Pharmaceutical compositions containing bispecific antibodies against CD3 and CD20, and their use

Abstract

This invention provides a stable pharmaceutical composition of a bispecific CD3×CD20 antibody suitable for both intravenous and subcutaneous administration. [Solution] A stable liquid pharmaceutical composition is provided, comprising a bispecific antibody in a concentration of 5 mg / mL to 150 mg / mL that binds to human CD3 and human CD20, and a pharmaceutically acceptable buffer, wherein the pH of the pharmaceutical composition is in the range of 5 to 6.5.

Description

[[Technical Field]]

[0001] The present invention relates to pharmaceutical compositions and unit dosage forms of bispecific antibodies against CD3 and CD20, and to uses thereof. [[Background Art]]

[0002] CD3 has been known for many years and has therefore been the subject of interest in many aspects. Specifically, antibodies raised against CD3 or the T cell receptor complex, of which CD3 is a part, are known. Antibodies raised against CD3 or the T cell receptor complex, of which CD3 is a part, are known.

[0003] A promising approach to improve targeted antibody therapy is by specifically delivering cytotoxic cells to antigen-expressing cancer cells. This concept of using T cells to efficiently kill tumor cells was described by Staerz et al., 1985, Nature 314:628-631. However, This concept of using T cells to efficiently kill tumor cells was described by Staerz et al., 1985, Nature 314:628-631. However, early clinical trials were rather disappointing, mainly due to low efficacy, severe adverse effects (cytokine storm) and immunogenicity of bispecific antibodies (Muller and Kontermann, 2010, BioDrugs 24:89-98), rather disappointing. Advances in the design and application of bispecific antibodies have partially overcome the initial barrier of cytokine storm and improved clinical efficacy without dose-limiting toxicity (Ga partially overcome the initial barrier of cytokine storm and improved clinical efficacy without dose-limiting toxicity (Ga rber, 2014, Nat. Rev. Drug Discov. 13:799-801; Lum and Thakur, 2011, BioDrugs 25:365-379 ). As described for catumaxomab, overcoming the initial barrier of cytokine storm is important for (Berek et al., 2014, Int. J Gynecol. Cancer 24(9):1583-1589; Mau-Soren Sen et al., 2015, Cancer Chemother. Pharmacol. 75:1065-1073), absence of Fc domain or It was erasing.

[0004] The CD20 molecule (also known as human B-leukocyte restriction antigen or Bp35) is hydrophobic and trans It is a membrane protein with a molecular weight of approximately 35 kD, and is located on pre-B and mature B leukocytes (Valent ine et al. (1989) J. Biol. Chem. 264(19):11282-11287;and Einfield et al., (1988) EMBO J. 7(3):711-717). CD20 is present in more than 90% of B cells derived from peripheral blood or lymphoid organs. CD20 is found on the surface, expressed in the early stages of pre-B cell development, and persists until plasma cell differentiation. It is present in both cellular and normal B cells. In particular, CD20 is found in 90% of B-cell non-Hodgkin lymphomas (NHL). Although it is expressed in over % of cells (Anderson et al. (1984), Blood 63(6):1424-1433), hematopoietic stem cells, pre-B It is not observed in cells, normal plasma cells, or other normal tissues (Tedder et al. (1985) J. Immunol.). 135(2):973-979).

[0005] To treat cancer, autoimmune diseases, and various immune diseases by targeting CD20 The method is publicly known in the field. For example, the chimeric CD20 antibody rituximab is non-Hodgkin Lymphoma (NHL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL) Human monoclonal CD20 is used or suggested to be used in the treatment of ulcer cancer. The antibody ofatumumab is particularly indicated for various CLL indications, follicular lymphoma (FL), and neuromyelitis optica. (NMO), used to treat diffuse and relapsing-remitting multiple sclerosis (RRMS), or Its use has been suggested.

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

[0007] WO2011028952, in particular, utilizes Xencor's XmAb bispecific Fc domain technology for CD3×CD2 The generation of a bispecific molecule is described.

[0008] WO2014047231 is REGN produced using Regeneron Pharmaceuticals' FcΔAdp technology. This document describes 1979 and other CD3×CD20 bispecific antibodies.

[0009] Sun et al. (2015, Science Translational Medicine 7, 287ra70) stated, "Nobu Intu Ho B cell-targeted anti-CD20 / CD3 T cell-dependent technology constructed using "knobs-into-holes" technology Describe the sex-bispecific antibodies.

