20 antibody rituximab has been shown to inhibit the activity of non-Hodgkin lymphoma. Neoplastic lymphoma (NHL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL) These cancers have been used or proposed for treatment of cancers such as The noclonal CD20 antibody ofatumumab is being used in a variety of CLL indications, including follicular lymphoma (FL), neuromyelitis optica (NMO), diffuse and relapsing-remitting multiple sclerosis (RRM) S)

[0006] Bispecific antibodies that bind to both CD3 and CD20 are known from the prior art.

[0007] WO 2011028952 (Patent Document 1) specifically uses Xencor's XmAb bispecific Fc domain technology describe the generation of CD3xCD20 bispecific molecules in vivo.

[0008] WO 2014047231 (Patent Document 2) describes a method for producing a fusion protein using Regeneron Pharmaceuticals' FcΔAdp technology. The authors describe REGN1979 and other CD3xCD20 bispecific antibodies that have been developed.

[0009] Sun et al. (2015, Science Translational Medicine 7, 287ra70 (non-patent document 11)) The B is a 3-inch 10-inch 10-watt 12-watt ... An anti-CD20 / CD3 T cell-dependent bispecific antibody that targets T cells is described.

[0010] WO 2016 / 110576, incorporated herein by reference, describes a bispecific CD3 The present invention provides a method for the preparation of a CD3xCD20 antibody according to WO 2016 / 110576 (Patent Document 3). The present invention relates to a pharmaceutical preparation comprising a bispecific antibody that binds to both CD3 and CD20, and is capable of expressing CD20. These may be useful in therapeutic situations in which specific targeting and T cell-mediated killing of expressing cells is desirable. Such bispecific antibodies may be of potential therapeutic value in the treatment of NHL, CLL, and other B-cell malignancies. Prior art CD3xCD20 bispecific antibodies currently in development in clinical trials are being investigated. The body has been administered via the intravenous (IV) route. Such an administration route is known to be too high High C of CD3xCD20 bispecific antibodies can be correlated with cytokine release levels max This could lead to This is because the target cells expressing CD20 and the T cells are cross-linked by the bispecific antibody. This leads to the release of cytokines, such as inflammatory cytokines, e.g., IL-6, TNF-alpha, This leads to the release of IL-8 or IL-1, which can cause symptoms such as fever, nausea, vomiting, and chills. Thus, despite the unique antitumor activity of bispecific antibodies, These immune mechanisms can have undesirable "side" effects, e.g., "priming cytokines" These reactions can lead to the induction of unwanted inflammatory responses known as “inflammatory responses or syndromes.” In certain circumstances, the patient may be treated with, for example, analgesics, antipyretics, and / or nonsteroidal anti-inflammatory drugs. It is necessary to administer the drug in combination with other drugs or as a premedication. Altering the systemic cytokine release profile of a T cell-redirecting bispecific antibody following immunotherapy There is an unmet need to prevent or reduce the incidence of rheumatoid arthritis. There is a need for therapeutic agents and pharmaceutical compositions of bispecific antibodies that bind to CD3 and CD20. Such compositions, unlike others, avoid or reduce the side effects of systemic cytokine release. Reduces T cell-mediated killing of CD20-expressing tumor cells while at the same time providing highly efficient T cell-mediated killing of CD20-expressing tumor cells The object of the present invention is to provide a method for the treatment of a pulmonary artery disease, comprising administering to a patient a therapeutically effective amount of the compound disclosed in WO 2016 / 110576 (Patent Document 3). The present invention provides a simple and stable pharmaceutical formulation of the D3xCD20 bispecific antibody. The CD3xCD20 antibodies and formulations thereof can be used at antibody concentrations ranging from about 0.5 mg / mL to about 60 mg / mL. or 120 mg / mL, or even as high as 150 mg / mL antibody concentrations. A further object of the present invention is to provide pharmaceutical formulations of CD3xCD20 bispecific antibodies. and wherein the formulation has a shelf life of at least 3 months, or at least 6 months or less. Both are stable for a longer period, such as 12 months. A further object is to provide a formulation that is stable over a temperature range, such as 20° C. to 25° C. The present invention provides a pharmaceutical formulation of a bispecific CD3xCD20 antibody suitable for both intravenous and subcutaneous administration. In many cases, the infusion / injection time is much shorter with SC compared to IV administration. It may be more convenient for the patient if the pharmaceutical formulation is administered subcutaneously. The present invention provides a pharmaceutical formulation of a bispecific CD3xCD20 antibody that is well tolerated at the SC injection site. A further object is to develop a method for the treatment of a patient's pulmonary edema by administering a subcutaneously administered anti-inflammatory drug to the patient, which reduces cytokine release processes in the patient's body. It is possible to provide a tumor suppressor that suppresses tumor cell proliferation and proliferation by inhibiting the proliferation of tumor cells expressing CD20. The present invention provides a pharmaceutical composition capable of providing cell-mediated killing. [Prior art documents]

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

[0013] The object of the present invention is to provide a method for the preparation of a first antigen-binding region derived from a CD3 antibody and a second antigen-binding region derived from a CD20 antibody. The present invention provides pharmaceutical compositions of novel bispecific antibodies comprising the original binding regions.

[0014] Compositions containing novel CD3xCD20 bispecific antibodies are useful for specific targeting of cells expressing CD20. and are useful in therapeutic situations in which T cell-mediated killing is desirable. The formulations are administered IV and subcutaneously. It is useful for both.

[0015] Thus, in a main aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. 50-120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. 20-40 mM acetate; c. 140-160 mM sorbitol; d. Surfactants The present invention relates to a pharmaceutical composition comprising: wherein the pH of the composition is 5 to 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0016] In a further aspect, the present invention relates to the use of the pharmaceutical composition of the present invention for subcutaneous administration. .

[0017] In a further aspect, the present invention relates to the use of the pharmaceutical composition of the present invention for intravenous administration. do.

[0018] In a further aspect, the present invention relates to the use of the pharmaceutical composition of the present invention for the treatment of cancer. .

[0019] In a further aspect, the present invention relates to a method of treating cancer in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of Administering to a subject a pharmaceutical composition of the present invention for a period of time sufficient to treat the cancer. Includes floors.

[0020] In yet a further aspect, the invention relates to a unit dosage form, the unit dosage form comprising: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of about 5 μg to about 50 mg, b. Acetate buffer and sorbitol in a ratio of 1:5 to 1:10, where the osmolality of the unit dosage form is The concentration of the acetate buffer is about 210 to about 250 and the pH is 5 to 6, for example about 5.5. Thor, as well as c. Surfactants Includes.

[0021] In yet another aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of about 5 μg to about 50 mg, b. acetate buffer at a pH of about 5.5 and a concentration of about 30 mM; c. sorbitol at a concentration of about 150 mM; d. Approximately 0.04% w / v Polysorbate 80 The present invention relates to a unit dosage form comprising:

[0022] In yet another aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. epcolitamab, or a biosimilar thereof, in an amount of about 0.5 mg to about 120 mg / mL; b. acetate buffer at a pH of about 5.5 and a concentration of about 30 mM; c. sorbitol at a concentration of about 150 mM; d. Approximately 0.04% w / v Polysorbate 80 The present invention relates to a pharmaceutical composition comprising or consisting of:

[0023] In yet another aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. epcolitamab, or a biosimilar thereof, in an amount of about 0.5 μg to about 120 mg; b. acetate buffer at a pH of about 5.5 and a concentration of about 30 mM; c. sorbitol at a concentration of about 250 mM; d. Approximately 0.04% w / v Polysorbate 80 The present invention relates to a unit dosage form comprising or consisting of:

[0024] These and other aspects and embodiments are described in more detail in the following sections. . [Brief description of the drawings]

[0025] [Figure 1] Solubility screening of Duobody-CD3xCD20 in different formulations. Duobody-CD3xCD20 was formulated in the indicated buffers and sequentially concentrated using a centrifugal concentrator at timed spin intervals. The concentration of each formulation was measured after spin intervals of 20, 50, 60, and 90 minutes. [Diagram 2] Viscosity of Duobody-CD3xCD20 (120-150 mg / mL) in the indicated formulations. The viscosity (cP) of concentrated Duobody-CD3xCD20 samples (120-150 mg / mL) in the indicated formulations was measured at different shear rates using a Wells-Brookfield Cone / Plate rheometer. [Figure 3-1] Mean blood cytokine levels per group of cynomolgus monkeys receiving either a single IV dose (0.1 or 1 mg / kg) or a single SC dose (0.1 or 1 mg / kg) of DuoBody-CD3xCD20. [Figure 3-2] Please refer to the description of Figure 3-1. [Figure 4] Effect of 4x repeated IV administration of DuoBody-CD3xCD20 on B cells in peripheral blood of cynomolgus monkeys. (A) Mean B cell counts (CD4-CD8-CD16-CD19+ cells) per dose group over time in peripheral blood of cynomolgus monkeys after 4 weekly IV administrations of DuoBody-CD3xCD20 (0.01, 0.1, and 1 mg / kg). (B) Mean B cell counts per dose group as a percentage of pre-administration B cell counts. B cell counts are shown as absolute cell counts (cells / μL). [Diagram 5] Effect of a single SC administration of DuoBody-CD3xCD20 on B cells in peripheral blood of cynomolgus monkeys. (A) Mean B cell counts per dose group over time in peripheral blood of cynomolgus monkeys following a single SC administration of DuoBody-CD3xCD20 (0.01, 0.1, 1, 10, or 20 mg / kg). (B) Mean B cell counts per dose group as a percentage of pre-treatment B cell counts. B cell counts are shown as absolute cell counts (cells / μL). [Figure 6] Effect of IV infusion of a priming dose and subsequent target dose of DuoBody-CD3xCD20 on peripheral blood B cells in cynomolgus monkeys. Mean B cell counts (CD4-CD8-CD16-CD19+ cells) over time in peripheral blood of cynomolgus monkeys receiving IV infusion as a priming dose (0.01 mg / kg) and one target dose (1 mg / kg; IV) the following day. B cell counts are shown as absolute cell counts (cells / μL). [Figure 7] Effect of 4x repeated IV administration of DuoBody-CD3xCD20 on B cells in lymph nodes of cynomolgus monkeys. (A) Mean B frequency (CD4-CD8-CD16-CD19+ cells as a percentage of the total lymphocyte population) by dose group over time in lymph nodes of cynomolgus monkeys after four weekly IV administrations of DuoBody-CD3xCD20 (0.01, 0.1, or 1 mg / kg). (B) Mean B cell frequency by dose group as a percentage of B cell frequency before administration. [Figure 8] Effect of a single SC administration of DuoBody-CD3xCD20 on B cells in lymph nodes of cynomolgus monkeys. (A) Mean B cell frequency (CD4-CD8-CD16-CD19+ cells as a percentage of the total lymphocyte population) by dose group over time in lymph nodes of cynomolgus monkeys following a single SC administration of DuoBody-CD3xCD20 (0.01, 0.1, 1, 10 or 20 mg / kg). (B) Mean B cell frequency by dose group as a percentage of B cell frequency before administration. [Figure 9] Effect of IV infusion of a priming dose and subsequent targeting dose of DuoBody-CD3xCD20 on B cells in lymph nodes of cynomolgus monkeys. Mean B cell frequency over time (CD4-CD8-CD16-CD19+ cells as a percentage of the total lymphocyte population) in lymph nodes of cynomolgus monkeys receiving IV infusion of a priming dose (0.01 mg / kg) followed by one targeting dose (1 mg / kg; IV) the following day. [Figure 10]B cell depletion and recovery in cynomolgus monkey spleen and lymph nodes after IV treatment with DuoBody-CD3xCD20. Top panel: Cynomolgus monkey 1 was treated with 0.01 mg / kg DuoBody-CD3xCD20 as a priming dose on day 1 and a target dose of 1 mg / kg on day 2. The animal was euthanized on day 29 according to schedule. Peripheral blood B cell counts had not recovered at the time of necropsy. Bottom panel: Cynomolgus monkey 2 was treated with four weekly doses of 1 mg / kg DuoBody-Cd3xCD20. After peripheral blood B cell recovery was observed, the animal was euthanized on day 148. Frozen sections of lymph nodes and spleens were stained with CD19-specific antibodies to detect B cells (brown staining). Hematoxylin was used to detect cell nuclei (blue staining). [Figure 11] Effect of 5x repeated IV administration of DuoBody-CD3xCD20 on B cells in peripheral blood of male cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) by dose group over time in peripheral blood of male cynomolgus monkeys after 5 weekly IV administrations of saline or 0.01, 0.1 or 1 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts by dose group as a percentage of pre-treatment B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 12] Effect of 5x repeated IV administration of DuoBody-CD3xCD20 on peripheral blood B cells in female cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) by dose group over time in peripheral blood of female cynomolgus monkeys following 5 weekly IV administrations of saline or 0.01, 0.1 or 1 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts by dose group as a percentage of pre-treatment B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 13]Effect of a single IV infusion of DuoBody-CD3xCD20 on B cells in peripheral blood of male cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) by dose group over time in peripheral blood of male cynomolgus monkeys following a single IV infusion of 0.1 or 1 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts by dose group as a percentage of pre-dose B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 14] Effect of a single IV infusion of DuoBody-CD3xCD20 on peripheral blood B cells in female cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) by dose group over time in peripheral blood of female cynomolgus monkeys following a single IV infusion of 0.1 or 1 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts by dose group as a percentage of pre-dose B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 15] Effect of SC injection of DuoBody-CD3xCD20 on B cells in peripheral blood of male cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) per dose group over time in peripheral blood of male cynomolgus monkeys following SC injection of 0.1, 1 or 10 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts per dose group as a percentage of pre-dose B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 16] Effect of SC injection of DuoBody-CD3xCD20 on B cells in peripheral blood of female cynomolgus monkeys. (A) Mean B cell counts (CD45+CD4-CD8-CD16-CD19+ cells) per dose group over time in peripheral blood of female cynomolgus monkeys following SC injection of 0.1, 1 or 10 mg / kg DuoBody-CD3xCD20. (B) Mean B cell counts per dose group as a percentage of pre-dose B cell counts. Numbers of B cells are shown as % of gated lymphocytes. [Figure 17-1](A) Individual plasma concentration profiles in cynomolgus monkeys after IV administration of DuoBody-CD3xCD20. (B) Individual plasma concentration profiles in cynomolgus monkeys after SC administration of DuoBody-CD3xCD20. Plasma concentration profiles of DuoBody-CD3xCD20 were measured after a single SC injection of DuoBody-CD3xCD20 at dose levels of 0.01, 0.1, 1, 10, or 20 mg / kg. (C) Group mean plasma concentration profiles in cynomolgus monkeys after either IV infusion or SC injection. [Figure 17-2] Please refer to the description of Figure 17-1. [Figure 18A] Design fits of HMWP and purity (main peak) data for samples stored at 40° C. for 8 weeks, with the factor levels of NaCl and pH having a significant effect on the results. In FIG. 18A, pH is plotted against NaCl concentration and SEC HMW (%), and as shown, optimal results (low HMW%) were obtained with low NaCl (e.g., 0) and high pH (e.g., 5.3-5.5). Similarly, in FIG. 18B, pH is plotted against NaCl concentration and SEQ Purity (%), and as shown, optimal results (high SEQ Purity %) were obtained with low NaCl (e.g., 0) and high pH (e.g., 5.3-5.5). [Figure 18B] Please see the legend to Figure 18A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention definition The term "immunoglobulin" refers to a class of structurally related glycoproteins, They consist of two pairs of polypeptide chains: one pair of light (L) low molecular weight chains and one pair of heavy (H) chains. All four chains are connected to each other by disulfide bonds. The structure is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W. , ed., 2nd ed. Raven Press, NY (1989)). A chain typically comprises a heavy chain variable region (herein VH or V H and the heavy chain constant region (Herein, CH or C H The heavy chain constant region is typically composed of CH1, C The hinge region is made up of three domains: CH1, CH2, and CH3 of the heavy chain. The region between the main chain and the main chain is highly flexible. The light chain bond is part of the interaction between the two heavy chains of an IgG molecule. Each light chain typically has The variable region (referred to herein as VL or V L and a light chain constant region (abbreviated herein as CL or C L The light chain constant region typically consists of one domain, CL. The VH and VL regions are regions of hypervariability (i.e., hypervariable regions, which have different sequences and and / or the conformation of the structure-restricted loops may be hypervariable, These are also called complementarity determining regions (CDRs), and between them are called framework regions (FRs). Each VH and VL typically has three CDRs and four FRs, flanked by more conserved regions. From the amino terminus to the carboxy terminus, they are composed of FR1, CDR1, FR2, , CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196 , 901- (See also 917 (1987)). Unless otherwise indicated or contradicted by context, In this study, CDR sequences are identified according to the IMGT rules (Brochet X., Nucl Acids Res. 2008;36 : W503-508 and Lefranc MP., Nucleic Acids Research 1999;27:209-212; Internet (See also http: / / www.imgt.org / , the http address of the Unless inconsistent, references to amino acid positions in the constant regions herein are by EU numbering. (Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(1):78-85; Ka bat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 For example, SEQ ID NO: 15 herein is Amino acid positions 118 to 447 of the IgG1 heavy chain constant region are shown according to numbering.

[0027] As used herein, the term "amino acid corresponding to position ..." refers to a human IgG1 heavy chain. Refers to the position number of an amino acid in the chain. Corresponding amino acid in other immunoglobulins The positions can be found by alignment with human IgG1. An amino acid or segment in one sequence corresponds to an amino acid or segment in another sequence. "Corresponding" refers to standard sequence alignment tools such as ALIGN, ClustalW, or similar. The program will then select the other amino acid or segment, typically with default settings. and at least 50%, at least 80%, at least 90% with a human IgG1 heavy chain; or At least 95% identity. How to identify a sequence or a segment of a sequence The choice of whether to align the sequences and thereby determine the positions within the sequences that correspond to the amino acid positions of the present invention is up to the discretion of the individual. It is believed to be well known in the art.

[0028] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, This refers to a fragment of the phospholipid molecule, or any derivative thereof, that is an antigen under typical physiological conditions. and has a half-life of at least about 30 minutes, at least about 45 minutes, At least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, At least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6 , 7 days or more, or any other relevant functionally defined period (e.g., induce, promote, enhance, or cause a physiological response associated with an antibody binding to an antigen) and / or for a period of time sufficient for the antibody to recruit effector activity. The heavy and light chains of the immunoglobulin molecule are separated into a large number of fragments over a long period of time, such as a period sufficient to cause the fragmentation of the heavy and light chains of the immunoglobulin molecule. The variable region of the chain contains a binding domain that interacts with an antigen. The term "antibody binding region" refers to the region that interacts with an antigen, and includes both the VH region and the VL region. As used herein, the term antibody includes not only monospecific antibodies, but also bispecific antibodies. Also included are multispecific antibodies that contain a number of different antigen-binding regions, such as three or more. The constant regions of antibodies (Abs) are involved in the functioning of various cells of the immune system (e.g., effector cells and classical cells). Complement activation is mediated by host tissues and immune cells, including components of the complement system, such as C1q, the first component in the complement activation pathway. As mentioned above, the present invention relates to a method for the treatment of various types of cancer. In this document, the term antibody is used unless otherwise specified or clearly contradicted by the context. As long as the antibody is an antigen-binding fragment, i.e., it retains the ability to specifically bind to an antigen, It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include: (i) Fab' or is a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or (ii) a F(ab')2 fragment, with a disulfide bridge at the hinge region; (iii) a bivalent fragment comprising two Fab fragments linked together; and (iv) a fragment essentially consisting of a VH and a CH1 domain. (iv) an Fv fragment, consisting essentially of the VL and VH domains of one arm of an antibody; , (v) consisting essentially of a VH domain, also called a domain antibody (Holt et al.; Trends Biotechnol. 2003 Nov; 21 (11):484-90), dAb fragments (Ward et al., Nature 341 , 544-546 (1989)); (vi) camelid antibodies or nanobodies (Revets et al.; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24), and (vii) isolated complementarity determining regions In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes. The VL and VH regions are encoded by a single nucleotide polymorphism (SNP) and then combined with each other to form a monovalent molecule. Alternatively, the two proteins may be linked together by a synthetic linker that can be combined into one protein chain to form These are known as single chain antibodies or single chain Fv (scFv) and are described, for example, in Bird et al., Science2 42 , 423-426 (1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988). Unless otherwise stated or clearly indicated by the context, single chain antibodies such as Such fragments are generally included within the meaning of antibody, but These, taken together and individually, are unique features of the present invention and have distinct biological properties and These and other useful antibody fragments in the context of the present invention, as well as Bispecific formats of such fragments are discussed further herein. The term antibody also refers to polyclonal, monoclonal, or IgG antibodies, unless otherwise specified. (mAb), antibody-like polypeptides, chimeric and humanized antibodies, and enzymatic cleavage, peptide The antigen-specific peptides may be provided by any known technique, including peptide synthesis, and recombinant methods. It should also be understood to include antibody fragments (antigen-binding fragments) that retain the ability to bind. The antibody produced can be of any isotype. The term "isotype" refers to the class of immunoglobulins encoded by heavy chain constant region genes. As used herein, the term "immunoglobulin" refers to any of the following antibody classes (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM). When referring to a particular isotype, e.g., IgG1, the term refers to that particular isotype. The antibody is not limited to a specific type sequence, e.g., a specific IgG1 sequence, but rather is Used to indicate closer similarity to an isotype, e.g. IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies of the present invention are naturally occurring, including the diversity in the constant region. The antibody may be a sequence variant of the IgG1 antibody of the present invention.

[0029] As used herein, the term "monoclonal antibody" refers to an antibody molecule of single molecular composition. A monoclonal antibody composition refers to a preparation of antibodies that bind to a particular epitope. Thus, the term "human monoclonal antibody" refers to a The variable and constant regions are derived from human germline immunoglobulin sequences and exhibit two binding specificities. A human monoclonal antibody refers to an antibody that contains human heavy chain transgenes and light chain transgenes. Transgenic or transchromosomal non-human animals having a genome containing an introduced gene, e.g. The hybridomas produced by the hybridomas contain fusions of B cells from the transgenic mice with immortalized cells. It can be done.

[0030] The term "bispecific antibody" or "bs" or "bsAb" in the context of the present invention means This refers to an antibody that has two distinct antigen-binding regions defined by different antibody sequences. The heterologous antibodies can be in any format.

[0031] As used herein, the terms "half molecule," "Fab arm," and "arm" refers to one heavy chain-light chain pair.