[0010] WO2016 / 110576, incorporated herein by reference, provides a bispecific CD3×CD20 antibody. The present invention relates to a stable pharmaceutical formulation of the CD3×CD20 antibody of WO2016 / 110576. The bispecific antibody that binds to it specifically targets CD20-expressing cells and prevents T cell death. Such bispecific antibodies may be useful in the desired treatment setting, and such bispecific antibodies are used for NHL, CLL, and It is being investigated for the potential treatment of other B-cell malignancies. It is being developed in clinical trials. The conventional CD3×CD20 bispecific antibody is administered via the intravenous (IV) route. This type of administration route is related to CD3×CD20 bispecific antibodies. max The price may increase. , excessively high levels of cytokine release, that is, CD20 and T cells induced by bispecific antibodies Crosslinking of expressed target cells triggers cytokine release, for example, pro-inflammatory cytokines. This can lead to the release of steroids (e.g., IL-6, TNF-α, or IL-8), causing symptoms such as fever, nausea, vomiting, and chills. This may cause harmful effects. Therefore, the unique antitumor activity of bispecific antibodies Nevertheless, these immunological mechanisms of action can have undesirable "side" effects, namely, for example Induction of an undesirable inflammatory response known as "first-dose cytokine response or syndrome." This can induce [unclear]. In such circumstances, the patient may take, for example, analgesics, antipyretics, and / or non-stimulants. Adjunctive therapy or premedication with teroid anti-inflammatory drugs is necessary. Therefore, human Alternatively, upon administration to animals, systemic cytokine release of T cells that retarget bispecific antibodies occurs. The need to modify or reduce the output profile has not been met. Therefore, CD3 and C Further antibody formulations and pharmaceutical compositions of bispecific antibodies that bind to D20, i.e., systemic cysts To avoid or reduce the side effects of tokine release, it can be administered in different ways, but at the same time, A composition that enables highly efficient T-cell-mediated death of tumor cells expressing CD20 is needed. The key point is that the present invention relates to the CD3 × CD20 bispecific antibody disclosed in WO2016 / 110576. It is a simple and stable pharmaceutical formulation with high antibody concentrations of approximately 60 mg / mL or 120 mg / mL. It remains stable across a wide range of antibody concentrations, even at high antibody concentrations of 150 mg / mL or more. The present invention aims to provide CD3 × CD20 antibodies and formulations. A further object of the present invention is to provide at least 3 For a period of a month, or even longer, for example, over a period of at least 6 months or at least 12 months, the weather is calm. The objective is to provide a pharmaceutical formulation of a CD3 × CD20 bispecific antibody. Furthermore, the present invention provides a pharmaceutical formulation of a CD3 × CD20 bispecific antibody. The objective is to provide a stable formulation over a temperature range, for example, from 2°C to 25°C. The objective is to develop a bispecific CD3×CD20 antibody suitable for both intravenous and subcutaneous administration. The objective is to provide the drug. In many cases, subcutaneous administration is more effective than intravenous administration. Because the time is much shorter, subcutaneous administration of pharmaceutical preparations is sometimes more convenient for patients. A further object of the present invention is to provide a bispecific CD3×CD20 antibody that is well acceptable at the subcutaneous injection site. The objective is to provide a pharmaceutical formulation for the body. A further objective is to administer subcutaneously to patients' cytokinesiology. It can reduce the release profile, but at the same time, it can affect tumor cells that express CD20. By providing a pharmaceutical composition that can bring about highly efficient T cell death, be. [Overview of the project]

[0011] The object of this invention is to combine a first antigen-binding region derived from a CD3 antibody and a second antigen-binding region derived from a CD20 antibody. The objective is to provide a novel pharmaceutical composition of a bispecific antibody containing a binding region.

[0012] A novel composition containing a CD3×CD20 bispecific antibody specifically targets cells expressing CD20. It is useful in therapeutic situations where targeted administration or T-cell-mediated cell death is desired. Such preparations are administered intravenously. It is useful for both oral and subcutaneous administration.

[0013] Therefore, in its main aspects, the present invention relates to a pharmaceutical composition comprising the following: a. Bispecific antibodies that bind to human CD3 and human CD20 at concentrations of 50-120 mg / mL. b. 20-40 mM acetic acid, c. 140-160 mM sorbitol, Here, the pH of the composition is 5 to 6, and the bispecific antibody is The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 Includes.