[0032] A bispecific antibody is a half molecule "derived from" a first antibody, and a half molecule "derived from" a second antibody. When describing a bispecific antibody as including a half-molecule antibody, the term "derived from" is intended to include the bispecific antibody. The antibody is prepared by isolating the half molecules from each of the first antibody and the second antibody by any known method. This statement indicates that the bispecific antibody was produced as a result of recombination. In this context, "recombinant" is not intended to be limited by any particular recombinant method, and therefore , for example by half molecule exchange (also known as "controlled Fab arm exchange") In addition, recombination at the nucleic acid level and / or simultaneous recombination of two half molecules in the same cell To produce the bispecific antibodies described herein below, including recombinantly by expression, This includes all methods of

[0033] The term "monovalent antibody" in the context of the present invention refers to an antibody molecule having the ability to bind to one molecule of an antigen. This means that it has potency and therefore has no ability to cross-link antigens or cells.

[0034] When used in the context of antibodies, the term "full length" refers to an antibody that is not a fragment and is a specific antibody. All domains normally found in the natural isotype of a given isotype, e.g., VH, CH of an IgG1 antibody. 1, CH2, CH3, hinge, VL, and CL domains.

[0035] As used herein, unless the context indicates differently, the term "Fc region" refers to a The term "antibody region" refers to an antibody region consisting of the Fc sequences of two heavy chains of an immunoglobulin, the Fc sequences comprising at least It contains a hinge region, a CH2 domain, and a CH3 domain.

[0036] As used herein, "heterodimeric interaction between a first CH3 domain and a second CH3 domain" The term refers to the first CH3 region and the second CH3 region in a first-CH3 / second-CH3 heterodimeric protein. Refers to the interaction with the H3 region.

[0037] As used herein, "homodimeric interaction between a first CH3 domain and a second CH3 domain" refers to The term refers to a first CH3 region in a first-CH3 / first-CH3 homodimeric protein and a separate first CH 3 domain, and the second CH3 domain in 2-CH3 / 2-CH3 homodimeric proteins and another second CH3 domain.

[0038] As used herein, the use of "binding" in the context of an antibody binding to a given antigen. The term typically refers to antibodies, e.g., by BioLayer Interferometry (BLI) technology. As determined on an Octet HTX instrument, using as ligand and antigen as analyte, approximately 10 - 6 M or less, say 10 -7 M or less, e.g., about 10 -8 M or Less than about 10 -9 M or less, about 10 -10 M or less, or about 10 - 11 M or lower K D and the antibody binds with an affinity corresponding to K binding to non-specific antigens (e.g., BSA, casein) that are not the same antigen or a closely related antigen D than , at least 10 times lower, for example at least 100 times lower, for example at least 1,000 times lower , e.g. at least 10,000 times lower, e.g. at least 100,000 times lower K D With an affinity corresponding to , binds to a given antigen. The K D The lower the amount of antibody, the K D Depending on the bond, K D Very If the binding K D is the K for binding to nonspecific antigens. D Amounts lower than are at least As used herein, the antibody may be highly specific (i.e., the antibody is highly specific). "K D The term "M" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. When used in D is inversely proportional to the affinity, i.e., the higher the affinity, the lower the K D A lower affinity refers to a higher K D This refers to the following.

[0039] In a preferred embodiment, the antibodies of the present invention are isolated. "Antibody" shall mean an antibody that is substantially free of other antibodies having different antigen specificities. In a preferred embodiment, the isolated bispecific antibody that specifically binds to CD20 and CD3 is In addition, the antibody is substantially free of monospecific antibodies that specifically bind to CD20 or CD3.

[0040] As used herein, the term "CD3" refers to a member of the T cell coreceptor protein complex. CD3 refers to the human cluster of differentiation protein 3, which is a portion of the chromosome that is composed of four separate chains. Because CD3 is found in other species, the term "CD3" is used to refer to human CD3, unless the context indicates otherwise. In mammals, this complex is not limited to the CD3γ (gamma) chain (human CD3γ chain UniProt KB / Swiss-Prot No P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No Q95LI7 ), CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No P04234, or cynomolgus monkey Monkey CD3δ UniProtKB / Swiss-Prot No Q95LI8), two CD3ε (epsilon) chains (human CD3 ε UniProtKB / Swiss-Prot No P07766;Cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No Q95L I5; or rhesus CD3ε UniProtKB / Swiss-Prot No G7NCB9), and CD3ζ-chain (Z Human CD3ζ UniProtKB / Swiss-Prot No. P20963, Cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No. P20963 These chains are expressed in a molecule known as the T cell receptor (TCR). TCR molecules and CD3 molecules generate activation signals in T lymphocytes. Together they form the TCR complex.

[0041] "CD3 antibody" or "anti-CD3 antibody" refers to an antibody specific to the antigen CD3, specifically human CD3ε (epsilon). It is an antibody that specifically binds to

[0042] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No P118 36) is a gene that is naturally expressed by cells, including tumor cells, or that is associated with the CD20 gene or cDNA. Any variant, isoform, and / or expression of CD20 expressed on NA-transfected cells. Species homologs include rhesus CD20 (Macaca mulatta) and species homologs. mulatta); UniProtKB / Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (Macaca fascicularis Examples include macaca fascicularis; UniProtKB No G7PQ03.

[0043] A "CD20 antibody" or "anti-CD20 antibody" specifically binds to the antigen CD20, particularly human CD20. It is an antibody.

[0044] "CD3xCD20 antibody", "anti-CD3xCD20 antibody", "CD20xCD3 antibody", or "anti-CD20xCD3 antibody" " is a bispecific antibody, containing two different antigen-binding regions, one specific for the antigen CD20. and the other specifically binds to CD3.

[0045] As used herein, the term "DuoBody-CD3xCD20" refers to an IgG1 bispecific CD3xCD 20 antibody, wherein the CD3 binding Fab arm is a VH as defined in SEQ ID Nos. 6 and 7, respectively. and VL sequence, the constant light chain sequence as set forth in SEQ ID NO:22, and SEQ ID NO:19(FEAL). wherein the CD20 binding Fab arm comprises the constant heavy chain sequence as set forth in SEQ ID NO: 13 and and 14, the constant light chain sequence as set forth in SEQ ID NO:23, and the VH and VL sequences of SEQ ID NO:20. (FEAR) constant heavy chain sequence. This bispecific antibody comprises the constant heavy chain sequence as defined in WO 2016 / 110576. It may be prepared as described above.

[0046] In a preferred embodiment, the bispecific antibodies of the invention are isolated. In this case, an "isolated bispecific antibody" is substantially free of other antibodies having different antigen specificities. (e.g., an isolated antibody that specifically binds CD20 and CD3) Bispecific antibodies are substantially free of monospecific antibodies that specifically bind to CD20 or CD3. ).

[0047] The present invention relates to a functional barrier domain of the VL region, the VH region, or one or more CDRs of each of the antibody examples. The functional barrier of a VL, VH, or CDR used in the context of an antibody is also provided. The antibody is designed to be compatible with the affinity and / or specificity / selectivity of a "reference" or "parent" antibody. At least a substantial percentage of selectivity (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) In some cases, such antibodies may be able to retain the parental antibody (or more). They may associate with greater affinity, selectivity, and / or specificity than antibodies.

[0048] Such functional variants typically retain significant sequence identity with the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences. (i.e., % homology = number of identical positions / total number of positions x 100), and the optimal alignment of the two sequences is Taking into account the number of gaps that need to be introduced for the insertion, and the length of each gap, The percent identity between two nucleotide or amino acid sequences can be calculated using, for example, , E. Meyers and W. Miller, Compu The PAM120 residue weight table was generated by the algorithm of T. Appl. Biosci 4, 11-17 (1988). Using the side table, the gap length penalty was set to 12 and the gap penalty was set to 4. In addition, the percent identity between two amino acid sequences may be determined according to the Needleman a The sequence may be determined using the algorithm of Wunsch, J. Mol. Biol. 48, 444-453 (1970). .

[0049] Exemplary variants include those that combine the VH and / or VL and / or CDR regions of a parent antibody sequence with It includes those that differ primarily in conservative substitutions, e.g., 10 of the substitutions in the variants, e.g. 9, 8, 7, 6, 5, 4, 3, 2, or 1 are conservative amino acid residue replacements.

[0050] In the context of the present invention, conservative substitutions are made within the classes of amino acids represented in the table below. Can be defined as: Classes of Amino Acid Residues for Conservative Substitutions TIFF2024156853000001.tif76136

[0051] In the context of the present invention, unless otherwise indicated, the following notation is used to describe mutations: i) An amino acid substitution at a given position is indicated, for example, K409R, which is a substitution of the lysine at position 409. ii) for certain variants, any Certain three-letter or one-letter codes are used to represent amino acid residues, including Xaa and X. Thus, a substitution of lysine at position 409 with arginine is written as K409R, and Substitution of lysine at position 409 with any amino acid residue is written as K409X. In the case of a loss, it is indicated by K409*.

[0052] In the context of the present invention, "competition" (or "blocking" or "cross-blocking") ) refers to the tendency of a particular molecule to bind to a particular binding partner, as compared to the tendency of another molecule to bind to that binding partner. The binding of two or more anti-CD20 antibodies to CD20 is significantly reduced in the presence of , may be determined by any suitable technique.

[0053] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. An epitope is a grouping of molecules, usually amino acids or sugar side chains, on a surface. They usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The binding to the conformational epitope and non-conformational epitope is lost in the presence of denaturing solvents, whereas the binding to the non-conformational epitope is lost in the presence of denaturing solvents. The latter is distinguished by the fact that binding to the latter is not lost. The amino acid residues and other amino acid residues not directly involved in binding, e.g., specific antigen-binding peptides The amino acid residues that are effectively blocked or covered by the The group may be within the footprint of a specific antigen-binding peptide.

[0054] As used herein, the term "chimeric antibody" refers to an antibody whose variable regions are derived from a non-human species ( It refers to an antibody whose constant region is derived from a different species, such as a rodent, and whose constant region is derived from a different species, such as a human. Chimeric monoclonal antibodies for therapeutic use are developed to reduce the immunogenicity of the antibodies. When used in the context of a mAb, the term "variable region" or "variable domain" refers to Refers to the region that contains the CDRs and framework regions of both the heavy and light chains of an immunoglobulin Chimeric antibodies are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, N ew York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies are genetically or enzymatically engineered recombinant antibodies. The production of chimeric antibodies is within the knowledge of one of ordinary skill in the art, and therefore the chimeric antibodies of the present invention may be used in combination with other chimeric antibodies. The generation of antibodies may be carried out in ways other than those described herein.

[0055] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody. The antibody has a high level of sequence homology with human antibody constant domains and human variable domains. and a non-human variable domain modified to contain The six non-human antibody complementarity determining regions (CDRs) were then aligned to the homologous human acceptor framework. This can be achieved by connecting the region (FR) to the In order to fully reconstitute the binding affinity and specificity of the parent antibody, the parent antibody (i.e. Substitution of framework residues from a non-human antibody into human framework regions (reverse Structural homology modeling can be used to identify fragments that are important for the binding properties of an antibody. It may be useful to identify amino acid residues within the framework region. contains a non-human CDR sequence, one or more amino acid backmutations in the non-human amino acid sequence It may comprise predominantly human framework regions, and fully human constant regions. Optionally, additional amino acid modifications, not necessarily back mutations, may be used to improve affinity and bioactivity. Humanized antibodies may be obtained that have favorable characteristics, such as biological properties.

[0056] As used herein, the term "human antibody" refers to an antibody that is produced using human germline immunoglobulin sequences. A human antibody refers to an antibody having variable and constant regions derived from human germline immune systems. It may contain amino acid residues not encoded by the immunoglobulin sequence (e.g., in vitro By random or site-specific mutagenesis or by in vivo somatic mutation, (In which mutations have been introduced.) However, as used herein, the term "human antibody" , by substituting CDR sequences derived from the germline of another mammalian species, such as mouse, with human framework sequences. The human monoclonal antibodies of the present invention are not intended to include antibodies that are not related to conventional monoclonal antibodies. Clonal antibody techniques, e.g., the standard method of Kohler and Milstein, Nature 256: 495 (1975) They can be produced by a variety of techniques, including cell hybridization. Although somatic cell hybridization procedures are preferred, other methods for producing monoclonal antibodies are also possible. For example, viral or cancer-induced transformation of B lymphocytes, or licensure of human antibody genes Phage display techniques using human monoclonal antibodies can be used. The preferred animal system for preparing antibody-secreting hybridomas is the murine system. The production of hybridomas in culture is a well-established procedure. Immunization Protocol Techniques for isolating and fusing immunized splenocytes are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Monoclonal antibodies carry parts of the human immune system rather than the mouse or rat system. can be generated using transgenic or transchromosomal mice or rats Thus, in one embodiment, human germline immunoglobulin sequences are used in place of the animal's immunoglobulin sequences. Human antibodies are obtained from transgenic animals such as mice or rats carrying globin sequences. In such embodiments, the antibodies are derived from human germline immunoglobulin sequences introduced into the animal. However, the final antibody sequence is determined by the human germline immunoglobulin sequence undergoing somatic hypermutation. , and are the result of further modification by affinity maturation via the endogenous animal antibody machinery. See, e.g., Mendez et al. 1997 Nat Genet. 15(2):146-56. The term "reducing environment" refers to the reduction of a substrate, here a cysteine ​​residue in the hinge region of an antibody. Refers to the conditions or circumstances under which a group is more likely to be reduced rather than oxidized.

[0057] As used herein, the term "recombinant host cell" (or simply "host cell") The present invention relates to a cell line comprising an expression vector, such as an expression vector encoding an antibody of the present invention, Recombinant host cells include, for example, CHO, CHO-S, HEK, HEK293, H Transfectomas such as EK-293F, Expi293F, PER.C6, or NS0 cells, and lymphomas Examples include globular cells.

[0058] The term "treatment" refers to the treatment or administration of any medicine that relieves, ameliorates, arrests, or cures a symptom or disease state. refers to the administration of an effective amount of a therapeutically active antibody of the invention for eradication (cure) purposes.

[0059] The term "effective amount" or "therapeutically effective amount" refers to the dosage required to achieve the desired therapeutic effect. The therapeutically effective amount of an antibody is determined based on the individual's disease state, age, and duration of administration. factors such as the individual's age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual's body. A therapeutically effective amount may also vary depending on any toxic or detrimental effects of the antibody or antibody portion. This is an amount in which the therapeutically beneficial effect outweighs the therapeutically beneficial effect.

[0060] As used herein, the term "buffer" refers to a pharma- ceutically acceptable buffer. The term "buffer" refers to a compound that maintains the pH value of a solution, e.g., within an acceptable range. Agents that inhibit the release of acetylcholine include, but are not limited to, acetate, histidine, Tris® (Tris( Hydroxymethyl)aminomethane, citrate, succinate, glycolate, etc. Generally, as used herein, a "buffer" refers to a solution having a pH in the range of about 5 to about 6, preferably or has a suitable pKa and buffering capacity for a pH of about 5.5.

[0061] As used herein, a "surfactant" is a surfactant typically used in pharmaceutical formulations to Surfactants are compounds that prevent the adsorption or aggregation of drugs on surfaces. Reduces the surface tension (interfacial tension) between liquids or between liquids and solids. For example, Exemplary surfactants that can be used are those that are present at very low concentrations (e.g., 5% w / v or less, e.g., 3% w / v or less, for example 1% w / v or less, for example 0.4% w / v or less , e.g. less than 0.1% w / v or less, e.g. 0.04% w / v), Surfactants are amphiphilic, meaning that they are usually hydrophilic. They consist of both hydrophobic and lipophilic groups and therefore form micelles or have the ability to form similar self-assembled structures. These include glycerol monooleate, benzethonium chloride, sodium docusate, Lipids, Polyethylene Alkyl Ether, Sodium Lauryl Sulfate, and Tricaprylyl Tricaprylin (anionic surfactant); benzalkonium chloride, citrimide (ci trimide), cetylpyridinium chloride, and phospholipids (cationic surfactants); and Contains alpha tocopherol, glycerol monooleate, myristyl alcohol, phosphorus Lipids, poloxamer, polyoxyethylene alkyl ether, polyoxyethylene castor Oil derivatives, polyoxyethylene sorbintan fatty acid esters, polyoxy Ethylene sterarates, polyoxyl hydroxystearates, polyoxyl polysorbate 20 or polysorbate 80; Propylene glycol dilaurate, propylene glycol monolaurate, sorbitan Esters Sucrose Palmitate, Sucrose Stearate, Tricaprylin, and TP GS (nonionic and zwitterionic surfactants).

[0062] The "diluents" of interest herein are pharma- ceutically acceptable (safe for administration to humans). and non-toxic) and useful for preparing dilutions of pharmaceutical compositions. Such dilutions of the compositions of the present invention dilute only the concentration of the antibody, and the buffer and stabilizer are Thus, in a preferred embodiment, no diluent is present in the pharmaceutical composition of the present invention. Further exemplary diluents include sterile ethyl acetate, ethyl acetate diluents, and ethyl acetate buffers in the same concentrations as those used in the preparation of the pharmaceutical composition. Water, bacteriostatic water for injection (BWFI), pH buffer, preferably acetate buffer, sterile saline , Ringer's solution, or dextrose solution. In a preferred embodiment, the diluent is It comprises or consists essentially of acetate buffer and sorbitol.

[0063] The terms "pharmaceutical composition" and "pharmaceutical formulation" are used interchangeably herein. Used.

[0064] Specific Aspects of the Invention In a main aspect, the present invention provides a method for producing ... liquid crystal display comprising the steps of: a. 0.5 to 120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. 20-40 mM acetate; c. 140-160 mM sorbitol; d. Surfactants wherein the pH of the composition is 5 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0065] In another main aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: a. about 0.50 to about 120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. about 20 to about 40 mM acetate; c. about 140 to about 160 mM sorbitol d. Approximately 0.03-0.05% w / v Polysorbate 80 and providing a pharmaceutical composition comprising: wherein the pH of the composition is about 5 to about 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1, VH-CDR2: SEQ ID NO: 2, VH-CDR3: SEQ ID NO: 3, VL-CDR1: SEQ ID NO: 4, VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8, VH-CDR2: SEQ ID NO: 9, VH-CDR3: SEQ ID NO: 10, VL-CDR1: SEQ ID NO: 11, VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0066] In another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: a. about 0.50 to about 120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. about 20 to about 40 mM acetate; c. about 140 to about 160 mM sorbitol d. Approximately 0.03-0.05% w / v Polysorbate 80 The present invention provides a pharmaceutical composition, comprising: wherein the pH of the composition is about 5 to about 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1, VH-CDR2: SEQ ID NO: 2, VH-CDR3: SEQ ID NO: 3, VL-CDR1: SEQ ID NO: 4, VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8, VH-CDR2: SEQ ID NO: 9, VH-CDR3: SEQ ID NO: 10, VL-CDR1: SEQ ID NO: 11, VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0067] In another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: a. about 0.50 to about 120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. about 20 to about 40 mM acetate; c. about 140 to about 160 mM sorbitol d. Approximately 0.03-0.05% w / v Polysorbate 80 and providing a pharmaceutical composition comprising: wherein the pH of the composition is about 5 to about 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1, VH-CDR2: SEQ ID NO: 2, VH-CDR3: SEQ ID NO: 3, VL-CDR1: SEQ ID NO: 4, VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8, VH-CDR2: SEQ ID NO: 9, VH-CDR3: SEQ ID NO: 10, VL-CDR1: SEQ ID NO: 11, VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0068] This provides a simple yet stable pharmaceutical composition. A further advantage is that the composition is suitable for both IV and SC administration. The compounds are stable, and in particular bispecific antibodies are stable over a wide range of antibody concentrations. Therefore, the formulation can be used at concentrations as low as about 4 μg / mL of antibody in the composition. Phase I doses in clinical trials have ranged from as high as 120 mg / mL or even higher. The composition may be used in titration studies and may be used in subsequent stages of clinical trials and ultimately in Such formulations may even be used in formulations that are commercially available in the United States. It is surprising that it is stable at temperatures ranging from 2°C to 25°C or even higher. The composition of the present invention has a long shelf life of at least 3 months when stored at 2°C to 8°C. For example, stable for at least 6 months, or at least 9 months, or at least 12 months. be.

[0069] In one embodiment of the composition of the invention, the first binding domain of the bispecific antibody that binds to CD3 has SEQ ID Contains the VH and VL sequences of NOs:6 and 7.

[0070] In a further embodiment of the composition of the invention, the second binding region of the bispecific antibody that binds to CD20 comprises , including the VH and VL sequences of SEQ ID: 13 and 14.

[0071] In a further embodiment of the pharmaceutical composition of the invention, the bispecific antibody is DuoBody-CD3xCD20. do.

[0072] In a preferred embodiment of the composition of the invention, the bispecific antibody is an IgG1 antibody. Bispecific antibodies may alternatively be of the IgG2, IgG3, or IgG4 antibody isotype, or of the IgG1, For example, the first heavy chain may be an IgG1 antibody. and the second heavy chain may be of the IgG4 isotype.

[0073] In a further embodiment of the composition of the invention, the bispecific antibody comprises a first heavy chain and a second heavy chain. Fc region, which is reduced compared to a bispecific antibody comprising a wild-type IgG1 Fc region. The bispecific antibody has been modified to have effector functions. and have a reduced ability to bind to human Fc-gamma receptors and human complement component C1q, As a result, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and and their ability to induce Fc-mediated effector functions such as complement-dependent cytotoxicity (CDC). Thus, the bispecific antibodies of the present invention with reduced effector function are 20 expressing cells. In other words, such bispecific antibodies activate T cells only in the presence of the The body induces antibody-mediated FcR-dependent CD3 cross-linking and subsequent target-independent T cell activation. Does not lead.

[0074] In another embodiment of the pharmaceutical composition of the present invention, the bispecific antibody has a wild-type IgG1 Fc region. The binding of C1q to the antibody is at least 70%, at least 80%, or less than that of a bispecific antibody. F that has been modified to reduce F by at least 90%, at least 95%, at least 97%, or 100%. c region, where C1q binding is determined by ELISA.

[0075] The bispecific antibodies described herein can be used in any of the methods described, for example, in WO 2011 / 131746 and in Labrijn AF e t al., (2013) PNAS 110(13): 5145-5150, using DuoBody® technology. It can be produced according to the platform (Genmab A / S). a first monospecific antibody half containing two heavy chains and two light chains, and a second monospecific antibody half containing two heavy chains and two light chains. The resulting heterodimer can then be combined with one half of a second monospecific antibody containing the light chain of The dimer comprises one heavy chain and one light chain derived from a first antibody and one heavy chain derived from a second antibody. and one light chain paired with the first monospecific antibody. If the specific antibodies recognize separate epitopes on separate antigens, such as CD3 and CD20, The resulting heterodimer is a bispecific antibody against CD3 and CD20.