[0014] In a further embodiment, the present invention relates to the use of the pharmaceutical composition of the present invention for subcutaneous administration.

[0015] In a further embodiment, the present invention relates to the use of the pharmaceutical composition of the present invention for intravenous administration.

[0016] In a further embodiment, the present invention relates to the use of the pharmaceutical composition of the present invention for the treatment of cancer.

[0017] In a further embodiment, the present invention is a method for treating cancer in a subject, which requires The subject is a method comprising administering the pharmaceutical composition of the present invention for a sufficient amount of time to treat cancer. To relate to.

[0018] Further aspects of the present invention relate to unit dose formulations including the following: a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 It contains approximately 5 μg to 50 mg of bispecific antibodies. b. Acetate buffer and sorbitol in a ratio between 1:5 and 1:10, where the unit dose The osmotic pressure of the dosage form is approximately 210 to 250, and the pH is approximately 5.5.

[0019] In yet another embodiment, the present invention relates to a unit dose dosage form including: a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 It contains approximately 5 μg to 50 mg of bispecific antibodies. b. Acetate buffer solution at a concentration of approximately 30 mM, c. Sorbitol at a concentration of approximately 150 mM, Here, the pH is approximately 5.5.

[0020] The above and other aspects and embodiments will be described in detail below. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows a solubility screening of Duobody-CD3×CD20 in different formulations. Duobody-CD3×CD20 was formulated in the buffer shown and then continuously concentrated using a centrifugal concentrator at time-spin intervals. The concentration of each formulation was measured after spin intervals of 20, 50, 60, and 90 minutes.

[0022] [Figure 2] Figure 2 shows the viscosity of Duobody-CD3×CD20 (120–150 mg / mL) in the illustrated formulations. The viscosity (cP) of concentrated Duobody-CD3×CD20 samples (120–150 mg / mL) was measured at various shear rates for the illustrated formulations using a Wells-Brookfield Cone / Plate Rheometer.

[0023] [Figure 3A] Figure 3A shows the mean cytokine concentrations in the blood of cynomolgus monkeys after a single intravenous (0.1 or 1 mg / kg) or subcutaneous (0.1 or 1 mg / kg) administration of DuoBody-CD3×CD20. [Figure 3B] Figure 3B shows the mean cytokine concentrations in the blood of cynomolgus monkeys after a single intravenous (0.1 or 1 mg / kg) or subcutaneous (0.1 or 1 mg / kg) administration of DuoBody-CD3×CD20.

[0024] [Figure 4] Figure 4 shows the effects of four repeated intravenous administrations of DuoBody-CD3×CD20 on B cells in the peripheral blood of cynomolgus monkeys. (A) Mean B cell count (CD4-CD8-CD16-CD19+ cells) in the peripheral blood of cynomolgus monkeys over time, for each administration group, after four weekly intravenous administrations of DuoBody-CD3×CD20 (0.01, 0.1, or 1 mg / kg). (B) Percentage (%) of the mean B cell count for each administration group relative to the B cell count before administration. B cell count is expressed as absolute cell count (cells / μL).

[0025] [Figure 5] Figure 5 shows the effect of a single subcutaneous administration of DuoBody-CD3×CD20 on B cells in the peripheral blood of cynomolgus monkeys. (A) Mean B cell count in the peripheral blood of cynomolgus monkeys over time for each administration group after a single subcutaneous administration of DuoBody-CD3×CD20 (0.01, 0.1, 1, 10, or 20 mg / kg). (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. B cell count is expressed as absolute cell count (cells / μL).

[0026] [Figure 6] Figure 6 shows the effect of intravenous infusion of the target dose of DuoBody-CD3×CD20 on B cells in the peripheral blood of cynomolgus monkeys after initial dose administration. The mean B cell count over time (CD4-CD8-CD16-CD19+ cells) in the peripheral blood of cynomolgus monkeys administered an initial dose (0.01 mg / kg) followed by an intravenous infusion of one target dose (1 mg / kg; IV) one day later. B cell counts are expressed as absolute cell counts (cells / μL).