[0076] DuoBody technology consists of monospecific antibodies that each contain a heavy chain constant region with one point mutation in the CH3 domain. By virtue of point mutations, the CH3 domains of the resulting bispecific antibody are The interaction is stronger than the interaction between the CH3 domains of both monospecific antibodies. Single point mutations in heterologous antibodies can be used in the EU According to the index numbering, residues 366, 368, 370, and 371 of the CH3 domain of the heavy chain constant region are In addition, one point mutation is present in one monospecific antibody at position 399, 405, 407, or 409. are present at different residues compared to the other monospecific antibody. The antibody may include the mutation F405L (i.e., a mutation of residue 405 from phenylalanine to leucine). However, the other monospecific antibody has the mutation K409R (i.e., a lysine to arginine mutation at residue 409). The heavy chain constant region of the monospecific antibody may comprise an IgG1, IgG2, IgG3, or IgG4 isotype. The antibody may be of the human IgG1 isotype, and may be a bispecific antibody produced by DuoBody technology. Heterologous antibodies may retain Fc-mediated effector functions or may have the Fc-mediated effector functions described herein. The Fc region may be further mutated to reduce Fc-mediated effector functions as described above. .

[0077] Fc regions may have a lysine at the C-terminus. The source of this lysine may vary depending on the antigen-binding domain from which these Fc regions are derived. During the production of recombinant antibodies in cell culture, this terminal The terminal lysine can be proteolytically cleaved by endogenous carboxypeptidases. This results in a constant region with the same sequence but without the C-terminal lysine. For manufacturing purposes, this terminal lysine is encoded by a lysine-free antibody. The DNA encoding the terminal lysine can be omitted from the sequence. The antibody and the antibody produced from the non-encoding nucleic acid sequence are substantially identical in sequence and function. However, this is because, for example, when using antibodies produced in a CHO-based production system, This is because the degree of processing of is typically high (Dick, LW et al. Biotechnol. Bioeng. 2008;100: 1132-1143).

[0078] Thus, in another embodiment of the pharmaceutical composition of the present invention, the bispecific antibodies each comprise at least The first and second heavy chains each include at least a hinge region, a CH2 region, and a CH3 region, The heavy chain consists of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain. At least one of the amino acids at a position corresponding to a position selected from the group consisting of: In the second heavy chain, T366, L368, K370, D399, F405, Y407, and K40 in the human IgG1 heavy chain 9 (according to the EU numbering system) At least one amino acid in the first heavy chain is substituted at the same position in the second heavy chain. It has not been done.

[0079] In yet another embodiment of the pharmaceutical composition of the present invention, (i) a polypeptide corresponding to F405 in a human IgG1 heavy chain is provided. the amino acid at position A is replaced with L in the first heavy chain of the bispecific antibody, The amino acid at the position corresponding to K409 in the G1 heavy chain is R in the second heavy chain of the bispecific antibody. or (ii) an amino acid at a position corresponding to K409 in a human IgG1 heavy chain is substituted with and in the first heavy chain is R, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain.

[0080] In one embodiment the bispecific antibody of the pharmaceutical composition comprises a first constant heavy chain and a second constant heavy chain of the bispecific antibody. and the second constant heavy chain, at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain. (EU index numbering), L234 is replaced by F (L234F) and L235 is replaced by E (L235E) to enhance Fc-mediated efficacy of the antibody. The function of the vector is reduced.

[0081] In a further embodiment, the bispecific antibody of the pharmaceutical composition comprises a first constant domain of the bispecific antibody. In both the first and second constant heavy chains, D265 is A at the position corresponding to D265 in human IgG1. It may be further substituted, such as substituted (D265A).

[0082] In another embodiment, the bispecific antibody of the pharmaceutical composition comprises the first constant heavy chain and Both the first and second constant heavy chains contain three substitutions: L234F+L235E+D265A.

[0083] In another embodiment, the bispecific antibody of the pharmaceutical composition comprises the first constant heavy chain and Both the first and second constant heavy chains contain three substitutions L234F+L235E+D265A, and the first constant heavy chain contains a F405L substitution. and the second constant heavy chain further comprises a K409R substitution, or vice versa. The first constant heavy chain contains the substitutions L234F+L235E+D265A+F405L (also referred to herein as the "FEAL" mutation). and the second constant heavy chain comprises the substitutions L234F+L235E+D265A+K409R (referred to herein as "FEAR"). mutation), or the first constant heavy chain contains the substitutions L234F+L235E+D265A+K409R, and the second The constant heavy chain comprises the substitutions L234F+L235E+D265A+F405L. The first and second constant heavy chains are of the IgG1 isotype, but each contains the substitution L234F+ L235E+D265A+F405L and L234F+L235E+D265A+K409R. Thus, one aspect of the present invention In the present invention, the bispecific antibody of the pharmaceutical composition comprises a first heavy chain constant region of SEQ ID NO: 19 and a second heavy chain constant region of SEQ ID NO: or a first heavy chain constant region of SEQ ID NO: 20 and a second heavy chain constant region of SEQ ID NO: 20. In another embodiment, the bispecific antibody comprises a second heavy chain constant region of SEQ ID NO: 19. At least 90% sequence identity, e.g., at least 91%, with the amino acid sequence of O 19 and 20 For example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, For example, those with at least 99% sequence identity but containing the FEAR and FEAL amino acids described above. , including the heavy chain constant region.

[0084] The first and second light chains of the bispecific antibody of the composition preferably comprise a first light chain constant region and and a second light chain constant region. The light chain constant region is of the lambda subtype. In a preferred embodiment of the invention, the CD3 binding arm may be of the kappa subtype. The constant region of the light chain of the CD20-binding arm is of the lambda subtype, and the constant region of the light chain of the CD20-binding arm is of the In one embodiment the light chain (VL+CL) of the CD3 binding arm is of the kappa subtype. The light chain of the CD20 binding arm has the sequence of SEQ ID NO:24, and the light chain of the CD20 binding arm has the sequence of SEQ ID NO:25.

[0085] In further highly preferred aspects, the pharmaceutical compositions, methods, uses, and the bispecific CD3xCD20 antibody in unit dosage form are shown in SEQ ID NOs. 26 and 24, respectively. a first binding arm comprising a binding region that binds to human CD3, said binding arm having defined heavy and light chains; and human CD2 having heavy and light chains as set forth in SEQ ID NOs. 27 and 25, respectively. 0. These chains are represented by SEQ ID NOs. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 2, and 23, and the CDRs, VHs, and VHs set forth in SEQ ID NOs. 1-14, and VL sequences. Pharmaceutical compositions, methods, uses, and unit dosage forms described herein In a further aspect, variants of such antibodies are envisaged. The heterologous antibody is epcolitamab (CAS 2134641-34-0), or a biosimilar thereof.

[0086] The concentration of the bispecific antibody in the pharmaceutical composition may be from about 0.5 mg / mL to about 200 mg / mL. In one embodiment, the concentration of the bispecific antibody is about 0.5 to about 120 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is about 1 to about 110 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is about 1 to about 60 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is about 10 to about 30 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is about 12 to about 24 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 5 mg / mL. In one embodiment, the concentration of the bispecific antibody is about 6 mg / mL. The concentration of the antibody is about 7 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 8 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 9 mg / mL. In another embodiment, the concentration of the bispecific antibody is about 10 mg / mL. The concentration of the specific antibody is about 11 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is In another embodiment of the invention, the concentration of the bispecific antibody is about 13 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 14 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 15 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 17 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 18 mg / mL. In one embodiment, the concentration of the bispecific antibody is about 19 mg / mL. The concentration of the isomeric antibody is about 20 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about In another embodiment of the invention, the concentration of the bispecific antibody is about 22 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 23 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 24 mg / mL. The concentration is about 25 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 26 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 27 mg / mL. In an embodiment, the concentration of the bispecific antibody is about 28 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 30 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 35 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 40 mg / mL. The concentration of the bispecific antibody is about 45 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is In another embodiment of the invention, the concentration of the bispecific antibody is about 50 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 55 mg / mL. In one embodiment, the concentration of the bispecific antibody is about 60 mg / mL. The concentration of the antibody is about 65 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is about 70 mg / mL. g / mL.

[0087] In a further embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is between 50 and 120 mg / kg. In another embodiment of the present invention, the concentration of the bispecific antibody is 50 to 110 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is 50 to 100 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is 50 to 90 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is 50 to 70 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 60 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 70 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 80 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 90 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 100 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody in the pharmaceutical composition is 110 mg / mL. In another embodiment, the concentration of the bispecific antibody in the pharmaceutical composition is 120 mg / mL. In another embodiment, the concentration of the bispecific antibody in the pharmaceutical composition is 130 mg / mL. In another embodiment, the concentration of the bispecific antibody in the pharmaceutical composition is 140 mg / mL. In an embodiment, the concentration of the bispecific antibody in the pharmaceutical composition is 150 mg / mL.

[0088] The pharmaceutical compositions of the present invention adjust the pH to optimize the therapeutic efficacy and stability of the bispecific antibody. Acetate buffers are used to control the concentration of the acetate in the injection It may be produced by mixing sodium acetate trihydrate with acetic acid in water. In one embodiment, the acetate buffer may be adjusted to 20% by adding sodium chloride. In one embodiment of the invention, the acetate buffer is present in the composition at a concentration of from about 10 mM to about 40 mM. In another embodiment of the invention, the concentration of acetate buffer in the composition is 25 mM. In another embodiment of the invention, the concentration of acetate buffer in the composition is 26 mM. In one embodiment, the concentration of acetate buffer in the composition is 27 mM. In another embodiment of the present invention, the concentration of acetate in the composition is 28 mM. The concentration of the buffer is 29 mM. In another embodiment of the present invention, the acetate buffer in the composition In another embodiment of the invention, the concentration of acetate buffer in the composition is 31 mM. In another embodiment of the present invention, the concentration of acetate buffer in the composition is 32 mM. In another embodiment of the present invention, the concentration of acetate buffer in the composition is 33 mM. In another embodiment of the present invention, the concentration of acetate buffer in the composition is 34 mM. In another embodiment of the present invention, the acetate buffer in the composition has a concentration of 35 mM. In one embodiment of the invention, the pharmaceutical composition contains an additional buffer. In another embodiment, the pharmaceutical composition does not contain an additional buffer. The inventors have found that acetate buffers improve the stability of bispecific antibodies compared to histidine buffers. We have found that the bispecific antibodies of the invention are surprisingly well suited for immunoprecipitation at 30 mM, pH 5. After storage at 40°C for 8 weeks in histidine buffer, 14.2% of high molecular weight particles were formed. However, after 8 weeks of storage at 40°C in 30 mM acetate buffer, pH 5.5, the high molecular weight particles were 5.8%. It was found that no carboxyl group was formed (see Example 3).

[0089] In one embodiment of the present invention, the pH of the pharmaceutical composition is in the range of 5 to 6. It will be appreciated that it is preferred to include a buffer suitable for the purpose of In another embodiment, the pH of the pharmaceutical composition is in the range of 5.2 to 5.8. In another embodiment, the pH of the pharmaceutical composition is in the range of 5.3 to 5.5. In another embodiment of the present invention, the pH of the pharmaceutical composition is in the range of 5.4 to 5.6. In another embodiment, the pH of the pharmaceutical composition is about 5.5. In another embodiment, the pH of the pharmaceutical composition is about It is about 5.4.

[0090] The pharmaceutical composition of the present invention may further comprise a polyol as a "stabilizer", which They can interact with the charged groups of amino acid side chains, thereby improving inter- and intramolecular correlation. Preferably, the pharmaceutical composition of the present invention is The composition may further include sorbitol as a "stabilizer," which interacts with the charged groups of the amino acid side chains. can act, thereby reducing the potential for inter- and intra-molecular interactions. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 100 mM to 250 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 150 mM to 250 mM. In another embodiment, sorbitol is present at a concentration of 130 mM to 200 mM. In one embodiment, sorbitol is present at a concentration of 140 mM to 160 mM. In the pharmaceutical composition, sorbitol is present at a concentration of 140 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 146 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 147 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 148 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 149 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 150 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 151 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 153 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 154 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 155 mM. Sorbitol is present in the pharmaceutical composition at a concentration of 160 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 170 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 190 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 200 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 210 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 220 mM. Sorbitol is present in the pharmaceutical composition at a concentration of 230 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 240 mM. It is present in a concentration of 250 mM.

[0091] As mentioned above, the pharmaceutical composition of the present invention further comprises a polyol as a "stabilizer." which can interact with the charged groups of the amino acid side chains, thereby forming intermolecular and reducing the potential for intramolecular interactions. In one embodiment, the polyol is present in the pharmaceutical composition at a concentration of 100 mM to 300 mM. In another embodiment, the polyol is present in the pharmaceutical composition at a concentration of 140 mM to 260 mM. In another embodiment, the polyol is present at a concentration of 140 mM to 160 mM. In another embodiment, the polyol is present at a concentration of from 240 mM to 260 mM.

[0092] In one embodiment, the osmolality (mOsm / kg) of the pharmaceutical composition is 200 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 210 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 220 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 240 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 250 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 260 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is In another embodiment, the osmolality of the pharmaceutical composition is 290 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 300 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 310 mOsm / kg. The osmolality is 320 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 3 30 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 340 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 350 mOsm / kg. The osmolality of the pharmaceutical composition is 360 mOsm / kg. In another embodiment, the osmolality of the pharmaceutical composition is 380 mOsm / kg.

[0093] For example, it may be preferable to have an osmolality of less than 600 mOsm / kg, which , such osmolality is widely accepted as being well tolerated upon subcutaneous administration. Thus, in another embodiment, the osmolality (mOsm / kg) of the pharmaceutical composition is from 2 The range of the blood glucose level is 00 to 600 mOsm / kg, and more preferably 200 to 450 mOsm / kg. In this case, an osmolality in the range of 220-380 mOsm / kg is selected.

[0094] In one embodiment of the invention, the concentration ratio of acetate buffer to sorbitol in the pharmaceutical composition is 1: In one embodiment of the present invention, the concentration ratio of acetate buffer to sorbitol is 1:5 to 1:10. In another embodiment of the invention, the concentration ratio of acetate buffer to sorbitol is 1:6. In another embodiment of the invention, the concentration ratio of acetate buffer to sorbitol is 1:7. In another embodiment of the present invention, the concentration ratio of acetate buffer to sorbitol is 1:8. In an embodiment, the concentration ratio of acetate buffer to sorbitol is 1:9. The acetate buffer to sorbitol concentration ratio is 1:10.

[0095] The pharmaceutical composition of the present invention further comprises a surfactant. In one embodiment, the surfactant is a glycolipid. Cerol monooleate, benzethonium chloride, sodium docusate, phospholipids, poly Ethylene alkyl ether, sodium lauryl sulfate, and tricaprylin, benzoyl chloride Zalkonium, Citrimide, Cetylpyridinium chloride, Phospholipids, Alpha Tocopherol , glycerol monooleate, myristyl alcohol, phospholipids, poloxamer, Polyoxyethylene alkyl ether, polyoxyethylene castor oil derivatives, polyoxy Ethylene sorbitan fatty acid ester, polyoxyethylene stearate, polyoxyl Hydroxystearate, polyoxylglyceride, polysorbate, propylene glycol Dilaurate, Propylene Glycol Monolaurate, Sorbitan Ester Sucrose Selected from the group including palmitate, sucrose stearate, tricaprylin, and TPGS. It is selected.

[0096] In a preferred embodiment, the surfactant is a polysorbate. It is resorbate 20. In another preferred embodiment, it is polysorbate 80.

[0097] In one embodiment of the present invention, the surfactant is present at a concentration of about 0.005% to 0.4% w / v. In one embodiment, the surfactant is present at a concentration of about 0.01 to 0.1% w / v. The surfactant is present at a concentration of about 0.01 to 0.09% w / v. In one embodiment of the present invention, the surfactant In one embodiment of the present invention, the surfactant is present at a concentration of about 0.01 to 0.06% w / v. In one embodiment of the invention, the surfactant is present at a concentration of about 0.01% w / v. In one embodiment of the invention, the surfactant is present at a concentration of about 0.02% w / v. In one particular embodiment, the surfactant is present in a concentration of about 0.03% w / v. The surfactant is present at a concentration of about 0.04% w / v. In one embodiment of the invention, the surfactant is present at a concentration of about 0. In one embodiment of the invention, the surfactant is poly(Aminopropyl) at a concentration of 0.04% w / v. The present inventors found that the inclusion of a surfactant reduces the physical properties of the bispecific antibody. We found that the stability was improved and the level of visible particles was significantly reduced. It was found that this is more important for larger batches of formulations than for smaller batches. .

[0098] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 20-40 mM acetate c. 140-160 mM sorbitol d. 0.005%-0.4% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0099] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 20-40 mM acetate c. 140-160 mM sorbitol d. 0.005%-0.4% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It essentially consists of:

[0100] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 20-40 mM acetate c. 140-160 mM sorbitol d. 0.005%-0.4% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0101] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 5-60 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0102] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 5-60 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It essentially consists of wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0103] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 10-50 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It essentially consists of wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0104] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 12-24 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It essentially consists of wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0105] In another embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 5-60 mg / mL, e.g., 10-50 mg / mL, e.g., 12-24 mg / mL of a bispecific antibody b. 30 mM acetate buffer c. 150 mM sorbitol d. 0.04% w / v surfactant, preferably a polysorbate, such as polysorbate 20 or Polysorbate 80, e.g. Polysorbate 80 It essentially consists of wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0106] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 5-60 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0107] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 10-50 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0108] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 12-24 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0109] In another embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 5-60 mg / mL, e.g., 10-50 mg / mL, e.g., 12-24 mg / mL of a bispecific antibody f. 30 mM acetate buffer g. 150 mM Sorbitol h. 0.04% w / v surfactant, preferably a polysorbate, such as polysorbate 20 or Polysorbate 80, e.g. Polysorbate 80 It consists of: wherein the CD3-binding Fab arms of the bispecific antibodies are set forth in SEQ ID Nos. 6 and 7, respectively. VH and VL sequences, and the constant heavy chain sequence as set forth in SEQ ID NO: 19 (FEAL), The 0-binding Fab arms were based on the VH and VL sequences of SEQ ID NO: 13 and 14, respectively, and SEQ ID NO: The heavy chain constant sequences are as defined in FEAR 20 (FEAR).

[0110] In one embodiment, the pharmaceutical composition comprises 30 mM acetate, 150 mM sorbitol, pH 5.5, and 0.5% ethanol. The drug concentrate (DuoBodyCD3xCD20) was formulated in 0.04% w / v polysorbate 80. The condensate was diluted with diluent prior to administration to give bispecific antibody concentrations ranging from 2 μg / mL to 24 mg / mL. The concentrate may be diluted with a diluent on the day before administration to obtain a concentration of 2 μg / mL to 24 mg / mL of the bispecific antibody. The concentrate may be diluted with a diluent prior to administration on the day of administration to a concentration of 2 μg / mL to 24 μg / mL. The concentrate may be diluted with a diluent immediately prior to administration to provide a bispecific antibody concentration of 2 μg / mL. In one embodiment, the diluent formulation may have a bispecific antibody concentration of 30 μg / mL to 24 mg / mL. M acetate, 150 mM sorbitol, pH 5.5, and 0.04% w / v polysorbate 80. Other suitable pharma- ceutically acceptable diluents, as described herein, may be envisaged.

[0111] In another embodiment, the diluent is a commercially available diluent. Highly preferred diluents are suitable for injection. A sodium chloride solution such as 0.9% NaCl (w / v) in water. Such diluents are used herein. It is very useful for diluting the CD3xCD20 bispecific antibody in a pharmaceutical composition as defined in For example, the present invention can be formulated using a 0.9% sodium chloride solution (w / v) in water suitable for injection. The epcolitamab contained in the pharmaceutical solution can be diluted.

[0112] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0113] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It essentially consists of wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0114] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 0.5-120 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0115] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 2-8 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0116] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 2-8 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0117] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 40-80 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0118] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 40-80 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0119] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. Epcolitamab from 0.5 to 120 mg / mL b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0120] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. Epcolitamab from 0.5 to 120 mg / mL b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0121] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 2-8 mg / mL epcolitamab b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0122] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 2-8 mg / mL epcolitamab b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0123] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 40-80 mg / mL epcolitamab b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0124] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. 40-80 mg / mL epcolitamab b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0125] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and a. Epcolitamab at 5, 48, or 60 mg / mL b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0126] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 0.5-120 mg / mL of bispecific antibody j. 28-32 mM acetate k. 145-155 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0127] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 2-8 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0128] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 2-8 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0129] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 40-80 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0130] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 40-80 mg / mL of bispecific antibody f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0131] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 0.5-120 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0132] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 0.5-120 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0133] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 2-8 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0134] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 2-8 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0135] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 40-80 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0136] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. 40-80 mg / mL epcolitamab f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0137] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and e. Epcolitamab at 5, 48, or 60 mg / mL f. 28-32 mM acetate g. 145-155 mM sorbitol h. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0138] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and m. 0.5-120 mg / mL of bispecific antibody n. 28~32 mM acetate o. 140~260 mM sorbitol p. 0.02-0.06% w / v surfactant, preferably polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0139] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 2-8 mg / mL of bispecific antibody j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0140] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 2-8 mg / mL of bispecific antibody j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0141] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 40-80 mg / mL of bispecific antibody j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Including, wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0142] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 40-80 mg / mL of bispecific antibody j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of: wherein the bispecific antibody comprises the heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. It contains a CD20-binding arm having a CD20-binding domain and a light chain.

[0143] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 0.5-120 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0144] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 0.5-120 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0145] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 2-8 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0146] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 2-8 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0147] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 40-80 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 Includes.

[0148] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. 40-80 mg / mL epcolitamab j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0149] In one embodiment of the invention, the pharmaceutical composition has a pH of about 5.5, and i. Epcolitamab at 5, 48, or 60 mg / mL j. 28-32 mM acetate k. 140-260 mM sorbitol l. 0.02-0.06% w / v surfactant, preferably a polysorbate, e.g. polysorbate 20 or polysorbate 80, e.g. polysorbate 80 It consists of:

[0150] In one embodiment, the pharmaceutical composition is prepared as described herein, e.g., with 30 mM acetate, Formulated and administered in 150 mM sorbitol, pH 5.5, and 0.04% w / v polysorbate 80 The bispecific antibody may be pre-diluted with a diluent to a desired concentration. For example, the antibody may be diluted with 2 μl of The antibody may be diluted to a bispecific antibody concentration ranging from 200 µg / mL to 24 mg / mL, for example in 30 mM acetic acid. Formulated in 0.01% saline, 150 mM sorbitol, pH 5.5, and 0.04% w / v polysorbate 80. The agent comprises a bispecific antibody, such as epcolitamab, and as defined herein. The concentration of the active ingredient may range from 2 to 8 mg / mL, and may be adjusted to a concentration of 100 μg / mL to 2 mg / mL using an appropriate diluent prior to administration. In one embodiment, the diluent formulation is 30 mM acetate, 150 mM sol. Bitol, pH 5.5, and 0.04% w / v polysorbate 80. In one embodiment, the diluent formulation The buffer consisted of 30 mM acetate, 250 mM sorbitol, pH 5.5, and 0.04% w / v polysorbate 80. In another aspect, the diluent is a pharma- ceutically acceptable diluent. A pharma- ceutically acceptable diluent is 0.9% w / v sodium chloride in water. The antibody is diluted to a selected concentration so that an appropriate volume can be administered to the patient, e.g., subcutaneously. The enabling of administration is performed several days prior to administration. In one embodiment, the dilution is prepared the day before administration. In another embodiment, dilutions of the antibody product are prepared on the day of administration.