[0027] [Figure 7]Figure 7 shows the effects of four repeated intravenous administrations of DuoBody-CD3×CD20 on B cells in the lymph nodes of cynomolgus monkeys. (A) Mean B cell frequency (proportion of CD4-CD8-CD16-CD19+ cells relative to the total lymphocyte population) in the lymph nodes of cynomolgus monkeys over time, after four weekly intravenous administrations of DuoBody-CD3×CD20 (0.01, 0.1, or 1 mg / kg) for each administration group. (B) Proportion of mean B cell frequency for each administration group compared to the B cell frequency before administration.

[0028] [Figure 8] Figure 8 shows the effect of a single subcutaneous administration of DuoBody-CD3×CD20 on B cells in the lymph nodes of cynomolgus monkeys. (A) Mean B cell frequency (proportion of CD4-CD8-CD16-CD19+ cells relative to the total lymphocyte population) in the lymph nodes of cynomolgus monkeys over time after a single subcutaneous administration of DuoBody-CD3×CD20 (0.01, 0.1, 1, 10, or 20 mg / kg) for each administration group. (B) Proportion of mean B cell frequency for each administration group relative to the B cell frequency before administration.

[0029] [Figure 9] Figure 9 shows the effect of intravenous infusion of the target dose of DuoBody-CD3×CD20 on B cells in the peripheral blood of cynomolgus monkeys after initial dose administration. The mean B cell frequency over time in the peripheral blood of cynomolgus monkeys (percentage of CD4-CD8-CD16-CD19+ cells relative to the total lymphocyte population) was measured after initial dose administration (0.01 mg / kg) followed by intravenous infusion of one target dose (1 mg / kg; IV) one day later.

[0030] [Figure 10]Figure 10 shows the depletion and recovery of B cells in the spleen and lymph nodes of cynomolgus monkeys after IV treatment with DuoBody-CD3×CD20. Top figure: Cynomolgus monkey 1 was treated with DuoBody-CD3×CD20 as an initial dose of 0.01 mg / kg on day 1 and as a target dose of 1 mg / kg on day 2. The animal was euthanized on day 29 according to the schedule. Peripheral blood B cell counts had not recovered at necropsy. Bottom figure: Cynomolgus monkey 2 was treated with DuoBody-Cd3xCD20 at 1 mg / kg weekly for four times. The animal was euthanized on day 148 after B cell recovery was observed in the peripheral blood. Frozen sections of lymph nodes and spleen were stained with CD19-specific antibody to detect B cells (brown staining). Cell nuclei were detected using hematoxylin (blue staining).

[0031] [Figure 11] Figure 11 shows the effects of five repeated intravenous administrations of DuoBody-CD3×CD20 on B cells in the peripheral blood of male cynomolgus monkeys. (A) Mean B cell count over time (CD45+CD4-CD8-CD16-CD19+ cells) in the peripheral blood of male cynomolgus monkeys administered intravenously five times weekly using physiological saline or 0.01, 0.1, or 1 mg / kg of DuoBody-CD3×CD20 for each administration group. (B) Percentage of mean B cell count for each administration group relative to the B cell count before administration. B cell count is expressed as a percentage of gaited lymphocytes.

[0032] [Figure 12] Figure 12 shows the effects of five repeated intravenous administrations of DuoBody-CD3×CD20 on B cells in the peripheral blood of female cynomolgus monkeys. (A) Mean B cell count over time (CD45+CD4-CD8-CD16-CD19+ cells) in the peripheral blood of female cynomolgus monkeys administered intravenously five times weekly using physiological saline or 0.01, 0.1, or 1 mg / kg of DuoBody-CD3×CD20 for each administration group. (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. The B cell count is expressed as a percentage of gaited lymphocytes.

[0033] [Figure 13] Figure 13 shows the effect of a single intravenous infusion of DuoBody-CD3×CD20 on B cells in the peripheral blood of male cynomolgus monkeys. (A) Mean B cell count over time for each administration group in the peripheral blood of male cynomolgus monkeys after a single intravenous infusion using physiological saline or 0.1 or 1 mg / kg of DuoBody-CD3×CD20. (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. The B cell count is expressed as a percentage of gaited lymphocytes.

[0034] [Figure 14] Figure 14 shows the effect of a single intravenous infusion of DuoBody-CD3×CD20 on B cells in the peripheral blood of female cynomolgus monkeys. (A) Mean B cell count over time for each administration group in the peripheral blood of female cynomolgus monkeys after a single intravenous infusion using physiological saline or 0.1 or 1 mg / kg of DuoBody-CD3×CD20. (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. The B cell count is expressed as a percentage of gaited lymphocytes.