[0151] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5 at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM of a polyol; and (iv) a surfactant. In some embodiments, the surfactant comprises about 0.005% to 0.4% w / v concentration. In some embodiments, the buffer has a pH of about 5.3 to about 5.6.

[0152] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, at about 5 to about 60 mg / mL; 0 to 40 mM acetate buffer; (iii) about 140 to about 250 mM polyol; and (iv) a surfactant. In some embodiments, the surfactant is present in an amount of about 0.005% to 0. It is contained at a concentration of 4% w / v.

[0153] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, at about 5 to about 60 mg / mL; 0 to 40 mM acetate buffer, (iii) about 140 to about 250 mM polyol, and (iv) polysol. In some embodiments, the surfactant is about 0.001%. It is found in concentrations of 5% to 0.4% w / v.

[0154] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM sorbitol; and (iv) a surfactant. In some embodiments, the surfactant is present in an amount of about 0.005% to about 0.5%. It is contained at a concentration of 0.4% w / v.

[0155] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5 at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM of a polyol; and (iv) polysorbate In some embodiments, the surfactant is about 0.005% to about 0.5% by weight. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. do.

[0156] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5 at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM sorbitol, and (iv) a surfactant. In some embodiments, the surfactant is about 0.005% to 0.4% In some embodiments, the buffer has a pH of about 5.3 to about 5.6. .

[0157] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5 at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM sorbitol, and (iv) a surfactant. In some embodiments, the surfactant is about 0.005% to 0.4% In some embodiments, the buffer has a pH of about 5.3 to about 5.6. .

[0158] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, at about 5 to about 60 mg / mL; (iii) about 140 to about 250 mM sorbitol, and (iv) polysorbate. In some embodiments, the surfactant is about 0. It is contained in concentrations of 0.05% to 0.4% w / v.

[0159] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM of a polyol; and (iv) an interface. In some embodiments, the surfactant is about 0.005%. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. Yes.

[0160] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM polyol; and (iv) In some embodiments, the surfactant is about 0. It is contained in concentrations of 0.05% to 0.4% w / v.

[0161] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM polyol; and (iv) Polysorbate 80. In some embodiments, the surfactant is , at a concentration of approximately 0.005% to 0.4% w / v.

[0162] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM sorbitol, and (iv ) a surfactant. In some embodiments, the surfactant is about 0. Found in concentrations between .005% and 0.4% w / v.

[0163] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM of a polyol; and (iv) a poly Sorbate 80. In some embodiments, the surfactant comprises about In some embodiments, the buffer is present at a concentration of about 5.3 to about 5.6% w / v. The pH is

[0164] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM sorbitol, and (iv) about 20 to about 40 mM of a buffer containing In some embodiments, the surfactant is about 0.00 In some embodiments, the buffer is present at a concentration of about 5% to about 0.4% w / v. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. has.

[0165] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM sorbitol, and (iv) about 20 to about 40 mM of a buffer for Resorbate 80. In some embodiments, the surfactant is In some embodiments, the buffer is present at a concentration of about 5.3 to about 5. It has a pH of .6.

[0166] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM sorbitol, and (iv ) Polysorbate 80. In some embodiments, the surfactant is present at a concentration of approximately 0.005% to 0.4% w / v.

[0167] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 0.3 to about 6.5; (iv) about 140 to about 250 mM polyol; and (iv) a surfactant. The inhibitor is present at a concentration of about 0.005% to 0.4% w / v. In some embodiments, the buffer is at about It has a pH of 5.3 to about 5.6.

[0168] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 1.3 to about 5.6; In some embodiments, the present invention relates to a pharmaceutical composition comprising: The surfactant is included at a concentration of about 0.005% to 0.4% w / v.

[0169] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 1.3 to about 5.6; and (iv) polysorbate 80. The surfactant is contained at a concentration of about 0.005% to 0.4% w / v.

[0170] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 1.3 to about 5.6; In some embodiments, the pharmaceutical composition comprises: The surfactant is included at a concentration of about 0.005% to 0.4% w / v.

[0171] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 0.3 to about 6.5; (iv) about 140 to about 250 mM polyol; and (iv) polysorbate 80. The surfactant is present at a concentration of about 0.005% to 0.4% w / v. In some embodiments, the buffer has a pH of about 5.3 to about 5.6.

[0172] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 0.3 to about 6.5; and (iv) about 140 to about 250 mM sorbitol. and (iv) a surfactant. The active agent is present at a concentration of about 0.005% to 0.4% w / v. In some embodiments, the buffer comprises: It has a pH of about 5.3 to about 5.6.

[0173] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 0.3 to about 6.5; and (iv) about 140 to about 250 mM sorbitol. and (iv) polysorbate 80. In some embodiments, the surfactant is present at a concentration of about 0.005% to 0.4% w / v. The pH of the solution is about 5.3 to about 5.6.

[0174] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a light chain; (ii) about 5 to about 60 mg / mL of a bispecific antibody comprising a CD20-binding arm having a CD20-binding domain and a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 1.3 to about 5.6; In some embodiments, the pharmaceutical composition comprises: (i) glycerol; and (ii) polysorbate 80. In the present invention, the surfactant is present at a concentration of about 0.005% to 0.4% w / v.

[0175] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL epcolitamab, (ii) about 5.3 to about 6.5 mg / mL epcolitamab, (iii) about 140 to about 250 mM of a polyol; and (iv) In some embodiments, the surfactant is at about 0.00 In some embodiments, the buffer is present at a concentration of about 5% to about 0.4% w / v. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. has.

[0176] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL of epcolitamab, (ii) about 5.3 to about 5.6 (iii) about 140 to about 250 mM polyol, and iv) A pharmaceutical composition comprising a surfactant. In some embodiments, the surfactant is about 0. Found in concentrations between .005% and 0.4% w / v.

[0177] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL of epcolitamab, (ii) about 5.3 to about 5.6 mg / mL of epcolitamab, (iii) about 140 to about 250 mM polyol, and iv) A pharmaceutical composition comprising polysorbate 80. In some embodiments, a surfactant is present at a concentration of approximately 0.005% to 0.4% w / v.

[0178] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL of epcolitamab, (ii) about 5.3 to about 5.6 mg / mL of epcolitamab, (iii) about 140 to about 250 mM sorbitol, and and (iv) a surfactant. In some embodiments, the surfactant is It is found at a concentration of approximately 0.005% to 0.4% w / v.

[0179] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL epcolitamab, (ii) about 5.3 to about 6.5 mg / mL epcolitamab, (iii) about 140 to about 250 mM of a polyol; and (iv) The present invention relates to a pharmaceutical composition comprising polysorbate 80. In some embodiments, the surfactant is In some embodiments, the buffer is present at a concentration of about 5.3 to about 5. It has a pH of .6.

[0180] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL epcolitamab, (ii) about 5.3 to about 6.5 mg / mL epcolitamab, (iii) about 140 to about 250 mM sorbitol, and (iv ) a surfactant. In some embodiments, the surfactant is about 0.0 In some embodiments, the buffer has a p value of about 5.3 to about 5.6. Has H.

[0181] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL epcolitamab, (ii) about 5.3 to about 6.5 mg / mL epcolitamab, (iii) about 140 to about 250 mM sorbitol, and (iv ) Polysorbate 80. In some embodiments, the surfactant is In some embodiments, the buffer is present at a concentration of about 5.3 to about 0.005% to about 0.4% w / v. It has a pH of about 5.6.

[0182] In another aspect, the present invention provides a method for the preparation of a medicament for the treatment of a disease comprising administering to said patient (i) about 5 to about 60 mg / mL of epcolitamab, (ii) about 5.3 to about 5.6 mg / mL of epcolitamab, (iii) about 140 to about 250 mM sorbitol, and and (iv) polysorbate 80. In some embodiments, the surfactant The agent is included at a concentration of about 0.005% to 0.4% w / v.

[0183] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5, (iii) about 140 to about 250 mM of a polyol; and (iv) a surfactant. In some embodiments, the surfactant is present in an amount of about 0.005% to 0. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. do.

[0184] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, (iii) about 140 to about 250 mM of a polyol; and (iv) an interface. In some embodiments, the surfactant is about 0.005%. Found at a concentration of ~0.4% w / v.

[0185] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, (iii) about 140 to about 250 mM of a polyol; and (iv) a poly Sorbate 80. In some embodiments, the surfactant comprises about It is found at a concentration of 0.005% to 0.4% w / v.

[0186] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, (iii) about 140 to about 250 mM sorbitol; and (iv) about 20 to about 40 mM acetate buffer. In some embodiments, the surfactant is about 0.00 It is found in concentrations of 5% to 0.4% w / v.

[0187] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5, (iii) about 140 to about 250 mM of a polyol; and (iv) a polysol. In some embodiments, the surfactant is about 0.001%. In some embodiments, the buffer is present at a concentration of about 5% to about 0.4% w / v. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. has.

[0188] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5, (iii) about 140 to about 250 mM sorbitol; and (iv) a surfactant. In some embodiments, the surfactant is present in an amount of about 0.005% to about 0.5%. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. do.

[0189] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.0 to about 6.5, (iii) about 140 to about 250 mM sorbitol; and (iv) a surfactant. In some embodiments, the surfactant is present in an amount of about 0.005% to about 0.5%. In some embodiments, the buffer has a pH of about 5.3 to about 5.6. do.

[0190] In another aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i) (1) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL-CDR2 comprising GTN, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 5. (1) a human CD3 binding domain comprising a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8; VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10 VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and DAS. and VL-CDR3 comprising an amino acid sequence comprising SEQ ID NO: 12. (ii) a bispecific antibody having a pH of about 5.3 to about 5.6, (iii) about 140 to about 250 mM sorbitol, and (iv) about 20 to about 40 mM acetate buffer. Resorbate 80. In some embodiments, the surfactant is It is found at a concentration of approximately 0.005% to 0.4% w / v.

[0191] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM of a polyol; and (iv) In some embodiments, the surfactant is about 0. In some embodiments, the buffer is present at a concentration of about 5.3 to about 5.6 w / v. Has a pH.

[0192] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM polyol, and iv) a surfactant. In some embodiments, the surfactant is It is found at a concentration of approximately 0.005% to 0.4% w / v.

[0193] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM polyol, and iv) Polysorbate 80. In some embodiments, the surfactant The agent is included at a concentration of about 0.005% to 0.4% w / v.

[0194] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM sorbitol, and In some embodiments, the surfactant is , at a concentration of approximately 0.005% to 0.4% w / v.

[0195] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM of a polyol; and (iv) Polysorbate 80. In some embodiments, the surfactant is In some embodiments, the buffer is present at a concentration of about 5.3 to about 0.005% to about 0.4% w / v. It has a pH of about 5.6.

[0196] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM sorbitol, and (iv ) a surfactant. In some embodiments, the surfactant is about 0. In some embodiments, the buffer is present at a concentration of about 5.3 to about 5.6% w / v. The pH is

[0197] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.0 to about 6.5; (iii) about 140 to about 250 mM sorbitol, and (iv ) Polysorbate 80. In some embodiments, the surfactant In some embodiments, the buffer is present at a concentration of about 5.3 to about 0.005% to about 0.4% w / v. ~Has a pH of about 5.6.

[0198] In another aspect, the present invention provides a method for the preparation of a medicament for the detection of a medicament for ... (1) a human CD3 binding domain comprising a VL having the amino acid sequence of SEQ ID NO: 7; and A human C13 comprising a VH having an amino acid sequence of SEQ ID NO: 13, and a VL having an amino acid sequence of SEQ ID NO: 14. (ii) a bispecific antibody comprising a D20 binding domain at a pH of about 5.3 to about 5.6; (iii) about 140 to about 250 mM sorbitol, and and (iv) polysorbate 80. In some embodiments, the surfactant is The inhibitor is present at a concentration of about 0.005% to 0.4% w / v.

[0199] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to 40 mM buffer having a pH of about 5.3 to about 6.5; In some embodiments, the present invention relates to a pharmaceutical composition comprising: The surfactant is present at a concentration of about 0.005% to 0.4% w / v. In some embodiments, the buffer is , and has a pH of about 5.3 to about 5.6.

[0200] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 5.3 to about 5.6; In some embodiments, the pharmaceutical composition comprises: The surfactant is present at a concentration of about 0.005% to 0.4% w / v.

[0201] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 5.3 to about 5.6; In some embodiments, the pharmaceutical composition comprises: Typically, the surfactant is present at a concentration of about 0.005% to 0.4% w / v.

[0202] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 5.3 to about 5.6; In some embodiments, the present invention relates to a pharmaceutical composition comprising: The surfactant is contained at a concentration of about 0.005% to 0.4% w / v.

[0203] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to 40 mM buffer having a pH of about 5.3 to about 6.5; and (iv) polysorbate 80. In some embodiments, the surfactant is present at a concentration of about 0.005% to 0.4% w / v. The pH of the solution is about 5.3 to about 5.6.

[0204] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 5.3 to about 6.5; In some embodiments, the pharmaceutical composition comprises: The surfactant is present at a concentration of about 0.005% to 0.4% w / v. In some embodiments, the buffer has a pH of about 5.3 to about 5.6.

[0205] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM buffer having a pH of about 5.3 to about 6.5; In some embodiments, the pharmaceutical composition comprises: (i) glycerol; and (ii) polysorbate 80. In some embodiments, the surfactant is present at a concentration of about 0.005% to 0.4% w / v. The fur has a pH of about 5.3 to about 5.6.

[0206] In another aspect, the present invention provides a method for the preparation of a medicament comprising the steps of: (i) a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively; and A CD3 binding arm having a light chain and a heavy chain as set forth in SEQ ID NOs. 27 and 25, respectively. and a CD20-binding arm having a light chain; (iii) about 20 to about 40 mM acetate buffer having a pH of about 5.3 to about 5.6; (iv) polysorbate 80. In embodiments, the surfactant is included at a concentration of about 0.005% to 0.4% w / v.

[0207] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 6.5 mg / mL epcolitamab; (iii) about 140 to about 250 mM of a polyol; and (iv ) a surfactant. In some embodiments, the surfactant is about 0.0 In some embodiments, the buffer has a p value of about 5.3 to about 5.6. Has H.

[0208] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 5.6 (iii) about 140 to about 250 mM polyol; and and (iv) a surfactant. In some embodiments, the surfactant is It is found at a concentration of approximately 0.005% to 0.4% w / v.

[0209] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 5.6 (iii) about 140 to about 250 mM polyol; and and (iv) polysorbate 80. In some embodiments, the surfactant The agent is included at a concentration of about 0.005% to 0.4% w / v.

[0210] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 5.6 (iii) about 140 to about 250 mM sorbitol, and and (iv) a surfactant. In some embodiments, the surfactant is , at a concentration of approximately 0.005% to 0.4% w / v.

[0211] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 6.5 mg / mL epcolitamab; (iii) about 140 to about 250 mM of a polyol; and (iv ) Polysorbate 80. In some embodiments, the surfactant is In some embodiments, the buffer is present at a concentration of about 5.3 to about 0.005% to about 0.4% w / v. It has a pH of about 5.6.

[0212] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 6.5 mg / mL epcolitamab; (iii) about 140 to about 250 mM sorbitol, and iv) A pharmaceutical composition comprising a surfactant. In some embodiments, the surfactant is about 0. In some embodiments, the buffer is present at a concentration of about 5.3 to about 5.6% w / v. The pH is

[0213] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 6.5 mg / mL epcolitamab; (iii) about 140 to about 250 mM sorbitol, and iv) A pharmaceutical composition comprising polysorbate 80. In some embodiments, a surfactant In some embodiments, the buffer is present at a concentration of about 5.3 to about 0.005% to about 0.4% w / v. ~Has a pH of about 5.6.

[0214] In another aspect, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) about 0.5 to about 120 mg / mL epcolitamab; (ii) about 5.3 to about 5.6 (iii) about 140 to about 250 mM sorbitol, and and (iv) polysorbate 80. In some embodiments, the surfactant The inhibitor is present at a concentration of about 0.005% to 0.4% w / v.

[0215] The composition preferably has a shelf life of at least 6 months at a storage temperature of 2-8°C, e.g. 5°C. For example, for pharmaceutical use over a period of at least 9 months, or at least 12 months. In one embodiment, the pharmaceutical composition has a storage temperature of 2 to 8°C, e.g., 5°C, of ​​1 The pharmaceutical composition is stable for pharmaceutical use for over 2 months. In one embodiment, the pharmaceutical composition is stable for pharmaceutical use for over 2 to 8 months. 1. Stable for pharmaceutical use for up to 18 months at a storage temperature of 5° C., e.g. In one embodiment, the pharmaceutical composition has a shelf life of 24 months at a storage temperature of 2-8°C, e.g., 5°C. In one embodiment, the pharmaceutical composition is stable for pharmaceutical use over a period of time ranging from 2 to 8° C., e.g. For example, it is stable for pharmaceutical use for up to 30 months at a storage temperature of 5°C. In one embodiment, the pharmaceutical composition has a shelf life of 36 months at a storage temperature of 2-8°C, e.g., 5°C. It is stable for pharmaceutical use.

[0216] In another embodiment, the pharmaceutical composition does not include hyaluronidase.

[0217] In a preferred embodiment, the pharmaceutical composition does not contain significant amounts of sodium chloride. It is understood that the formulation should not contain large amounts of sodium chloride added. For example, the pH of a buffer, such as the preferred acetate buffer, can be adjusted, optionally with NaOH and / or Alternatively, it may be adjusted using HCl solution, where addition of NaOH solution is generally sufficient to set the pH. The pharmaceutical composition may preferably contain less than 20 mM sodium chloride. , a bispecific antibody as described herein, acetate buffer, sorbitol, a surfactant, and pH, the pharmaceutical composition further comprises a small amount, i.e., less than 20 mM, of sodium chloride. and wherein more preferably, no sodium chloride is added to the pharmaceutical composition.

[0218] In another embodiment, the pharmaceutical composition may further comprise arginine. Bispecific antibodies, acetate buffer, sorbitol, detergent, and pH were as described in The pharmaceutical composition may further comprise low amounts of arginine, i.e., less than 60 mM.

[0219] Thus, arginine can be included in the composition, for example, up to about 60 mM / ml. For example, arginine may be included at a concentration of about 20 mM.

[0220] In a preferred embodiment, the pharmaceutical composition is a subcutaneous composition or is used for subcutaneous administration. However, the pharmaceutical composition of the present invention may also be administered intravenously. Thus, in one embodiment, the pharmaceutical composition is an intravenous composition, or the pharmaceutical composition is administered intravenously. The advantage of the present invention is that the pharmaceutical composition can be administered subcutaneously and intravenously. The present invention is suitable for both intravenous and intravenous administration.

[0221] In one aspect of the invention, the pharmaceutical composition is for use in the treatment of cancer. In an embodiment, the pharmaceutical composition is for use in the treatment of a B cell malignancy.

[0222] In another embodiment, the pharmaceutical compositions of the present invention are useful for preventing and treating T cell-mediated immune responses, inflammation, and microcirculation. It may be used to induce border remodeling.

[0223] In certain embodiments, the pharmaceutical compositions are useful for treating and preventing various CD20-related diseases in vivo. Examples of CD20-related diseases include, among others, But especially B-cell lymphomas, such as non-Hodgkin's lymphoma (NHL), B-cell leukemia, and and immune disorders, such as autoimmune disorders, including those listed below.

[0224] In one embodiment, the pharmaceutical composition of the invention is for use in the treatment of NHL or B-cell leukemia. That is why.

[0225] In one embodiment, the pharmaceutical compositions of the invention are used to treat CD20 antibody resistant NHL or B cell leukemia, e.g. For example, rituximab- or ofatumumab-resistant NHL or B-cell leukemia, e.g., Rituximab-resistant It is used to treat citabine-resistant non-aggressive B-cell lymphoma.

[0226] In one embodiment, the pharmaceutical compositions of the present invention are used to treat acute lymphoblastic leukemia (ALL), such as relapsed or is used to treat refractory ALL.

[0227] In one embodiment, the pharmaceutical compositions of the invention are used to treat CLL, e.g., relapsed or refractory CLL. It is used.

[0228] In one embodiment, the pharmaceutical composition of the present invention is used to treat FL, such as relapsed or refractory FL. It is something that will be done.

[0229] The present invention further provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof The method includes administering the pharmaceutical composition for a sufficient period of time to treat the cancer.

[0230] The present invention further provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof administering the pharmaceutical composition subcutaneously for a period of time sufficient to treat the cancer. .

[0231] The present invention further provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof administering the pharmaceutical composition intravenously for a period of time sufficient to treat the cancer. In one aspect of the invention, the cancer treated by this method is a B-cell malignancy, such as NHL, CLL, ALL, or FL. Tumors, or CD20 antibody-resistant NHL, or B-cell leukemia, e.g., rituximab or ovo Fatumumab-resistant NHL or B-cell leukemia, such as aggressive rituximab-resistant It is a non-small cell B-cell lymphoma.

[0232] In one embodiment, the pharmaceutical composition of the present invention is in unit dosage form. The unit dose is a liquid unit dose.

[0233] In one aspect of the invention, the unit dosage form comprises: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of about 5 μg to about 50 mg, b. acetate buffer, sorbitol, and a surfactant, having a pH of about 5.5; Includes.

[0234] In one embodiment of the unit dosage form, the acetate buffer and sorbitol are mixed in a concentration ratio of 1:5 to 1:10, for example. For example, in concentration ratios of 1:6, 1:7, 1:8, or 1:9.

[0235] In a further embodiment, the osmolality of the unit dosage form is from about 210 to about 250, e.g., 220, 230, 240, 260, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 42 0, or 250 mOsm / kg. In a further embodiment, the osmolality of the unit dosage form is about 200 to The osmolality is about 600, more preferably about 220 to about 380. Therefore, the osmolality is 220 to 600, or 220 to 380 mM, for example, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 29 0, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, or about 380 mOsm / kg The following may be selected:

[0236] In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of about 5 μg to about 50 mg, b. acetate buffer at a concentration of about 30 mM; c. sorbitol at a concentration of about 150 mM and a pH of about 5.5; and d. Approximately 0.04% w / v Polysorbate 80 The present invention relates to a unit dosage form comprising:

[0237] The compositions in unit dosage form can be the same as the pharmaceutical compositions defined herein.

[0238] In one embodiment of the above-mentioned unit dosage form, the amount of bispecific antibody is from about 40 μg to about 40 mg, for example 5 0 μg~40 mg.

[0239] In one embodiment of the unit dosage form, the amount of bispecific antibody is from about 40 μg to about 30 mg. In another embodiment, the amount of bispecific antibody is about 150 μg. The amount of bispecific antibody is about 200 μg. In another embodiment of the unit dosage form, In another embodiment of the unit dosage form, the amount of bispecific antibody is about 300 μg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 350 μg. In another embodiment, the amount of bispecific antibody is about 400 μg. The amount of bispecific antibody is about 450 μg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 600 μg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 700 μg. In one embodiment, the amount of bispecific antibody is about 800 μg. In another embodiment of the unit dosage form, the bispecific antibody is The amount of specific antibody is about 900 μg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 2 mg. In another embodiment of the dosage form, the amount of bispecific antibody is about 3 mg. The amount of bispecific antibody is about 4 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 6 mg. In another embodiment of the dosage form, the amount of bispecific antibody is about 7 mg. The amount of bispecific antibody is about 8 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 10 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 11 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 12 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 14 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 15 mg. In an embodiment, the amount of bispecific antibody is about 16 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 18 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 19 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 20 mg. The amount of bispecific antibody is about 21 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 23 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 24 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 25 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 27 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 28 mg. In an embodiment, the amount of bispecific antibody is about 29 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 30 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 32 mg. In another embodiment, the amount of bispecific antibody is about 33 mg. The amount of specific antibody is about 34 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 36 mg. In another embodiment of the dosage form, the amount of bispecific antibody is about 37 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 38 mg. The amount is about 39 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 40 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 41 mg. In one embodiment, the amount of bispecific antibody is about 42 mg. The amount of antibody is about 43 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 44 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 45 mg. In another embodiment, the amount of bispecific antibody is about 46 mg. The amount of specific antibody is about 47 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is In another embodiment of the unit dosage form, the amount of bispecific antibody is about 49 mg. In another embodiment of the dosage form, the amount of bispecific antibody is about 50 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 51 mg. The amount is about 52 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 53 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 54 mg. In one embodiment, the amount of bispecific antibody is about 55 mg. The amount of antibody is about 56 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 57 mg. In another embodiment of the unit dosage form, the amount of bispecific antibody is about 58 mg. In another embodiment, the amount of bispecific antibody is about 59 mg. The amount of specific antibody is approximately 60 mg.

[0240] In one embodiment of the unit dosage form of the invention, the first binding region of the bispecific antibody that binds to human CD3 is a SE Q ID: Second binding of a bispecific antibody comprising the VH and VL sequences of 6 and 7 and binding to human CD20 In a preferred embodiment of the unit dosage form of the present invention, the region comprises the VH and VL sequences of SEQ ID: 13 and 14. In a preferred embodiment, the bispecific antibody is DuoBody-CD3xCD20 as described above. In the dosage form, the bispecific antibody comprises a heavy chain as set forth in SEQ ID NOs. 26 and 24, respectively. and a CD3 binding arm having a light chain, as set forth in SEQ ID NOs. 27 and 25, respectively. In a further preferred embodiment, the monoclonal antibody of the present invention comprises a CD20 binding arm having a heavy chain and a light chain that binds the CD20. In the dosage form, the bispecific antibody is epcolitamab, or a biosimilar thereof. .

[0241] In another embodiment, the total volume of the unit dosage form of the present invention is from about 0.3 mL to about 3 mL, for example, from 0.3 mL to 3 In another embodiment, the total volume of a unit dosage form of the present invention is 0.5 mL. The total volume of the unit dosage form of the present invention is 0.8 mL. In another embodiment, the total volume of the unit dosage form of the present invention is In another embodiment, the total volume of the unit dosage form of the present invention is 1.2 mL. In another embodiment, the total volume of the unit dosage form of the present invention is 1.5 mL. The total volume is 1.7 mL. In another embodiment, the total volume of the unit dosage form of the present invention is 2 mL. In one embodiment, the total volume of the unit dosage form of the present invention is 2.5 mL. Such unit dosage forms are suitable for subcutaneous administration. In one embodiment, the preferred volume for sc injection from the unit dosage form is about 1 mL. In another preferred embodiment, the volume for sc injection from the unit dosage form is about 0.8 mL.

[0242] In embodiments where the unit dosage form is for IV administration, the volume is typically larger, such as 10 mL to 500 mL. In one embodiment, the volume of the unit dosage form is 20 mL. In one embodiment, the volume of the unit dosage form is In one embodiment, the volume of the unit dosage form is 80 mL. In one embodiment, the volume of the unit dosage form is 50 mL. The volume is 100 mL. In one embodiment, the volume of the unit dosage form is 150 mL. The volume of the unit dosage form is 200 mL. In one embodiment, the volume of the unit dosage form is 250 mL. In one embodiment, the volume of the unit dosage form is 300 mL. In one embodiment, the volume of the unit dosage form is 350 mL. In one embodiment, the volume of the unit dosage form is 400 mL. In one embodiment, the volume of the unit dosage form is 450 mL. In one embodiment, the volume of the unit dosage form is 500 mL.

[0243] The unit dosage form may be prepared by dissolving the pharmaceutical composition of the present invention in a solution, for example, made from acetate buffer and sorbitol. and having a pH of 5.5. Commercially available pharma- ceutically acceptable diluents are preferred. Only the concentration of the bispecific antibody is affected by dilution. The diluent contains the same surfactants, buffers, and solvents as the pharmaceutical composition so that the diluent is affected by the Alternatively, it may be preferable to have a concentration of 100% by weight ... As shown, even with the use of a diluent such as a 0.9% w / v solution of sodium chloride in water suitable for injection, good.

[0244] In another aspect, the invention provides a container or receptacle containing a unit dosage form as described herein. Further providing

[0245] The present invention further provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of and administering a unit dosage form as described herein for a period of time sufficient to treat the cancer. In one aspect, the present invention relates to a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of In one aspect, the present invention provides a method for treating cancer in a subject, comprising administering the unit dosage form subcutaneously to the subject. For a method of treating, the method comprises the steps of intravenously administering to a subject in need thereof a unit dosage form. Includes.

[0246] In another aspect, the present invention relates to the above-mentioned unit dosage form for use in the treatment of cancer. In another embodiment, the unit dosage form is for subcutaneous administration. In another embodiment, the unit dosage form is for intravenous administration.

[0247] The present invention relates to a. Pharmaceutical Compositions Described Herein b. Diluents, including acetate, sorbitol, and polysorbate 80 c. Receptacles for unit dosage forms d. Instructions for dilution and / or use It also relates to kits of parts, including:

[0248] It is preferred that the concentration ratio of acetate to sorbitol be equal in the diluent and in the pharmaceutical composition. I wish.

[0249] In one aspect of the invention, the kit-of-parts comprises: i. i. Bispecific antibodies such as DuoBody-CD3xCD20 at 5-60 mg / mL ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. 0.04% w / v Polysorbate 80 Including, v. pH is 5.5; Pharmaceutical compositions, ii. a. 30 mM acetate buffer b. 150 mM sorbitol c. 0.04% w / v Polysorbate 80 A diluent comprising iii. a receptacle for the unit dosage form; and iv. Instructions for dilution and / or use Includes.

[0250] In one aspect of the invention, the kit-of-parts comprises: i. i. a bispecific antibody, such as epcolitamab or a biosimilar thereof, at 5-60 mg / mL; ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. 0.04% w / v Polysorbate 80 Including, v. pH is 5.5; Pharmaceutical compositions, ii. a receptacle for the unit dosage form; and iii. Instructions for dilution and / or use Includes.

[0251] In one aspect of the invention, the kit-of-parts comprises: i. i. a bispecific antibody, such as epcolitamab or a biosimilar thereof, at 5-60 mg / mL; ii. 30 mM acetate buffer iii. 250 mM sorbitol iv. 0.04% w / v Polysorbate 80 Including, v. pH is 5.5; Pharmaceutical compositions, ii. a diluent, iii. a receptacle for the unit dosage form; and iv. Instructions for dilution and / or use Includes.

[0252] In one aspect, the present invention further relates to a method for preparing the pharmaceutical compositions described herein. The method further comprises: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Bispecific antibodies at 5-120 mg / mL b. 3.53 mg / mL sodium acetate trihydrate c. 0.24 mg / mL acetic acid or 0.32 mg / mL 75% acetic acid solution d. 27.3 mg / mL sorbitol e. 0.4 mg Polysorbate 80 in water for injection; and adjusting the pH to 5.5 by adding sodium hydroxide. Includes.

[0253] In one embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 5 mg / mL. In one embodiment of the method for preparing the pharmaceutical composition, a. is 10 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 12 mg / mL. In another embodiment of the method, a. is 15 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 20 mg / mL. In one embodiment, a is 24 mg / mL. In another embodiment of the method for preparing a pharmaceutical composition of the present invention, In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 40 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 48 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a. is 50 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the invention, a. is 60 mg / mL. In another embodiment of the method of preparing the pharmaceutical composition, a. is 120 mg / mL. In another embodiment of the method of preparing the composition, a. is 200 mg / mL.

[0254] The present invention further relates to a method of preparing the unit dosage forms described herein, the method comprising the steps of: a. i. The following CDR sequences: VH-CDR1: SEQ ID NO: 1, VH-CDR2: SEQ ID NO: 2, VH-CDR3: SEQ ID NO: 3, VL-CDR1: SEQ ID NO: 4, VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8, VH-CDR2: SEQ ID NO: 9, VH-CDR3: SEQ ID NO: 10, VL-CDR1: SEQ ID NO: 11, VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, 5-120 mg / mL, e.g., 12 mg or 24 mg, of a bispecific antibody comprising ii. 3.53 mg / mL sodium acetate trihydrate iii. 0.32 mg / mL in 75% acetic acid iv. 27.3 mg / mL sorbitol v. 0.04% w / v Polysorbate 80 in water for injection, and vi. Adjusting the pH to 5.5 by adding sodium hydroxide. preparing a pharmaceutical composition by b. i. 3.53 mg / mL sodium acetate trihydrate ii. 0.32 mg / mL in 75% acetic acid iii. 27.3 mg / mL sorbitol iv. 0.04% w / v Polysorbate 80 in water for injection; and v. Adjust the pH to 5.5 by adding sodium hydroxide; preparing a diluent; c. mixing the pharmaceutical composition with a diluent to the desired bispecific antibody concentration in a unit dosage form. Includes.

[0255] In a further aspect, the present invention relates to a pharmaceutical composition or unit dosage form obtainable by the above-mentioned method. Pertaining to shape.

[0256] Aspects 1. a. 0.5 to 120 mg / mL of a bispecific antibody that binds to human CD3 and human CD20; b. 20-40 mM acetate; c. 140-160 mM sorbitol; d. Surfactants 1. A pharmaceutical composition comprising, or consisting essentially of, wherein the pH of the composition is 5 to 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Includes.

[0257] 2. The first binding region of the bispecific antibody that binds to CD3 has the VH and VL sequences of SEQ ID: 6 and 7. at least 90% sequence identity with the VH and VL sequences of SEQ ID: 6 and 7, for example VH and VL sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. 2. The pharmaceutical composition of embodiment 1, comprising:

[0258] 3. The second binding region of the bispecific antibody that binds to CD20 comprises the VH and VL of SEQ ID: 13 and 14. At least 90% sequence identity with the VH and VL sequences of SEQ ID: 13 and 14, for example. VH and VL sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. 3. The pharmaceutical composition of embodiment 1 or 2, comprising the sequence

[0259] 4. The pharmaceutical composition of any one of aspects 1 to 3, wherein the bispecific antibody is an IgG1 antibody.

[0260] 5. The bispecific antibody comprises a lambda light chain constant region, such as the light chain constant region of SEQ ID Nos. 22 and 23. and a kappa light chain constant region. and a second light chain.

[0261] 6. The bispecific antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, the Fc region being a wild-type antibody. Modified to have reduced effector function compared to bispecific antibodies containing IgG1 Fc regions. 20. The pharmaceutical composition of any one of the preceding aspects, wherein

[0262] 7. The bispecific antibody has a higher affinity for C1q than a bispecific antibody having a wild-type IgG1 Fc region. Binding to the antibody is at least 70%, at least 80%, at least 90%, at least 95%, Both contain Fc regions modified to reduce C1q binding by 97% or 100% by ELISA. 3. The pharmaceutical composition of any one of the preceding aspects, wherein the pharmaceutical composition is

[0263] 8. The bispecific antibodies each comprise at least a hinge region, a CH2 region, and a CH3 region. The first and second heavy chains include T366, L368, K370 in human IgG1 heavy chain, , D399, F405, Y407, and K409 At least one of the amino acids in the second heavy chain is replaced with T366, L368 in the human IgG1 heavy chain. , K370, D399, F405, Y407, and K409 At least one of the amino acids is substituted, and the first heavy chain and the second heavy chain are substituted at the same position. 20. The pharmaceutical composition of any one of the preceding aspects, wherein

[0264] 9. (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain; and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain. or (ii) an amino acid at a position corresponding to K409 in a human IgG1 heavy chain is and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is R in the second heavy chain. The pharmaceutical composition of any one of the preceding embodiments, wherein

[0265] 10. Position L234 in the human IgG1 heavy chain, both in the first and second heavy chains of the bispecific antibody and the positions corresponding to L235 are F and E, respectively. chemical composition.

[0266] 11. Position L234 in the human IgG1 heavy chain, both in the first and second heavy chains of the bispecific antibody Any of the above embodiments, wherein the positions corresponding to L1, L235, and D265 are F, E, and A, respectively. Any one of the pharmaceutical compositions.

[0267] 12. The first and second constant heavy chains of the bispecific antibody are both human IgG1 heavy chains. The positions corresponding to positions L234, L235, and D265 are F, E, and A, respectively, and the first The position of the constant heavy chain corresponding to F405 in the human IgG1 heavy chain is L, and the position of the second constant heavy chain corresponding to F405 in the human IgG1 heavy chain is L. IgG1 heavy chain, wherein the position corresponding to K409 in the composition.

[0268] 13. The first and second constant heavy chains have an amino acid sequence that is at least 90% identical to that of SEQ ID NO:16. 4. The pharmaceutical composition of any one of the preceding aspects, comprising an amino acid sequence having the following structure:

[0269] 14. The first and second constant heavy chains comprise the amino acid sequences of SEQ ID Nos. 19 and 20, respectively. , The pharmaceutical composition of any one of the above aspects.

[0270] 15. The bispecific antibody comprises a heavy chain and a light chain as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. 14. The pharmaceutical composition of any one of embodiments 1-13, comprising a CD20 binding arm having a CD20-binding domain and a light chain.

[0271] 16. The method of any one of claims 1 to 10, wherein the bispecific antibody is epcolitamab or a biosimilar thereof. 3. Any one of the pharmaceutical compositions of claim 3.

[0272] 17. a. is 0.5-120 mg / mL, for example 1.0-60 mg / mL, or for example 5-30 mg / mL, For example, 5 mg / mL, or 6 mg / mL, or 7 mg / ml, or 8 mg / ml, or 9 mg / ml, or is 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or is 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or is 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or is 25 mg / ml, or 26 mg / ml, or 27 mg / ml, or 28 mg / ml, or 29 mg / ml, or The pharmaceutical composition of any one of the preceding embodiments, for example 30 mg / ml.

[0273] 18. b. is 25 to 35 mM, for example, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, Any one of the above embodiments, wherein the concentration is 32 mM, 33 mM, 34 mM, or 35 mM, preferably 30 mM. A pharmaceutical composition comprising:

[0274] 19. c. is 145-155 mM, for example 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM , 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, preferably 150 mM. Any one of the pharmaceutical compositions.

[0275] 20. Any of the above embodiments, wherein the pH is 5.3 to 5.6, such as 5.4 to 5.6, for example about 5.5. or a pharmaceutical composition.

[0276] 21. The surfactant is glycerol monooleate, benzethonium chloride, sodium dodecyl sulfate, Cusart, phospholipids, polyethylene alkyl ether, sodium lauryl sulfate, and Tricaprylin, benzalkonium chloride, citrimide, Cetylpyridinium chloride, and phospholipids, alpha tocopherol, glycerol monohydrate Oleate, myristyl alcohol, phospholipids, poloxamer, polyoxyethylene aldehyde Kyl ether, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan ( sorbintan fatty acid esters, polyoxyethylene sterarates, polyoxyethylene sterarates Xylhydroxystearate, polyoxylglyceride, polysorbate, propylene Glycol dilaurate, propylene glycol monolaurate, sorbitan esters A group that includes clos palmitate, sucrose stearate, tricaprylin, and TPGS 2. The pharmaceutical composition of any one of the preceding aspects, selected from:

[0277] 22. The pharmaceutical composition of any one of the above embodiments, wherein the surfactant is a polysorbate.

[0278] 23. The above, wherein the surfactant is polysorbate 20 or 80, for example polysorbate 80. The pharmaceutical composition of any one of the preceding embodiments.

[0279] 24. The surfactant is about 0.005% to 0.4% w / v, for example about 0.01 to 0.1% w / v, for example about 0.01 ~0.09% w / v, for example about 0.01-0.06% w / v, for example about 0.01-0.05% w / v, for example 0.02% w / v, or 0.03% w / v, or 0.04% w / v, or 0.05% w / v, preferably 0.04% w / v. 4. The pharmaceutical composition of any one of the preceding aspects, comprising

[0280] 25. The composition has a pH of 5.4 to 5.6, for example about 5.5, and a. 0.5-120 mg / mL of bispecific antibody b. 20-40 mM acetate c. 140-160 mM sorbitol d. 0.005%-0.4% w / v of a surfactant, preferably a polysorbate, such as polysorbate 2 0 or polysorbate 80, e.g. polysorbate 80 23. The pharmaceutical composition of any one of the preceding aspects, comprising, or consisting essentially of:

[0281] 26. The composition has a pH of 5.4 to 5.6, for example about 5.5, and a. 5-60 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 23. The pharmaceutical composition of any one of the preceding aspects, comprising, or consisting essentially of:

[0282] 27. The pharmaceutical composition of embodiment 25 or 26, wherein a. is 10 to 50 mg / mL.

[0283] 28. The drug of embodiment 25 or 26, wherein a. is 12 to 24 mg / mL, for example 12 mg / mL or 24 mg / mL. chemical composition.

[0284] 29. The composition has a pH of about 5.5, and a. 5-60 mg / mL of bispecific antibody b. 30 mM acetate c. 150 mM sorbitol d. 0.04% w / v surfactant, preferably a polysorbate, such as polysorbate 20 or Polysorbate 80, e.g. Polysorbate 80 23. The pharmaceutical composition of any one of the preceding aspects, comprising, or consisting essentially of:

[0285] 30. The pharmaceutical composition of embodiment 29, wherein a. is 10 to 50 mg / mL.

[0286] 31. The method of embodiment 29 or 30, wherein a. is 12 to 24 mg / mL, for example 12 mg / mL, or 24 mg / mL. Pharmaceutical compositions.

[0287] 32. The pharmaceutical composition of any one of the preceding aspects, wherein the composition does not comprise hyaluronidase. .

[0288] 33. The pharmaceutical composition of any one of the above aspects, wherein the composition is a subcutaneous composition.

[0289] 34. The pharmaceutical composition of any one of the above aspects, wherein the composition is an intravenous composition.

[0290] 35. The pharmaceutical composition of any one of the above embodiments, wherein the composition is used to treat cancer.

[0291] 36. The pharmaceutical composition of any one of the above aspects, wherein the composition is used for subcutaneous administration.

[0292] 37. The pharmaceutical composition of any one of aspects 1 to 32 above, wherein the composition is used for intravenous administration.

[0293] 38. The pharmaceutical composition of any one of aspects 1 to 37 above, in a unit dosage form.

[0294] 39. The composition has a shelf life of at least 6 months, even at a storage temperature of 2-8°C, e.g. 5°C. stable for pharmaceutical use for at least 9 months, or at least 12 months. The pharmaceutical composition of any one of the preceding aspects.

[0295] 40. Use of a pharmaceutical composition according to any one of aspects 1 to 33 for subcutaneous administration.

[0296] 41. Use of a pharmaceutical composition according to any one of aspects 1 to 32 for intravenous administration.

[0297] 42. The use of any one of aspects 40 or 41, for the treatment of cancer.

[0298] 43. Treating cancer by administering to a subject in need thereof a pharmaceutical composition according to any one of aspects 1-39. 46. ​​A method of treating cancer in a subject, comprising administering for a period of time sufficient to

[0299] 44. The method of embodiment 43, wherein the composition is administered subcutaneously or intravenously.

[0300] 45. The method of any one of aspects 43 or 44, wherein the cancer is a B-cell malignancy.

[0301] 46. ​​A unit dosage form comprising: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of 5 μg to 50 mg, b. Acetate buffer and sorbitol in a ratio of 1:5 to 1:10, where the osmolality of the unit dosage form is Acetate buffer and sorbitol, with a concentration of 210-250 and a pH of 5.4-5.6, c. Surfactants A unit dosage form comprising, or consisting essentially of.

[0302] 47. a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of 5 μg to 50 mg, b. Acetate at a concentration of about 30 mM at a pH of about 5.5 c. sorbitol at a concentration of about 150 mM, and d. Approximately 0.04% w / v Polysorbate 80 45. The unit dosage form of embodiment 44, comprising, or consisting essentially of:

[0303] 48. The first binding region of a bispecific antibody that binds to human CD3 comprises the VH and VL of SEQ ID: 6 and 7. The second binding region of the bispecific antibody that binds to human CD20 and comprises a VL sequence is represented by SEQ ID: 13 and 1 The unit dosage form of any one of embodiments 46 or 47, comprising four VH and VL sequences.

[0304] 49. The bispecific antibody comprises a first and a second constant region heavy chain of SEQ ID NOs: 19 and 20, respectively. The unit dosage form of embodiment 48, comprising:

[0305] 50. The bispecific antibody comprises a heavy chain and a light chain as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy chain and a CD3 binding arm having the sequences set forth in SEQ ID NOs. 27 and 25, respectively. The unit dosage form of embodiment 48, comprising a CD20 binding arm having a CD20 binding domain and a light chain.