[0035] [Figure 15] Figure 15 shows the effects of SC administration of DuoBody-CD3×CD20 on B cells in the peripheral blood of male cynomolgus monkeys. (A) Mean B cell count over time for each administration group in the peripheral blood of male cynomolgus monkeys administered SC using physiological saline or 0.1, 1, or 10 mg / kg of DuoBody-CD3×CD20. (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. The B cell count is expressed as a percentage of gaited lymphocytes.

[0036] [Figure 16]Figure 16 shows the effects of SC administration of DuoBody-CD3×CD20 on B cells in the peripheral blood of female cynomolgus monkeys. (A) Mean B cell count over time for each administration group in the peripheral blood of female cynomolgus monkeys administered SC using physiological saline or 0.1, 1, or 10 mg / kg of DuoBody-CD3×CD20. (B) Percentage of the mean B cell count for each administration group relative to the B cell count before administration. The B cell count is expressed as a percentage of gaited lymphocytes.

[0037] [Figure 17AB] Figure 17 (A) Individual plasma concentration profiles in cynomolgus monkeys after intravenous administration of DuoBody-CD3×CD20. (B) Individual plasma concentration profiles in cynomolgus monkeys after SC administration of DuoBody-CD3×CD20. Plasma concentration profiles of DuoBody-CD3×CD20 were measured after a single SC injection of DuoBody-CD3×CD20 at doses of 0.01, 0.1, 1, 10, or 20 mg / kg. [Figure 17C] Figure 17(C) Group-mean plasma concentration profiles after intravenous or subcutaneous injection in cynomolgus monkeys. [Modes for carrying out the invention]

[0038] definition The term "immunoglobulin" refers to two sets of polypeptide chains, one set of light (L) low molecular weight chains and one set These four chains, all consisting of heavy (H) chains, are interconnected by disulfide bonds. It refers to a class of structurally related glycoproteins. The structure of immunoglobulins is well-defined. It is attached. For example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Ra See ven Press, NY (1989). Simply put, each heavy chain is typically a heavy chain variable region (specified herein). So VH or V H(abbreviated as CH or C) and heavy chain constant region (referred to herein as CH or C) H (Abbreviated as) It is composed of three domains: CH1, CH2, and CH3. The region is the area between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bond in the hinge region is part of the interaction between the two heavy chains in the IgG molecule. Each light chain typically has a light chain variable region (VL or V in this specification). L (and omitted) and the light chain steady region ( Here, CL or C L It consists of (and abbreviated). The light chain constant region is usually composed of one domain CL. The VH and VL regions are regions of hypervariability (structurally defined loop arrays and / or It is further subdivided into a highly variable region (which may be highly variable in form) and a complementary determination region (CDR). It is also called a framework area (FR), and there are scattered areas that are more preserved than the framework area. VH and VL typically consist of three CDRs and four FRs, arranged in the direction from the amino terminus to the carboxyl terminus. They are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J.) Mol. Biol. 196 (See also 901-917 (1987)). Unless otherwise specified or inconsistent with the context. To the extent that it is used herein, CDR sequences are identified according to the IMGT rules (Brochet X., Nucl Acid). s Res. 2008;36: W503-508 and Lefranc MP., Nucleic Acids Research 1999;27:209-212 ; See also the internet http address http: / / www.imgt.org / ). Unless otherwise specified or written Insofar as it does not contradict the pulse, when referring to the amino acid position in the steady region in this invention, the EU number Similar to the ring (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., sequences of Proteins of Immunological Interest, Fifth Edition. 19 (NIH Publication No. 91-3242). For example, Sequence ID No. 15 in this specification shows the IgG monohelic constant region. This shows amino acid positions 118-447 according to the EU numbering system for the region.

[0039] As used herein, the term "amino acid corresponding to the position..." refers to the amino acid in the human IgG monochain. This refers to the amino acid position number. The corresponding amino acid position in other immunoglobulins is indicated by It can be found by alignment with IgG1. Therefore, amino acids of a different sequence A sequence of amino acids or segments that "corresponds" to an acid or segment is a standard sequence. Alignment programs, such as ALIGN, ClustalW, or similar, are usually the default settings. It is a substance that is used to align with other amino acids or segments, and At least 50%, at least 80%, at least 90%, or at least IgG single chain It has 95% identity. Align the sequence or segments within the sequence, thereby generating A method for determining the corresponding position within a sequence for an amino acid position related to a given amino acid is in the field of the present art. It is considered to be knowledge.