[0306] 51. The method of claim 48, wherein the bispecific antibody is epcolitamab, or a biosimilar thereof. Unit dosage form.

[0307] 52. The unit dosage form of any one of aspects 46 to 51, wherein the amount of the bispecific antibody is 40 μg to 40 mg. .

[0308] 53. The amount of bispecific antibody is 40 μg to 30 mg, e.g., 40 μg, 50 μg, 150 μg, 200 μg, μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 600 μg, 700 μg, 80 0 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg , 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, for example, 30 mg. Any one of 52 unit dosage forms.

[0309] 54. A unit according to any one of aspects 46 to 53, wherein the total volume is between 0.5 mL and 2 mL, for example 1 mL. Dosage form.

[0310] 55. The unit dosage form of embodiment 54, which is for subcutaneous administration.

[0311] 56. Any of aspects 46 to 53, wherein the total volume is 20 mL to 200 mL and the unit dosage form is for IV administration. or one unit dosage form.

[0312] 57. A method for treating cancer comprising administering to a subject in need thereof a unit dosage form according to any one of aspects 46 to 56. 46. ​​A method of treating cancer in a subject, comprising administering to a subject a therapeutically effective amount of a compound comprising the steps of:

[0313] 58. The unit dosage form of any one of aspects 46-56, for use in treating cancer.

[0314] 59. A container comprising a unit dosage form of any one of aspects 46 to 56.

[0315] 60. a. The pharmaceutical composition of any one of embodiments 1 to 32. b. Diluents, including acetate, sorbitol, and polysorbate 80 c. Receptacles for unit dosage forms d. Instructions for dilution and / or use Includes kit of parts.

[0316] 61. a. The pharmaceutical composition of any one of embodiments 1 to 32. b. Receptacles for unit dosage forms c. Instructions for dilution and / or use Includes kit of parts.

[0317] 62. The ratio of concentrations of acetate, sorbitol, and polysorbate 80 in the diluent and pharmaceutical composition The kit-of-parts of embodiment 60, which is identical in structure.

[0318] 63. a. The pharmaceutical composition comprises: i. 5-60 mg / mL of bispecific antibody ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. 0.04% w / v Polysorbate 80 Including, v. pH is 5.5; b. If a diluent is included in the kit of parts: i. 30 mM acetate buffer ii. 150 mM sorbitol iii. 0.04% w / v Polysorbate 80 Including, c. a receptacle for the unit dosage form; and d. Instructions for dilution and / or use , A kit-of-parts according to any one of embodiments 60 to 62.

[0319] 64. a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, Bispecific antibodies at 5-120 mg / mL b. 3.53 mg / mL sodium acetate trihydrate c. 0.24 mg / mL acetic acid d. 27.3 mg / mL sorbitol e. 0.4 mg Polysorbate 80 in water for injection; and adjusting the pH to 5.5 by adding sodium hydroxide. 33. A method for preparing a pharmaceutical composition as defined in any one of embodiments 1 to 32, comprising:

[0320] 65. The method of embodiment 64, wherein a. is 12 mg / mL.

[0321] 66. The method of embodiment 64, wherein a. is 24 mg / mL.

[0322] 67. a. Preparing a pharmaceutical composition according to any one of the methods of embodiments 64 to 66. b. i. 3.53 mg / mL sodium acetate trihydrate ii. 0.32 mg / mL in 75% acetic acid iii. 27.3 mg / mL sorbitol iv. 0.4 mg Polysorbate 80 v. Sodium hydroxide to adjust the pH to 5.5 in water for injection. c. mixing the pharmaceutical composition with a diluent to the desired bispecific antibody concentration in a unit dosage form. A method for preparing a unit dosage form as defined in any one of aspects 46 to 56, comprising:

[0323] 68. A pharmaceutical composition obtainable by the method defined in any one of aspects 64 to 67. object or unit dosage form.

[0324] (Table 1) Sequence TIFF2024156853000002.tif153166TIFF2024156853000003.tif240166TIFF20241568530 00004.tif206166TIFF2024156853000005.tif223166TIFF2024156853000006.tif121166 EXAMPLES

[0325] The pharmaceutical composition of the present invention is prepared by mixing the components listed in Table 2. obtain.

[0326] Table 2. Composition of the DuoBody-CD3xCD20 pharmaceutical composition of the invention. TIFF2024156853000007.tif71167#75% acetic acid solution

[0327] Example 1: Stability of DuoBody-CD3xCD20 in different formulations Abbreviation TIFF2024156853000008.tif127131

[0328] material Unless otherwise stated, DuoBody-CD3xCD20 was formulated at 2 mg / mL.

[0329] method Thermal stability by fluorescence / static light scattering Conformation and colloidal stability were assessed using a UNit instrument (Unchained Labs) by fluorescence / static light diffusion. This was determined in conjunction with the SLS measurement, which uses increasing thermal stress to measure the temperature. Induce protein unfolding and aggregation, thereby affecting conformation and colloidal stability. The unfolded state transitions induced by increased heat stress are Changes in the surrounding environment after protein unfolding can cause Trp (and Tyr) It is detected by a change in the autofluorescence of the residues. When buried tryptophan residues are exposed, maximum emission occurs. The light wavelength shifts to longer wavelengths. The center of mass average (BCM), i.e. the fluorescence emission spectrum is equally divided. The wavelengths emitted by the proteins are plotted, showing the conformational changes of the proteins with temperature. The analysis was performed at the unfolding onset temperature (T onset ) and melting temperature (T m ), which provide the values ​​of Both are generated from the BCM curve. onset is the calculation time when the protein begins to unfold. Provides temperature. T m The value represents the transition from the folded to the unfolded state of the protein. It is the midpoint of the transition.

[0330] UNit measurements also provide SLS measurements to determine the colloidal stability of proteins. The sample was irradiated with laser light, which was scattered by molecules in the solution. The intensity of the static light scattering was This analysis is therefore useful for determining the molecular weight of proteins as they are induced by a temperature gradient. It is highly sensitive to agglutinations of small and large agglutinations. Static light scattering was measured at 473 nm to detect the concentration of protein. The aggregation onset temperature (T agg These data were used to determine These are best analyzed by the large change in intensity of the tandem. The formation of protein aggregates resulted in more light scattering. Between the SLS count data, 10 3 The change in scale is typically indicative of significant protein aggregation. The small change in SLS counts is believed to be due to partial aggregation, and the temperature gradient The lack of change in SLS counts during tilt indicates that protein aggregation is negligible. It is an indicator.

[0331] exterior Appearance was determined by visual assessment.

[0332] pH The pH was measured using a Mettler Toledo SevenMulti pH meter.

[0333] viscosity Viscosity was measured using a Wells-Brookfield Cone / Plate rheometer.

[0334] Osmolality Osmolality was measured using an osmometer.

[0335] Absorbance A 280 Protein concentration by Protein concentrations were determined by UV / Vis spectroscopy using an Agilent UV / Vis spectrophotometer (model 8453). (Determined by measuring absorbance at 280 nm (A280)).

[0336] Size Exclusion Chromatography (SEC) Size exclusion chromatography was performed on Agilent 1100 and 1200 HPLC systems at TOSOH, The separation was performed using a TSK-gel G3000SWxL (7.8 × 300 mm) column (Sigma, cat. no. 08541). did.

[0337] Imaged Capillary Isoelectric Focusing (icIEF) Imaged capillary isoelectric focusing using the iCE 3 Analyzer with PrinCE Autosampler carried out.

[0338] Microchip capillary electrochemical analysis under reduced and non-reduced conditions Electrophoresis - Sodium dodecyl sulfate Microchip capillary electrophoresis was performed using a Labchip GXII instrument according to the manufacturer's instructions. Electrophoresis (both reducing and non-reducing conditions) was performed.

[0339] Dynamic Light Scattering (DLS) Dynamic light scattering analysis was performed using a Wyatt DynaPro Plate Reader. DLS analysis was performed at room temperature. The size and aggregation of proteins were assessed. For DLS analysis, the time autocorrelation of scattered light was The function was determined and the mean of the molecules in solution was calculated based on a monoexponential cumulant fit of the data. The average size is calculated. Values ​​to be reported are polydispersity and hydrodynamic radius. If a protein sample is composed of a single distribution of nuclei, the sample is considered monodisperse, but if the sample is composed of multiple nuclei, the sample is considered monodisperse. If a sample contains a population of particle sizes, the sample is considered polydisperse. The percent particle distribution (%Pd) is a measure of the width of the particle size distribution; the higher the %Pd, the wider the particle distribution. It can therefore be seen that samples with high %Pd typically contain (large) aggregates. The hydrodynamic radius of a non-spherical protein particle is the radius of a sphere that has the same translational diffusion rate as the particle. The diffusion rate depends on the molecular weight and surface structure of the particles, as well as the concentration and type of ions in the formulation. The larger hydrodynamic radius in a monodisperse size distribution indicates larger aggregates in the solution. This can be attributed to the presence of higher oligomers (e.g., tetramers) rather than Cut.

[0340] Soluble Screening To determine the solubility of DuoBody-CD3xCD20, the material was first concentrated to low concentrations using a centrifugal concentrator. The solutions were formulated in the selected buffer at low starting concentrations. The eluate was concentrated sequentially by timed spins to a target concentration of >120 mg / mL. The protein concentration was measured.

[0341] result 1. Baseline Biophysical and Excipient Screening Perform initial biophysical screening and proceed to more detailed screening The buffer / pH / excipient combinations were selected to measure baseline biophysical and and excipient screening is performed using a wide range of buffer / pH / excipient conditions by fluorescence / SLS and DLS. This included screening the thermostability of DuoBody-CD3xCD20 (2 mg / mL) in combination with A list of the buffers used in the initial screen and their pH values ​​is provided in Table 3.

[0342] Table 3 shows the data obtained from the initial buffer screen. The buffer consisted of 30 mM acetate and 30 mM histidine, and the excipients (150 mM NaCl, 150 mM M arginine, 150 mM sorbitol, or 150 mM sucrose) Thermal stability was assessed using fluorescence / SLS measurements and DuoBody-CD3xCD was assayed using DLS. The aggregation of 20 (2 mg / mL) at room temperature was determined. Fluorescence / SLS analysis determined the melting temperature (T m ), Announcement Start of the call (T onset ), and T agg DLS analysis provided information on the polydispersity and We provided information on the molecular weight and hydrodynamic radius.

[0343] In the thermal stability analysis, T onset and T m acetate preparations (53-58°C and 60-62.5°C, respectively) ℃ range) were significantly higher than the corresponding histidine formulations (53-55℃ and 59-61℃ ranges, respectively). ) is slightly higher than the higher T onset Value and T m The value indicates that the protein is more thermally stable. It is an indicator of superiority. onset and T m The difference is small, but It is slightly less stable in the presence of nin, but is stable in sorbitol or sucrose. It can be noted that the T agg The decision was made based on the acetate preparations and In both the histidine and sorbitol formulations, the addition of NaCl or arginine resulted in no significant difference in the amount of sorbitol or sucrose in the sucrose formulation. Compared with formulations with or without excipients, T agg The temperature will be lower (59-60℃). Partial aggregation was observed at 66°C in acetate formulations with sorbitol or sucrose. However, no aggregation was observed in histidine buffer with these excipients.

[0344] DLS at room temperature showed that the molecular aggregation behavior was more pronounced in the presence of sucrose. The negative effects of the large mean radius and multimodal consistency were also shown. Bitol appears to induce a small increase in the mean radius and %Pd.

[0345] Based on data from initial screening, DuoBody-CD3xCD20 is excipient-free , acetate pH 5.5 buffer, histidine pH 6.0 buffer, and histidine pH 6.5 It was concluded that the sorbitol and sucrose were stable and monodisperse in the buffer. The addition of arginine and NaCl slightly increased the thermal stability. Based on the DLS results of the initial screening, Sucrose was excluded as an excipient for further solubility studies. Acetate pH with or without NaCl, 150 mM arginine, or 150 mM sorbitol The formulations selected were Histidine pH 5.5, Histidine pH 6.0, and Histidine pH 6.5.

[0346] Table 3. Initial baseline biophysical profile of DuoBody-CD3xCD20 (2 mg / mL) in the indicated formulations. and excipient screen results. Thermal Scan TIFF2024156853000009.tif149165 DLS TIFF2024156853000010.tif155128TIFF2024156853000011.tif119128

[0347] 2. Soluble Screening The second phase of the baseline biophysical screening study consisted of , pH / buffer combinations selected from the initial baseline biophysical screen The second screening test included a solubility screening of the same buffer. The list can be seen in Table 4.

[0348] Formulate materials in selected buffers to determine solubility in the presence of excipients The mixture was concentrated sequentially using a centrifugal concentrator at timed spin intervals. The concentrations of each formulation at 0 and after the 90 minute spin interval are shown. The acid salt formulation was the fastest concentrated to 150 mg / mL (50 min). The histidine formulation concentrated faster (50 min) but at a lower concentration (120 mg / mL). All formulations could be concentrated to 120 mg / mL, but this concentration was significantly lower than that of the acetate formulation. It took a long time to reach temperature (60-90 minutes).

[0349] Concentrated samples (final concentration 120-150 mg / mL) were analyzed by fluorescence / SLS and DLS (Table 4) and viscosity (Figure 2). was further analyzed.

[0350] Table 4 shows that formulations without excipients and with sorbitol had the highest T m Showing that T of formulations without excipients and with sorbitol agg Values ​​are for formulations with NaCl and Arg This was difficult to elucidate because the transition was not as clear as in the measurements taken at .

[0351] DLS data at RT show that the %Pd The variation in the mean hydrodynamic radius increases with concentration (>15% is considered polydispersity). This may be due to the viscous effect of the shrink material, which interferes with proper ranking of the formulation.

[0352] Figure 2 shows the viscosity (cP) of different formulations with and without sorbitol. The acetate formulation The histidine formulations without sorbitol showed viscosities ranging from 7.9 to 12.1 cP. The viscosity of the formulations was higher than that of the histidine salt formulations (range 28.4–79.9 cP). The viscosity of the glycerin formulation was reduced (in the range of 18-30 cP), whereas sorbitol reduced the viscosity of the acetate formulation. had no effect.

[0353] Table 4. Results for concentrated samples (DuoBody-CD3xCD20 [120-150 mg / mL] in the indicated formulation) Thermal Scan TIFF2024156853000012.tif183128 DLS TIFF2024156853000013.tif113128TIFF2024156853000014.tif149128

[0354] Additionally, acetate with or without sorbitol, pH 5.5, histidine with sorbitol, Male samples of concentrated samples in sorbitol buffer pH 6.0 and histidine with sorbitol pH 6.5 The molarity was measured using an osmometer and the results are shown in Table 5.

[0355] Acetate without sorbitol pH 5.5 osmolality ranged from 70 to 80 mOsm / kg The osmolality of acetate and histidine preparations with sorbitol is 220-230 mOs mOsm / kg, which is in the range of normal plasma osmolality (275–295 mOsm / kg; Rasouli 2016 C lin Biochem 49 (12):936-41).

[0356] Table 5. Osmolality of DuoBody-CD3xCD20 (120-150 mg / mL) in the indicated formulations. TIFF2024156853000015.tif75128

[0357] Based on the above results, 30 mM acetate pH 5.5 with 150 mM sorbitol was chosen as the optimal solution for DuoBody-CD It has been found that the acetate salt with sorbitol is a convenient formulation of 3xCD20. It exhibits comparable thermal stability to histidine formulations, while being most efficient at high concentrations (150 mg / mL). This was because it was the formulation that supported solubility and had the lowest viscosity.

[0358] 3. Real-time and acceleration stability Real-time stability of DuoBody-CD3xCD20 in 30 mM acetate, 150 mM sorbitol, pH 5.5. Qualitative analysis was performed using the assays described (appearance, pH, UV [A 280 ], SEC, icIEF, CE-SDS [reducing conditions and The study was evaluated at different time points ranging from 0 to 12 months using [non-reducing conditions].

[0359] Table 6 shows the results of DuoBody-CD3xCD20 (5 mg / mL) samples stored at 5±3°C for 0, 2, 3, or 6 months. The results of the stability test are shown.

[0360] Table 7 shows the results of DuoBody-CD3xCD20 (5 mg / mL) samples stored at 25°C for 0, 1, 2, 3, or 6 months. The results of the stability test of the material are shown.

[0361] Table 8 shows the results of DuoBody-CD3xCD20 (60 mg / mL) stored at 5±3°C for 0, 2, 3, 6, 9, or 12 months. ) The results of stability tests of the samples are shown.

[0362] Table 9 shows the results of DuoBody-CD3xCD20 (60 mg / mL) stored at 25±3° C. for 0, 1, 2, 3, or 6 months. The results of the stability test of the samples are shown.

[0363] After storage at 5 ± 3 °C and 25 ± 3 °C for 12 and 6 months, respectively, at concentrations of 5 mg / mL and All samples at 5 and 60 mg / mL were stable by all assays. Samples stored at 5 ± 3°C were stable for 6 months. At the 12-month or 13-month time point, none of the test methods showed any significant changes compared to the start of the study. As expected, minor purity profile changes were observed during accelerated stability testing. It was observed at 25±3°C by spectroscopic measurements, icIEF, reduced CE-SDS, and SEC studies.

[0364] Table 6. Example data from a stability study of DuoBody-CD3xCD20 (5 mg / mL) at 5 ± 3°C. TIFF2024156853000016.tif192149

[0365] Table 7. Example data from a stability study of DuoBody-CD3xCD20 (5 mg / mL) at 25 ± 3°C. TIFF2024156853000017.tif201170

[0366] Table 8. Example data from a stability study of DuoBody-CD3xCD20 (60 mg / mL) at 5 ± 3°C. TIFF2024156853000018.tif130167TIFF2024156853000019.tif165167

[0367] Table 9. Example data from a stability study of DuoBody-CD3xCD20 (60 mg / mL) at 25 ± 3°C. TIFF2024156853000020.tif245153

[0368] 4. Conclusion Results from analytical testing of DuoBody-CD3xCD20 in various formulations showed that 30 mM acetate , 150 mM sorbitol, pH 5.5 was the optimal formulation for this molecule. DuoBody-CD3xCD20 (in 30 mM acetate, 150 mM sorbitol, pH 5.5) was used for 5 and 60 min. The drug was shown to be stable at 5 ± 3°C for up to 12 months at 25 ± 3°C. Stability studies included 5 and 60 mg / mL DuoBody-CD3xCD20 (30 mM acetate, 150 mM sorbitol). In the case of ethanol, pH 5.5, only minor and expected changes were observed up to 6 months. Ta.

[0369] Example 2: Stability study of formulations of DuoBody-CD3xCD20 (5 mg / ml) with and without surfactant DuoBody-CD3xCD20 was formulated at 5 mg / mL as described in Example 1. To further stress the formulation, we performed freeze-thaw testing of the formulation. The samples were frozen in a -75°C chamber for at least 2 hours and then transferred to RT (23°C) for 2 hours. This procedure was repeated five times. In a formulation containing DuoBody-CD3xCD20 at 5 mg / ml in 50 mM sorbitol, pH 5.5, Some fine particles were observed. We added 0.04% w / v polysorbate 80 to this formulation. After five freeze-thaw cycles, only a few particles were visible. The samples were compared after 2, 4, 8, and 12 weeks at 2-8°C, 25°C, and 40°C. The results of these tests are shown in Table 10. The results show that the addition of 0.04% w / v polysorbate 80 The results showed that the level of visible particles was reduced, thereby improving the physical stability of the antibody. Similar results were observed for submicroscopic particles analyzed by microflow imaging. These data support the application of the bispecific antibody formulation of the present invention. This supports the addition of surfactants.

[0370] Table 10. Stability testing of DuoBody-CD3xCD20 (5 mg / mL) formulations with and without surfactant. Example of experimental data TIFF2024156853000021.tif183128

[0371] Example 3: With detergent, sorbitol, and histidine buffer or without surfactant Formulation of DuoBody-CD3xCD20 (5 mg / ml) with sorbitol, acetate buffer, and Stability testing A histidine-sorbitol formulation similar to those more commonly used in the industry was tested to confirm the efficacy of the present invention. We compared the results of 8 and 12 weeks of stressed conditions (40°C) with those of the previously preferred formulation. When analyzed by size exclusion chromatography after incubation for 1 h, The authors found that the acetate-based formulation was inferior to the acetate-based formulation herein. See Table 11. This indicates that under these conditions, the percentage of high molecular weight species (% HMW) is This is evident from the greater increase in the histidine formulation compared to the salt formulation.

[0372] Table 11. DuoBody-CD3xCD20 (5 mg / mL) with detergent but in different buffers ) Example of data on stability test of formulation TIFF2024156853000022.tif165145TIFF2024156853000023.tif96145

[0373] Example 4: Cancer treated with DuoBody-CD3xCD20 via intravenous (IV) and subcutaneous (SC) routes of administration Analysis of cytokines in blood of martins A dose-ranging finding (DRF) study of DuoBody-CD3xCD20 in female cynomolgus monkeys and Animals from a GLP toxicity study of DuoBody-CD3xCD20 in male and female cynomolgus monkeys were Blood samples were taken before, 2, 4, 6, 12, and 24 hours after administration. Transfer to a tube containing EDTA and spin at 3000 rpm (approximately 1500 g) for 10 min to obtain plasma. The plasma was transferred to a clean 0.5 mL polypropylene tube and diluted with - They were stored at 80°C until analysis.

[0374] Milliplex MAP NHP Cytokine Magnetic B used in a BioPlex 200 reader (BioRad) The lead panel (Millipore Cat. No. PRCYTOMAG-40K) was used according to the manufacturer's protocol. Analyze samples using IL-1β, IL-2, IL-6, IL-4, IL-8, IL-10, IL-12p40, IL-15, IFN The concentrations of TNF-γ, TNF-α, and MCP-1 were measured.

[0375] FIG. 3 shows the efficacy of a single IV dose (0.1 mg / kg / day) of DuoBody-CD3xCD20 in a pharmaceutical composition of the invention in a GLP toxicity study. The blood of animals receiving either a single SC dose (0.1 or 1 mg / kg) or Mean cytokine levels in the fluids are shown for each group.

[0376] Administration of DuoBody-CD3xCD20 significantly increased the expression of cytokines IL-1β, IL-4, IL-12p40, and IL-15. However, it induced only low levels of IL-1 (<150 pg / mL) of IL-1 (Figure 3A).

[0377] The cytokines IL-2, IL-6, IL-8, IL-10, IFNγ, TNFα, and MCP-1 were expressed using DuoBody-CD3x The induction was more pronounced after IV administration of CD20, reaching a peak 2–12 h after administration (Figure 3B Cytokine levels subsequently returned to baseline. This also indicates that peak levels were higher in the blood of animals receiving SC doses (0.1 and 1 mg / kg) than in the corresponding For IL-8 and IFN-γ, the peak levels after SC administration were lower than those after IV administration. The effect was both decreased and delayed compared to administration.

[0378] Similar findings were seen in the DRF study, although in this (smaller) study, IV There was no difference in IFNγ levels between SC- and SC-treated animals.

[0379] Example 5: 4x Repeat IV Infusions of DuoBody-CD3xCD20 in Cynomolgus Monkeys, with Priming Dose and Assessment of B cell depletion following a single IV dose or a single SC injection of Female cynomolgus monkeys were administered the formulation of the present invention (30 mM acetate, 150 mM sorbitol, pH 5.5). DuoBody-CD3xCD20 was administered as IV infusions (0.01, 0.1, or 1 mg / kg) weekly for 4 doses, as IV prime. A target dose of 1 mg / kg was administered as a single SC injection (0.01, 0.1 Subjects received either 1, 10, or 20 mg / kg of naphthalene per osmotic fluid as outlined below. TIFF2024156853000024.tif128152(The dates in the table reflect the actual test dates.)

[0380] This study is conducted under the Home Office's Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. In accordance with the European Convention on Chemical Substances (Council of Europe), Charles River Laboratories (Transcript of the Convention) The study was carried out at the

[0381] The crab-eating macaque, Macaca fascicularis, native to Mauritius, was bred for research purposes. Bioculture (Mauritius) Limited (Riviere des Angues, Mauritius) The samples were obtained from Noveprim (Mahébourg, Mauritius). The animals were kept in groups. and provided a high-quality environment.

[0382] Sampling A whole blood sample (approximately 0.5 mL) was collected from the femoral vein using a sterile hypodermic needle and sterile syringe. For immunophenotyping by flow cytometry, blood was diluted in sodium heparin. The mixture was transferred to a tube containing 0.1% ethanol and stored at room temperature until analysis within 48 hours.

[0383] The animals are placed under general anesthesia and a dissection is made over the lymph nodes using standard sterile surgical techniques. Biopsies (approximately 20 mg) were taken from superficial lymph nodes by endoscopic radiology. The samples were collected at Reece Memorial Memorial Institute (RPMI) and stored on wet ice until processing within 24 hours. Single cell suspensions were prepared using the Medimachine System (Becton) to mechanically disaggregate tissue. (See full CRL study report for details.) The cells were cultured in 2 mL Dulbecco's The cells were resuspended in phosphate-buffered saline (PBS; Gibco, cat. No. 14190).

[0384] At the time of autopsy, samples from lymph nodes and spleen were placed on a cork board in a uniform orientation and then Wrapped in miniature foil, individually labeled, flash frozen in liquid nitrogen, and set to maintain at -80°C. The tissue was then stored in a refrigerator awaiting immunohistochemical evaluation.

[0385] Flow cytometry Mixture of directly labeled antibodies (see below) in a round-bottom tube (Falcon, cat. no. 352052). The antibody mixture was selected so that CD19+ B cells could also be analyzed.

[0386] For peripheral blood immunophenotyping, 50 μL of anticoagulated whole blood was added to the antibody mixture. The red blood cells (RBCs) were lysed in 1x RBC lysis buffer and incubated for 20 min at RT protected from light. (eBioscience, cat. no. 00-4300-54) for 10 min at RT (or until RBC lysis is complete). The tubes were centrifuged at 300-500 g for 5 min at RT. The supernatant The supernatant was discarded and the cell pellet was resuspended in 0.5 mL PBS (Gibco, cat. No. 10010). Add 50 μL of Flow Count beads (Beckman Coulter, cat. no. 7546053) to each tube. Added.

[0387] For immunophenotyping of lymph node cells, 50 μL of cell suspension was added to the antibody mixture. Incubate on ice for 15 minutes protected from light. After incubation, add 0.5 mL of each tube. mL Dulbecco's PBS was added.

[0388] Dual laser 5 color Beckman Coulter FC500 or BD LSR Fortessa X-20 Flow Surgery Samples were analyzed using a cytometer. CD4-CD8-CD15-CD19+ events were classified as B cells. did.

[0389] The following antibody mixture was used: TIFF2024156853000025.tif33128 1 FITC: fluorescein isothiocyanate; PE: phycoerythin; ECD: electron coupling dye; BV: brilliant violet; V: violet; Cy: cyanine Pigment; APC: Allophycocyanin * The supplier changed to antibody catalogue during the test. The log number has been changed.

[0390] immunohistochemistry Frozen lymph nodes and spleens obtained at necropsy were sectioned and analyzed using standard immunohistochemistry. The cells were stained with anti-CD19 antibody (Abcam, cat. no. ab134114) using the procedure.

[0391] result Repeated IV and single SC administration, as well as IV administration with a priming dose, resulted in peripheral blood and This resulted in a dose-dependent depletion of B cells from lymph nodes (Figures 4-9). At 0.01 mg / kg, Two IV doses of induced B cell depletion to (almost) undetectable levels. The second and fourth doses resulted in partial to no depletion of B cells. The deficiency may be due to the formation of anti-drug antibodies. Repeated IV doses at 0.1 or 1 mg / kg were not completely Induce complete B cell depletion, with the onset of (partial) recovery after 21 days (0.1 mg / kg) or 42–11 days. The pharmaceutical composition of the present invention (30 mM acetate, 150 mM sorbitol) was administered at 1 mg / kg for 9 days. A single SC injection of DuoBody-CD3xCD20 in PBS (pH 5.5) at all dose levels significantly increased the circulating The lymph nodes were depleted of B cells to undetectable levels. B cell recovery was observed in all groups. At lower doses, the effect returned to baseline within a few weeks, whereas at higher doses, the effect returned to baseline within 70 days after administration. The IV administration of a priming dose (0.01 mg / kg) followed the next day by a target dose of 1 mg / kg Peripheral blood and lymph nodes were completely depleted of B cells on the scheduled autopsy day (day 29). In this study, depletion of B cells from lymph nodes and spleen was demonstrated by immunohistochemistry. This was confirmed by (Figure 10).

[0392] Example 6: After 5x repeated IV infusions and a single SC injection of DuoBody-CD3xCD20 in cynomolgus monkeys B cell depletion (GLP toxicity study) Male and female cynomolgus monkeys were administered DuoBody-CD3xCD20 in the pharmaceutical composition of the invention every 5 days. Weekly IV infusions (0.01, 0.1, or 1 mg / kg) and single IV infusions (0.1 or 1 mg / kg) Subjects received either intravenous or SC injection (0.1, 1, or 10 mg / kg) as outlined below. A control group receiving five weekly IV saline infusions was also included. TIFF2024156853000026.tif1341661qwx5 = 5 doses of IV once weekly (days 1, 8, 15, 22, and 29); End: day 36 (main study); Ended: Day 71 (recovered). IV SD = single IV dose (day 1); ending on day 36. SC 2x SD = DuoBody-CD3xCD20 and other SC administration of vehicle (30 mM acetate buffer, 150 mM sorbitol pH 5.5); Day 33.

[0393] This study is conducted under the Home Office's Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. In accordance with the European Convention on Chemical Substances (Council of Europe), Charles River Laboratories (Transcript of the Convention) The study was carried out at the

[0394] The crab-eating macaque, Macaca fascicularis, native to Mauritius, was bred for research purposes. All animals were obtained from LCL-Cynologics (Port Louis, Mauritius). They were accommodated in numbers and given a quality environment.

[0395] Whole blood samples and lymph node biopsies were taken as previously described.

[0396] B cells were quantified by flow cytometry as previously described with the following exceptions. carried out. For peripheral blood immunophenotyping, 50 μL of anticoagulated whole blood was added to the antibody mixture. Incubate for 30 minutes at +4°C protected from light. TruCount tubes (BD Biosiences) were used for data acquisition. · CD45+CD4-CD8-CD15-CD19+ events were classified as B cells. The following antibody mixture was used: TIFF2024156853000027.tif46128 1 FITC: fluorescein isothiocyanate; PE: phycoerythin; E CD: electron-binding dye; BV: brilliant violet; V: violet; Cy: cyanine Pigment; APC: allophycocyanin *During this study, CD45 V500 was temporarily replaced by CD45 BV711 due to supplier reasons. The CD45 V500 was reinstated after the supplier's situation was resolved. These were on different filters. Therefore, CD25 BV711 was temporarily substituted with CD25 V510.

[0397] result The results are shown in Figures 11 to 16. IV infusion of saline partially reduced the number of B cells in the peripheral blood. However, B cell counts returned to baseline within 3 weeks after the last infusion. Five weekly IV infusions of 100 mg / kg / day induced a dose-dependent depletion of B cells, with partial depletion in the lower dose group. A single IV infusion of 0.1 or 1 mg / kg resulted in complete depletion that lasted for a long time in the high dose group. Completely depletes B cells from peripheral blood, with depletion continuing until the day of necropsy (day 36) at the highest dose tested. SC administration of DuoBody-CD3xCD20 resulted in complete mobilization of B cells from peripheral blood at all doses tested. At the lowest dose, B cell levels were partially restored, but at 1 and 10 mg / mL In the kg group, complete B cell depletion persisted until the day of necropsy (day 33).

[0398] Example 7: Pharmacokinetics of DuoBody-CD3xCD20 in Cynomolgus Monkeys: Intravenous (IV) and Skin Subcutaneous (SC) route of administration material and method DuoBody formulated in 30 mM acetate buffer, 150 mM sorbitol, and pH 5.5 - The pharmacokinetic (PK) properties of CD3xCD20 were evaluated in cynomolgus monkeys administered intravenously (IV) and in a toxicology study. Both intravenous and subcutaneous (SC) routes of administration were evaluated and determined. Blood samples were collected from female cynomolgus monkeys. Dose-ranging finding (DRF) study of DuoBody-CD3xCD20 in cynomolgus monkeys and -CD3xCD20 GLP toxicity studies were performed on animals. The design and details of these studies are described in Example 4. PK evaluation was performed in animals receiving a single IV infusion or SC injection. DuoBody-CD3x Blood samples (approximately 0.5 mL each) were obtained from all animals to determine plasma concentrations of CD20. The plasma concentration of DuoBody-CD3xCD20 in cynomolgus monkeys in the DRF study was measured using Imperacer® Determined using a muno-PCR assay. DuoBody-CD3xCD in plasma of cynomolgus monkeys from a GLP toxicity study The concentrations of 20 were determined using single molecule counting (SMC) techniques. Details of these evaluations are given below. Provided in Section.

[0399] Imperacer® ImmunoPCR of PK of DuoBody-CD3xCD20 The concentration of DuoBody-CD3xCD20 in the plasma of cynomolgus monkeys in the DRF study was assessed using an advanced ultrasensitive immunoassay. Polymerase chain reaction (PCR) method, comprising the steps of: The Imperacer® method detects proteins using exponential amplification of markers. Briefly, an 8-point standard curve was prepared in 100% cynomolgus monkey plasma. The purified DuoBody-CD3xCD20, quality control (QC) samples, and (diluted) cynomolgus monkey test samples were , Imperacer® conjugate CHI-SAB1 A1 (Chimera, Dortmund, Germany) The samples were diluted with sample dilution buffer SDB6000 containing 100 mM NaCl (Cat. No. 11-272, Biotec GmbH). The His-tagged extracellular domain of CD3 (CD3ECDHis; Oral) was used to bind DuoBody-CD3xCD20. The antibodies were added to 96-well ELISA plates coated with ELISA kit (Genmab, Utrecht, India). The plates were washed, PCR master mix (Molzym, Cat no. C-022) was added, and the samples were then incubated on an Impera cer® RT-PCR Reader (Agilent Technologies / Chimera Enabled RT The immobilized DuoBody-CD3xCD20 was transferred to an Imperacer (registered trademark). can be detected during PCR amplification of the DNA marker contained in the detection conjugate Processing of the sequence-specific fluorescent probes in the PCR master mix results in the The fluorescence signal is directly related to the amount of DNA marker present and is reported as the ΔCt signal. When the real-time PCR signal generation is complete, the measured fluorescence data is The data were processed using the instrument software (MXPro; Chimera Biotec GmbH) and mathematical software (Micr The concentration of bound DuoBody-CD3xCD20 was calculated using the following formula: A nonlinear sigmoidal 4-parameter regression was used to fit the ΔCt signal to the log spiked ) Determined from a standard curve generated by plotting against DuoBody-CD3xCD20 concentration. The assay was developed and performed at Chimera Biotec GmbH, Dortmund. The LLOQ was 1.0 pg / mL in pure plasma.

[0400] DuoBody-CD3xCD20 PK Single Molecule Counting (SMC) Method Plasma DuoBody-CD3xCD20 concentrations in cynomolgus monkeys in a GLP toxicity study were measured using single molecule counting (SMC) method The SMC immunoassay detects DuoBody-CD3xCD20 molecules in cynomolgus monkey plasma. It is a fluorescent sandwich immunoassay technique that can measure

[0401] Briefly, calibrators, QCs, and test samples were filtered and magnetically coupled prior to use. The antibodies were injected with an anti-idiotypic antibody against the CD3 arm of DuoBody-CD3xCD20 (UM-IgG1mm-3005- 101-3-1-MP; Genmab, Utrecht, The Netherlands) and labeled according to the manufacturer's protocol. The filtered samples were then transferred to a coated magnetic The CD20 arm of DuoBody-CD3xCD20 was conjugated to a fluorochrome. Anti-idiotypic antibody against UM-IgG1mm-3001-2F2-Sab1.1(-FL); Genmab, The Netherlands After incubation, the particles were washed to remove unbound particles. The combined conjugate was removed. The magnetic particles with the analyte and conjugate bound thereto were then The magnetic particles were transferred to a clean plate and the remaining buffer was aspirated. The eluate was dissolved in elution buffer according to the manufacturer's protocol (Merck Millipore). The samples were then separated and the eluate was transferred to a 384-well plate containing neutralization buffer. (registered trademark) Single molecule measurement system (Merck / Millipore) was used to draw the molecule into the capillary. The fluorescent molecules emit light, and the signal that exceeds the threshold is regarded as a detection event. In addition, the amount of light in each event (event photons) and the total light The amount of (total photons) was measured.

[0402] The method was performed at the PRA Health Sciences Bioanalytical Laboratory in Assen, The Netherlands. During validation, the LLOQ was determined to be 0.100 ng / mL pure plasma. The upper limit of normal volume (ULOQ) was determined to be 50 ng / mL pure plasma.

[0403] result Dose range-finding study: single IV dose with priming dose Patients received a priming dose of 0.01 mg / kg DuoBody-CD3xCD20 on day 1, followed by 1 mg Cynomolgus monkeys (n = 2 females) receiving a target dose of DuoBody-CD3xCD20 at 100 mg / kg The plasma concentration profile of CD20 was measured. Both the priming and targeting doses were The dose was administered as an IV infusion at a volume of 10 mL / kg over 30 minutes. IV priming dose of 0.01 mg / kg After IV administration of DuoBody-CD3xCD20 as a target dose of 1 mg / kg (day 1) (day 2), Immediately at the end of the infusion of the mg / kg dose, max In the repeated IV infusion group, the same Order CL value (10.7-13.7 mL / day / kg) and V D values ​​(56.1–64.9 mL / kg) were observed. .

[0404] Individual plasma concentration profiles generated from the Imperacer immuno-PCR method are shown in FIG. 17A. Mean PK parameters are shown in Table 12. PK parameters were calculated only for the 1 mg / kg dose. Ta.

[0405] Table 12. IV Single Dose: Mean PK Parameters of DuoBody-CD3xCD20 in the DRF Study TIFF2024156853000028.tif28165

[0406] Dose range finding study: Single SC dose Following a single SC injection of DuoBody-CD3xCD20 at dose levels of 0.01, 0.1, 1, 10, or 20 mg / kg The plasma concentration profile of DuoBody-CD3xCD20 was measured (n = 2 females / group). SC injections were performed at 1 After SC administration, the C max reached. max Also is AUC 0-∞ A superproportional increase in exposure was observed up to a dose of 1 mg / kg. A proportional increase in exposure was observed with increasing dose from 1 mg / kg to 20 mg / kg. Individual plasma concentration profiles generated from the acer® immuno-PCR method are shown in FIG. Group mean PK parameters are shown in Table 13.

[0407] Absolute SC bioavailability (F) was calculated as the AUC inf , and 1 mg / kg AUC after first IV dose of g 0-∞ as a percentage of IV bioavailability This calculation gives a figure of 111%, which indicates perfect (100%) SC bioavailability at this dose. was shown.

[0408] Table 13. Single SC Dose: Mean PK Parameters of DuoBody-CD3xCD20 in the DRF Study TIFF2024156853000029.tif65165

[0409] GLP Toxicity Study: Single IV Dose DuoBody-CD3xCD following a single IV infusion of DuoBody-CD3xCD20 at dose levels of 0.1 or 1 mg / kg Twenty plasma concentration profiles were measured (3 monkeys / sex / group). Group averages generated from the SMC method The mean plasma concentration profiles are shown in Figure 17C, and group mean pharmacokinetic parameters are shown in Table 14. Systemic exposure to Body-CD3xCD20 (mean C max and AUC (0-t) (based on Dose-normalized estimates showed that the effect of DuoBody-CD3xCD20 on Systemic exposure increased more than dose-proportionally in both males and females over the dose range of 0.1 to 1 mg / kg. Median T max The time to infusion was consistently 0.5 hours (at the end of the infusion phase). There was a tendency for CL, VD, and VSS to decrease with increasing 1 / 2 at the higher dose of 1 mg / kg The induction was considered to be adequate (mean values ​​were 98 hours in males and 125 hours in females), and the excretion phase was stable in both sexes. was characterized for up to 840 hours compared with 168 or 336 hours at 0.1 mg / kg. Systemic exposure was roughly equivalent in males and females at 0.1 and 1 mg / kg, but was significantly higher in males at 0.1 mg / kg. Mean AUC of fed males (0-t) was higher in males than in females, probably more than in any other animal. This is an artifact of one animal that had a seven-fold higher total exposure. Taking this into account, C max and AUC (0-t) The female / male ratio ranged from 0.8 to 1.3 (0.1 mg / kg, AUC (0-t) is 0.3).

[0410] Table 14. IV Single Dose: Mean PK Parameters of DuoBody-CD3xCD20 in a GLP Toxicity Study TIFF2024156853000030.tif53165

[0411] Single SC dose DuoBody-CD3xCD20 after a single SC injection at dose levels of 0.1, 1, or 10 mg / kg The plasma concentration profile of CD3xCD20 was measured (3 monkeys / sex / group). SC injections were performed at 0.2 mL / The group mean plasma concentration profiles generated from the SMC method are shown in Figure 17C. Group mean pharmacokinetic parameters are shown in Table 15. Systemic exposure to DuoBody-CD3xCD20 (mean C m ax and AUC (0-t) (based on 100 mg / kg / day) increased with increasing SC dosing in males and females. According to dose-normalized estimates, C max In both males and females, the dose ratio was approximately the same at 0.1 to 1 mg / kg. The dose-normalized AUC(0-t ) was increased more than dose-proportionally at 0.1 to 10 mg / kg. Overall, the increase after SC administration was The effects were greater than dose-proportional in both males and females at 0.1 to 10 mg / kg. Median T max Is male In females, the T max No consistent trend was observed But that is individual T max This is because the variation between the values ​​was greater. 1 / 2 At high doses, the longest The elimination phase appeared to be best characterized in males and females. At 0.1 mg / kg, the levels were generally higher in males than in females, and at 1 and 10 mg / kg, the levels were similar in both sexes. Ri; C max and AUC (0-t) The female / male ratios at 0.1 mg / kg were 0.5 and 0.4, respectively, and at 1 mg / kg At 10 mg / kg, both parameters were 0.8, and at 10 mg / kg, both parameters were 1.0.

[0412] Table 15. Single SC Dose: Mean PK Parameters of DuoBody-CD3xCD20 in a GLP Toxicity Study TIFF2024156853000031.tif52167

[0413] In summary, after IV infusion of DuoBody-CD3xCD20, plasma concentrations increased until the end of the 30-minute administration phase. After SC administration, the increase was longer lasting, approximately 72 minutes after administration, and then decreased in a roughly biphasic manner. The peak was observed at 1 h and remained at a relatively stable level up to 168 h after administration. Concentrations then declined monophasically until the end of the 4-week sampling period. In the study, the maximum plasma concentrations after IV administration were significantly higher than those after SC administration.

[0414] Example 8 Humanized IgG1λ, C having heavy and light chain sequences shown in SEQ ID NOs. 26 and 24, respectively. D3ε specific antibody, IgG1-CD3-FEAL, and the heavy chains shown in SEQ ID NOs. 27 and 25, respectively. The two parent antibodies, IgG1-CD20-FEAR, with their respective long and light chain sequences, were synthesized using separate biological intermediates. The parent antibody was produced as a mammalian antibody using standard suspension cell culture and purification techniques. The Fab fragment was then produced in a Chinese hamster ovary (CHO) cell line. The CD3xCD20 antibody was produced by the cFAE method (Labrijn et al. 2013, Labrijn et al. After polishing / purification, the final product was purified to near 100% purity. The antibody concentration was calculated using the theoretical extinction coefficient ε = 1.597 mL mg-1 cm-1 at 280 nm. This bispecific antibody product has the international generic name epcolitamab. It was.

[0415] Epcolitamab formulation study Further formulation studies were performed on epcolitamab. Epcolitamab formulations were Each formulation was then sterile filtered, placed in a vial, and the buffer was then The vials were capped with stoppers. The tests were carried out at pH 4.8, 5.2, and 5.5, and at sorbitol. concentration (150 mM, 200 mM, 250 mM), sodium chloride concentration (0 mM, 35 mM, 70 mM), and Thirty-one variants of the formulation parameter space were included, including L-arginine concentrations (0 mM, 35 mM, and 70 mM). ation, where epcolitamab was at a concentration of 60 mg / mL and polysorbate 80 (PS80 The concentration of 0.04% (w / v) was unchanged. The formulations tested are listed in Table 16. Additional analyses were also performed on selected The epcolitamab concentration was 48 mg / mL using five selected formulations (data not shown). figure).