[0040] In the context of this invention, the term "antibody" (Ab) means immunoglobulin molecule, immunoglobulin component This refers to the child fragment, or any of its derivatives, under typical physiological conditions. For a considerable period of time, for example, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, less Each takes approximately 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, and about 24 hours. In addition, approximately 48 hours or more, approximately 3, 4, 5, 6, 7 days or longer, or other related matters. A functionally defined period (for example, a physiological response associated with an antibody binding to an antigen) Sufficient time for induction, promotion, enhancement, and / or regulation, and / or for the antibody to act as an effector - Having a half-life (sufficient time to recover activity, etc.) and the ability to specifically bind to an antigen. It has. The variable regions of the heavy and light chains of immunoglobulin molecules are binding zones that interact with antigens. Includes the main component. The term “antibody-binding region” as used herein refers to the region that interacts with the antigen. This refers to a region that includes both the VH and VL regions. As used herein, the term antibody Not only single-specific antibodies, but also multiple antibodies containing different antigens, for example, two or more, for example, three or more. This also includes multispecific antibodies that include a combined region. The constant region of an antibody (Abs) is a region that is present in various parts of the immune system. Cells (effector cells, etc.) and components of the complement system, such as C1q, that is, complement activation The binding of immunoglobulins to host tissues or factors, including the first components of the classical pathway. It may be involved. As stated above, the term antibody in this specification is used unless otherwise specified. Unless otherwise clearly contradictory to the context, it is an antigen-binding fragment, i.e., specific to the antigen. It contains an antibody fragment that retains the ability to bind to the antigen. The antigen-binding function of the antibody is the full-length antibody fragment. It has been shown that this may be carried out by antigens included in the term "antibody". Examples of binding fragments include: (i) Fab' or Fab fragment, VL, VH, CL A monovalent fragment consisting of the CH1 domain, or a monovalent antibody as described in WO2007059782 (Genmab); (ii)F(ab')2 fragment, 2 connected by a disulfide bridge in the hinge region (iii) A divalent fragment containing two Fab fragments; (iii) An Fd fragment essentially composed of VH and CH1 domains; (iv) Fv fragment essentially consisting of the VL and VH domains of a single arm of the antibody; (v) VH domain Essentially, it becomes (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90)dAb fragment (Ward et al., Nature 341 , 544-546 (1989)), also called domain antibody; (vi) Pipette antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111 -24); and (vii) isolated complementarity-determining region (CDR). Furthermore, the two domains VL of the Fv fragment. Although VH is encoded by separate genes, recombinant DNA can be used to synthesize them into phosphorus A single protein bound by Kerr, with the VL and VH regions pairing to form a monovalent molecule. It may be possible to make it possible to create it as a chain (single-chain antibody or single-chain Fv (scFv) and For example, Bird et al., Science 242 , 423-426 (1988) and Huston et al. al., PNAS USA 85 See 5879-5883 (1988). Such single-chain antibodies are not otherwise described. Unless otherwise specified or clearly indicated by the context, the term "antibody" includes... Fragments like these are generally included in the meaning of antibody, but although they are collective, each is independent. Therefore, this is a unique feature of the present invention, exhibiting different biological properties and usefulness. These and other useful antibody fragments in the pulse, as well as the bispecificity of such fragments Matt will be discussed further herein. Unless otherwise specified, the term antibody is known Polychromic compounds provided by technologies such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Monoclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, chimeric antibodies, humanized antibodies It may also include antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to the body and antigens. Please understand that the generated antibodies can be of any isotype. When used, the term "isotype" refers to the type encoded by the heavy chain constant region gene. The class of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or I This refers to (gM). When a specific isotype, such as IgG1, is described herein, this usage The term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but rather refers to the antibody within that sequence. Used to indicate that the isotype is closer to, for example, IgG1 than to other isotypes. Therefore, for example, the IgG1 antibody of the present invention includes variations in the constant region, which are naturally occurring. It may also be a sequence variant of the IgG1 antibody present.

[0041] A monoclonal antibody composition refers to a preparation of an antibody molecule with a single-molecule composition. The antibody composition exhibits a single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody derived from a human germline immunoglobulin sequence. This refers to a single antibody exhibiting binding specificity, possessing both a variable region and a constant region. (Human monoclonal antibody) The body has a genome containing human heavy chain transgenes and light chain transgenes. Obtained from transgenic or transchromosomal non-human animals such as transgenic mice. It can be produced by hybridomas created by fusing B cells with immortalized cells.