[0416] Table 16. Formulations tested in epcolitamab formulation studies TIFF2024156853000032.tif138170TIFF2024156853000033.tif125170

[0417] The samples were stored at 5±3°C and 40±2°C / 75% RH (relative humidity), respectively, for up to 8 weeks. Prior to analysis, selected formulations were further subjected to freeze / thaw (F / T) cycles or agitation conditions. Analytical tests performed included appearance (color, clarity, and particulates), pH, turbidity, and protein content. , DLS, SEC-HPLC, icIEF, CGE-SDS under reducing and non-reducing conditions, and osmolality The osmolality of the above formulations ranged from about 230 to about 530. The formulations tested were All were deemed suitable for subcutaneous administration to humans. The osmolality of some formulations was The appearance, pH, SEC-HPLC, and icIEF results are summarized below. The results of other test parameters support the conclusions of the other test methods used. I did.

[0418] Table 17. Osmolality of selected formulations TIFF2024156853000034.tif62167

[0419] Appearance Results All formulations tested were clear, slightly yellowish or slightly opalescent liquids. , and no visible particulates. No changes were observed after up to 8 weeks of storage. Table 18 Figure 1 shows the appearance results of selected formulations, where arginine and sorbitol levels are The rule has been changed.

[0420] Table 18: Appearance results TIFF2024156853000035.tif113167TIFF2024156853000036.tif161167Form.(Formulation); Cond.(Condition); SY(Slightly yellow); SO(Slightly opalescent); CL(Clear Liquid); F (no visible particles)

[0421] pH Results All formulations tested were at or close to the target pH. No changes were observed after storage at 40°C for up to 8 weeks. Results for all samples stored at 40°C for up to 8 weeks are shown in Table 19. You can see it.

[0422] Table 19. pH results for samples stored at 40°C for up to 8 weeks TIFF2024156853000037.tif224167TIFF2024156853000038.tif132167

[0423] HPLC-SEC results A decrease in monomer and an increase in high molecular weight (HMW) were observed in all samples after storage at 40°C for up to 8 weeks. Table 20 shows the HPLC-SEC results of selected formulations, where arginine and The levels of sorbitol and sorbitol were varied. The level of sorbitol had no significant effect on the results. As shown in Figure 18, increased levels of NaCl and decreased pH had no effect on the This results in a decrease in the nomer and an increase in HMW.

[0424] Table 20. SEC results for selected formulations and storage conditions TIFF2024156853000039.tif239167TIFF2024156853000040.tif33167

[0425] icIEF results As expected, the decrease in the main peak and the increase in the acidic variants were observed after storage at 40°C for up to 8 weeks. Table 21 shows the icIEF results for some selected formulations. Results are shown where the levels of arginine and sorbitol are varied. The levels of arginine did not have any significant effect on the results. It may have a small positive effect on ants.

[0426] Table 21. icIEF results for selected formulations and storage conditions TIFF2024156853000041.tif118170TIFF2024156853000042.tif165170

[0427] conclusion A change in sorbitol from 150 to 250 mM had a particular effect on the stability profile of the antibody. The decreased pH and addition of sodium chloride had no effect on the stability profile of the antibody. and was thought to have a negative impact.

[0428] Thus, the data from this experiment suggest that the range of 150–250 mM, inclusive, is optimal for antibodies. The sorbitol ranged from 5.3 to 5.5, with 30 mM acetate, 0.04% polysorbate 80, and Formulations with a pH range of 0.04% polysorbate 60 (P60) and 0.25% acetate are possible. Formulations containing a pH of 80 and a pH in the range of 5.3 to 5.5 may be envisaged. Further substitutes such as ginine have been shown to be effective in the formulation without adverse effects. , may be included in the formulation (e.g., up to about 58 mM).

[0429] In conclusion, the results obtained were consistent with those obtained with 30 mM acetate, 150 mM sorbitol, 0.04% polysorbate The formulation of Lubet 80, pH 5.5, is capable of delivering up to at least 60 mg / mL of epcolitamab, and The results support the optimal formulation at concentrations including 60 mg / mL. Optionally, additional amounts of substitutes may be added to increase the amount of the glycerol to a more physiological level. You might consider including it.

[0430] Example 9 Diluent 5 mg / mL of epcolitamab pharmaceutical composition was used for the preparation of the dilutions. It has been shown that product-specific diluents can be used for dilutions down to 5 μg / mL.

[0431] A commercially available diluent was also tested, namely 0.9% NaCl w / v in water suitable for injection. The agent was shown to be compatible with dilution up to at least about 80 μg / mL.

[0432] Testing included storage for up to 24 hours. Data showed that under the conditions tested, recovery There was no effect on the rate or stability profile.

Claims

1. a. 0.5-120 mg / mL of a bispecific antibody that binds human CD3 and human CD20; b. 20-40 mM acetate; c. 140-260 mM sorbitol; d. Surfactants A pharmaceutical composition comprising or consisting of: wherein the pH of the composition is between 5 and 6, and the bispecific antibody is The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, 23. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein c. is in the range of 140-160 mM sorbitol.

3. The first binding region of the bispecific antibody that binds to CD3 has the VH and VL sequences of SEQ ID: 6 and 7. At least 90% sequence identity, e.g., at least with the VH and VL sequences of SEQ ID: 6 and 7. VH and VL sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The pharmaceutical composition of claim 1 or 2.

4. The second binding region of the bispecific antibody that binds to CD20 is represented by the VH and VL sequences of SEQ ID: 13 and 14. at least 90% sequence identity with the VH and VL sequences of SEQ ID: 13 and 14, for example VH and VL sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

4. The pharmaceutical composition of any one of claims 1 to 3, comprising an array.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the bispecific antibody is an IgG1 antibody.

6. The bispecific antibody may comprise a lambda light chain constant region, such as the light chain constant regions of SEQ ID Nos. 22 and 23. and a kappa light chain constant region.

6. The pharmaceutical composition of any one of claims 1 to 5, comprising a light chain.

7. The bispecific antibody comprises an Fc region comprising a first and a second heavy chain, the Fc region comprising a wild-type IgG1 Modified to have reduced effector function compared to bispecific antibodies containing an Fc region 2. The pharmaceutical composition according to any one of the preceding claims,

8. The bispecific antibody has a higher affinity for C1q than a bispecific antibody having a wild-type IgG1 Fc region. Binding to the antibody is at least 70%, at least 80%, at least 90%, at least 95%, Both contain Fc regions modified to reduce C1q binding by 97% or 100%, respectively, by ELISA. The pharmaceutical composition of any one of the preceding claims, wherein the composition is determined by:

9. The bispecific antibodies each comprise at least a hinge region, a CH2 region, and a CH3 region. The first heavy chain comprises T366, L368, K370, D371, and D372 in a human IgG1 heavy chain. an amino acid at a position corresponding to a position selected from the group consisting of 399, F405, Y407, and K409 and in the second heavy chain, at least one of T366, L368 in the human IgG1 heavy chain is substituted. , K370, D399, F405, Y407, and K409 At least one amino acid is substituted, and the first heavy chain and the second heavy chain are not at the same position.

4. The pharmaceutical composition of any one of the preceding claims, which is unsubstituted.

10. (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain; and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain. or (ii) an amino acid at a position corresponding to K409 in a human IgG1 heavy chain is and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is R in the second heavy chain. The pharmaceutical composition of any one of the preceding claims, wherein

11. Positions L234 and L235 in the human IgG1 heavy chain of both the first and second heavy chains of the bispecific antibody The pharmaceutical composition according to any one of the preceding claims, wherein the positions corresponding to L235 and L236 are F and E, respectively. chemical composition.

12. Positions L234, L235 in the human IgG1 heavy chain of both the first and second heavy chains of the bispecific antibody Any of the preceding claims, wherein the positions corresponding to D265, D35, and D265 are F, E, and A, respectively. The pharmaceutical composition described in any one of claims 1 to 4.

13. Location of both the first and second constant heavy chains of the bispecific antibody in the human IgG1 heavy chain The positions corresponding to L234, L235, and D265 are F, E, and A, respectively, and the first stationary The position of the heavy chain corresponding to F405 in the human IgG1 heavy chain is L, and the position of the second constant heavy chain corresponding to F405 in the human Ig The pharmaceutical composition of any one of the preceding claims, wherein the position corresponding to K409 in the G1 heavy chain is R. composition.

14. The first and second constant heavy chains have at least 90% identity with the amino acid sequence of SEQ ID NO:

16.

2. The pharmaceutical composition of claim 1, comprising an amino acid sequence that binds to

15. The first and second constant heavy chains comprise the amino acid sequences of SEQ ID Nos. 19 and 20, respectively. A pharmaceutical composition according to any one of the preceding claims.

16. The bispecific antibody has heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy and light chain as set forth in SEQ ID NOs. 27 and 25, respectively.

15. The pharmaceutical composition of any one of claims 1 to 14, comprising a CD20 binding arm having a chain.

17. Claims 1 to 14, wherein the bispecific antibody is epcolitamab or a biosimilar thereof. The pharmaceutical composition according to any one of the preceding claims.

18. a. is 0.5-120 mg / mL, for example 1.0-60 mg / mL, or for example 5-30 mg / mL, e.g. For example, 5 mg / mL, 6 mg / mL, 7 mg / ml, 8 mg / ml, 9 mg / ml, or 10 mg / ml, or 11 mg / ml, or 12 mg / ml, or 13 mg / ml, or 14 mg / ml, or 15 mg / ml, or 16 mg / ml, or 17 mg / ml, or 18 mg / ml, or 19 mg / ml, or 20 mg / ml, or 21 mg / ml, or 22 mg / ml, or 23 mg / ml, or 24 mg / ml, or 25 mg / ml, or 26 mg / ml, or 27 mg / ml, or 28 mg / ml, or 29 mg / ml, 30 mg / ml , 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml , 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml , 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml , 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, or for example 60 mg / ml.

2. A pharmaceutical composition according to any one of the preceding claims.

19. b. is between 25 and 35 mM, for example, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM 3. The method according to claim 1, wherein the solubility of the solubility is 3 mM, 33 mM, 34 mM, or 35 mM, preferably 30 mM. A pharmaceutical composition comprising:

20. c. is 145 to 155 mM, for example, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, preferably 150 mM. The pharmaceutical composition described in any one of claims 1 to 4.

21. 2. Any one of the preceding claims, wherein the pH is between 5.3 and 5.6, such as between 5.4 and 5.6, for example about 5.

5. Item 3. A pharmaceutical composition according to item 2.

22. The surfactants are glycerol monooleate, benzethonium chloride, sodium doxa esters, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate, and triglycerides. Caprylin, Benzalkonium Chloride, Citrimide, Cetylpyridinium Chloride, and Li Lipids, Alpha Tocopherol, Glycerol Monooleate, Myristyl Alcohol , phospholipids, poloxamer, polyoxyethylene alkyl ether, polyoxyethylene Castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene Stellarate, polyoxyl hydroxystearate, polyoxyl glyceride, polysodium propylene glycol dilaurate, propylene glycol monolaurate, Sorbitan ester sucrose palmitate, sucrose stearate, tricaprylin 5. The pharmaceutical composition of claim 1, wherein the compound is selected from the group consisting of:

23. 2. The pharmaceutical composition of claim 1, wherein the surfactant is a polysorbate.

24. The surfactant is polysorbate 20 or 80, e.g., polysorbate 80. The pharmaceutical composition of any one of claims 1 to 5.

25. The surfactant is about 0.005% to 0.4% w / v, for example about 0.01 to 0.1% w / v, for example about 0.01 to 0. 0.09% w / v, for example about 0.01-0.06% w / v, for example about 0.01-0.05% w / v, for example 0.02% w / v , or 0.03% w / v, or 0.04% w / v, or 0.05% w / v, preferably 0.04% w / v.

20. The pharmaceutical composition of claim 19, comprising:

26. the composition has a pH of 5.3 to 5.8, or 5.4 to 5.7, for example about 5.5; and a. 0.5-120 mg / mL of bispecific antibody b. 20-40 mM acetate c. 140-260 mM sorbitol d. 0.005% to 0.4% w / v of a surfactant, preferably a polysorbate, e.g., polysorbate 2 0 or polysorbate 80, e.g. polysorbate 80 20. The pharmaceutical composition according to any one of the preceding claims, comprising or consisting of:

27. 27. The pharmaceutical composition of claim 26, wherein c. is 140-160 mM sorbitol.

28. the composition has a pH of 5.4 to 5.6, for example about 5.5; and a. 0.5-120 mg / mL of bispecific antibody b. 28-32 mM acetate c. 145-155 mM sorbitol d. 0.02-0.05% w / v surfactant, preferably a polysorbate, e.g., polysorbate 20 or polysorbate 80, e.g. polysorbate 80 20. The pharmaceutical composition according to any one of the preceding claims, comprising or consisting of:

29. The pharmaceutical composition of any one of claims 26 to 28, wherein a. is 5 to 60 mg / mL.

30. The pharmaceutical composition of claim 29, wherein a. is 12 to 24 mg / mL, e.g., 12 mg / mL or 24 mg / mL. Composition.

31. The pharmaceutical composition of any one of claims 1 to 28, wherein a. is 2 to 8 mg / mL.

32. The pharmaceutical composition of any one of claims 1 to 28, wherein a. is 30 to 90 mg / mL.

33. The pharmaceutical composition of any one of claims 1 to 28, wherein a. is 5 mg / mL.

34. The pharmaceutical composition of any one of claims 1 to 28, wherein a. is 60 mg / mL.

35. The composition has a pH of about 5.5; and e. 0.5-120 mg / mL of bispecific antibody f. 30 mM acetate g. 150 mM Sorbitol h. 0.04% w / v surfactant, preferably a polysorbate, such as polysorbate 20 or Polysorbate 80, e.g. Polysorbate 80 20. The pharmaceutical composition according to any one of the preceding claims, comprising or consisting of:

36. The composition has a pH of about 5.5; and i. 0.5-120 mg / mL of bispecific antibody j. 30 mM acetate k. 250 mM sorbitol l. 0.04% w / v surfactant, preferably a polysorbate, such as polysorbate 20 or Polysorbate 80, e.g. Polysorbate 80 20. The pharmaceutical composition according to any one of the preceding claims, comprising or consisting of:

37. The pharmaceutical composition of claim 35 or claim 36, wherein a. is 2 to 8 mg / mL.

38. The pharmaceutical composition of claim 35 or claim 36, wherein a. is 30 to 90 mg / mL.

39. The pharmaceutical composition of claim 35 or claim 36, wherein a. is 10 to 50 mg / mL.

40. Claim 35 or claim 36, wherein a. is 12 to 24 mg / mL, for example 12 mg / mL or 24 mg / mL. The pharmaceutical composition described.

41. 37. The pharmaceutical composition of claim 35 or claim 36, wherein a. is 5 mg / mL.

42. 37. The pharmaceutical composition of claim 35 or claim 36, wherein a. is 60 mg / mL.

43. The pharmaceutical composition of any one of the preceding claims, wherein the composition does not contain hyaluronidase. Composition.

44. 2. The pharmaceutical composition of claim 1, wherein the composition comprises less than 20 mM NaCl. .

45. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises less than 60 mM arginine. composition.

46. 2. The pharmaceutical composition of any one of the preceding claims, wherein the composition is a subcutaneous composition.

47. 2. The pharmaceutical composition of any one of the preceding claims, wherein the composition is an intravenous composition.

48. 2. The pharmaceutical composition of any one of the preceding claims, wherein the composition is used for the treatment of cancer.

49. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is for subcutaneous administration.

50. The pharmaceutical composition according to any one of claims 1 to 45, wherein the composition is for intravenous administration. thing.

51. 51. The pharmaceutical composition of any one of claims 1 to 50, in unit dosage form.

52. The composition has a shelf life of at least 6 months, for example at a storage temperature of 2-8°C, e.g., 5°C. stable for pharmaceutical use for at least 9 months, or at least 12 months.

2. The pharmaceutical composition according to any one of the preceding claims.

53. Use of the pharmaceutical composition according to any one of claims 1 to 46, which is for subcutaneous administration.

54. 46. ​​Use of the pharmaceutical composition according to any one of claims 1 to 45, which is for intravenous administration.

55. 55. The use of any one of claims 53 or 54, for the treatment of cancer.

56. A pharmaceutical composition according to any one of claims 1 to 52 is administered to a subject in need thereof to treat cancer.

46. ​​A method of treating cancer in a subject comprising administering to a subject a therapeutically effective amount of a compound comprising the steps of:

57. 57. The method of claim 56, wherein the composition is administered subcutaneously or intravenously.

58. 58. The method of any one of claims 56 or 57, wherein the cancer is a B-cell malignancy.

59. A unit dosage form comprising: a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of 5 μg to 120 mg, b. acetate buffer and sorbitol in a ratio of 1:5 to 1:10, wherein the maleic acid in said unit dosage form is The molarity is 200-600 and the pH is 5.4-5.

6. Acetate buffer and sorbitol And c. Surfactants The unit dosage form comprises or consists of:

60. a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of 5 μg to 120 mg, b. Acetate at a concentration of about 30 mM at a pH of about 5.5 c. sorbitol at a concentration of about 150 mM, and d. Approximately 0.04% w / v Polysorbate 80 60. The unit dosage form of claim 59, comprising or consisting of:

61. a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, a bispecific antibody in an amount of 5 μg to 120 mg, b. Acetate at a concentration of about 30 mM at a pH of about 5.5 c. sorbitol at a concentration of about 250 mM, and d. Approximately 0.04% w / v Polysorbate 80 60. The unit dosage form of claim 59, comprising or consisting of:

62. The first binding region of the bispecific antibody that binds to human CD3 has the VH and VL sequences of SEQ ID: 6 and 7. and a second binding region of the bispecific antibody that binds to human CD20, the second binding region comprising the V 62. The unit dosage form of any one of claims 59 to 61, comprising an H and a VL sequence.

63. The bispecific antibody comprises first and second constant region heavy chains of SEQ ID NOs: 19 and 20, respectively.

63. The unit dosage form of claim 62.

64. The bispecific antibody has heavy and light chains as set forth in SEQ ID NOs. 26 and 24, respectively. and a heavy and light chain as set forth in SEQ ID NOs. 27 and 25, respectively.

63. The unit dosage form of claim 62, comprising a CD20 binding arm having a chain.

65. 63. The method of claim 62, wherein the bispecific antibody is epcolitamab, or a biosimilar thereof. Unit dosage form.

66. The unit of any one of claims 59 to 65, wherein the amount of bispecific antibody is between 40 μg and 80 mg. Dosage form.

67. The amount of bispecific antibody is between 40 μg and 60 mg, for example, 40 μg, 50 μg, 100 μg, 150, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 45 0 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg , 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg , 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 2 9 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 m g, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, or for example 60 mg The unit dosage form of any one of claims 59 to 66.

68. The single composition according to any one of claims 59 to 67, wherein the total volume is between 0.5 mL and 2 mL, for example 1 mL. dosage form.

69. 70. The unit dosage form of claim 68, for subcutaneous administration.

70. 70. The unit dosage form of claim 69, wherein the volume of the unit dosage form administered is about 1.0 ml, or 0.8 ml. shape.

71. Any of claims 59-68, wherein the total volume is between 20 mL and 200 mL and the unit dosage form is for IV administration. A unit dosage form according to any one of claims 1 to 4.

72. A unit dosage form according to any one of claims 59 to 71 for treating cancer is administered to a subject in need thereof.

46. ​​A method of treating cancer in a subject, comprising administering for a period of time sufficient to

73. 72. The unit dosage form of any one of claims 59 to 71 for use in treating cancer.

74. A container comprising the unit dosage form of any one of claims 59-72.

75. a. A pharmaceutical composition according to any one of claims 1 to 52. b. Receptacles for unit dosage forms c. Instructions for dilution and / or use Kit of parts including:

76. a. A pharmaceutical composition according to any one of claims 1 to 52. b. Diluent c. Receptacles for unit dosage forms d. Instructions for dilution and / or use Kit of parts including:

77. a. A pharmaceutical composition according to any one of claims 1 to 52. b. Diluents, including acetate, sorbitol, and polysorbate 80 c. Receptacles for unit dosage forms d. Instructions for dilution and / or use Kit of parts including:

78. The ratio of concentrations of acetate, sorbitol, and polysorbate 80 in the diluent and pharmaceutical composition 78. The kit-of-parts of claim 77, wherein the parts are identical in size and shape.

79. a. The pharmaceutical composition comprises: i. 5-60 mg / mL of bispecific antibody ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. 0.04% w / v Polysorbate 80 Including, v. pH is 5.5; b. a receptacle for the unit dosage form; and c. Instructions for dilution and / or use The kit of parts according to any one of claims 75 to 78.

80. a. The following CDR sequences: VH-CDR1: SEQ ID NO: 1 VH-CDR2: SEQ ID NO: 2 VH-CDR3: SEQ ID NO: 3 VL-CDR1: SEQ ID NO: 4 VL-CDR2: GTN, and VL-CDR3: SEQ ID NO: 5 A first binding region that binds to human CD3, comprising: The following CDR sequences: VH-CDR1: SEQ ID NO: 8 VH-CDR2: SEQ ID NO: 9 VH-CDR3: SEQ ID NO: 10 VL-CDR1: SEQ ID NO: 11 VL-CDR2: DAS, and VL-CDR3: SEQ ID NO: 12 a second binding region that binds to human CD20, 0.5-120 mg / mL of a bispecific antibody comprising b. 3.53 mg / mL sodium acetate trihydrate c. 0.24 mg / mL acetic acid d. 27.3 mg / mL sorbitol e. 0.4 mg Polysorbate 80 in water for injection; and adjusting the pH to 5.5 by adding sodium hydroxide. A method for preparing a pharmaceutical composition as defined in any one of claims 1 to 47, comprising:

81. 81. The method of claim 80, wherein a. is 5 mg / mL.

82. 81. The method of claim 80, wherein a. is 12 mg / mL.

83. 81. The method of claim 80, wherein a. is 24 mg / mL.

84. 81. The method of claim 80, wherein a. is 48 mg / mL.

85. 81. The method of claim 80, wherein a. is 60 mg / mL.

86. a. preparing a pharmaceutical composition by the steps of the method of any one of claims 80 to 85. or providing a pharmaceutical composition as defined in any one of claims 1 to 45; b. Preparing a diluent c. Mixing the pharmaceutical composition with a diluent to the desired bispecific antibody concentration.

72. A method for preparing a unit dosage form as defined in any one of claims 59 to 71, comprising:

87. a. preparing a pharmaceutical composition by the steps of the method of any one of claims 80 to 85. or providing a pharmaceutical composition as defined in any one of claims 1 to 45; b. Regarding the substitute, the pharmaceutical composition prepared or provided in step a. Providing a diluent having the same or similar concentration as the product. c. Mixing the pharmaceutical composition with a diluent to the desired bispecific antibody concentration.

72. A method for preparing a unit dosage form as defined in any one of claims 59 to 71, comprising:

88. A drug obtainable by any of the methods defined in any one of claims 80 to 87. A pharmaceutical composition or unit dosage form.

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