[0042] In the context of this invention, the terms “bispecific antibody,” “bs,” or “bsAb” are different. This refers to an antibody that has two different antigen-binding regions, defined by its antibody sequence. Antibodies can be in any form.

[0043] As used herein, the terms “half-arm,” “Fab-arm,” and “arm” mean one This refers to a heavy chain-light chain pair.

[0044] The bispecific antibody consists of a half-subunit antibody "derived" from the first antibody and a half-subunit antibody "derived" from the second antibody. When it is stated that it contains molecular antibodies, the term "derived from" means that the bispecific antibodies are the aforementioned Using the halves obtained from antibodies 1 and 2, a double halves can be formed by any known method. This indicates that it is a bispecific antibody produced by recombination with an isogender antibody. In this context, The term "recombinant" is not intended to be limited to any specific method of recombination, but rather... Therefore, for example, by half-arm swapping (also known as "controlled Fab arm swapping") Recombination, as well as recombination at the nucleic acid level, and / or the formation of two halves in the same cell. To generate bispecific antibodies as described below in this specification, including recombination by co-expression. This includes all methods.

[0045] In the context of this invention, the term "monovalent antibody" refers to an antibody molecule binding to a single molecule of an antigen. This means that the antigen or cell cannot be transmitted.

[0046] The term "full length" is used in the context of antibodies, meaning that the antibody is not a fragment but the entire length of the natural body. For that isotype, all of the domains of a particular isotype that are usually found (for example) In the case of IgG1 antibodies, this indicates the presence of the VH, CH1, CH2, CH3, hinge, VL, and CL domains. .

[0047] Where used herein, unless otherwise inconsistent with the context, the term "Fc region" refers to immunoglobulins. This refers to the antibody region consisting of the Fc sequences of the two heavy chains of robulin, where the Fc sequences are at least This also includes the hinge region, the CH2 domain, and the CH3 domain.

[0048] As used herein, the term "heterodiode between the first CH3 region and the second CH3 region" Mer interaction refers to the first CH3 region in a heterodimer protein of the first CH3 and the second CH3. This refers to the interaction between the first CH3 region and the second CH3 region.

[0049] As used herein, the term "homodimeric between the first CH3 region and the second CH3 region" The interaction between the first CH3 region and the second CH3 region in the homodimer protein of the first CH3 and the second CH3 region is Interaction with the second CH3 region, and the second CH3 / second CH3 homodimer protein This refers to the interaction between one second CH3 region and another second CH3 region.

[0050] As used herein, the term " "Binding" typically refers to, for example, Octe when an antibody is used as a ligand and an antigen is used as an analyte t HTX instrument as determined by BIL (BioLayer Interferometry) technology, for example about 10 -6 M or less, for example 10 -7 M or less, for example about 10 -8 M or less, for example about 10 -9 M or less, about 10 -10 M or les s, or about 10 -11M or even lower K D d binding having an affinity corresponding to D, wherein the antibody binds to a non-specific antigen other than a given antigen or a closely related antigen (e.g., BSA, casei n) has a K D D that is at least 10-fold lower, for example at least 100-fold lower, for exam ple at least 1,000-fold lower, for example at least 10,000-fold lower, for example at least 100,000-fold lower affinity when binding to a given antigen. The K D D of the antibody is such that the K D D of the binding depend s on K D D and when the amount of K D D for binding to the antigen is lower, the K D D for binding to a non-specific antigen i s lower by at least 10,000-fold (i.e., the antibody is highly specific) . As used herein, the term "K D D" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction . As used herein, the terms affinity and K D D are inversely correlated. That is, higher affinity is intended to mean lower K D D, and lower affinity is intended to mean higher K D D .

[0051] In preferred embodiments, the antibody of the present invention is isolated. "Isolated antibodies" are antibodies that substantially do not contain other antibodies with different antigen specificities. This is the intended meaning. In a preferred embodiment, isolated which specifically binds to CD20 and CD3 These bispecific antibodies are further essentially monospecific antibodies that specifically bind to either CD20 or CD3. It is not included in the target.

[0052] The term "CD3" as used herein refers to a part of the T cell coreceptor protein complex. CD3 refers to the human differentiated 3-cluster protein, which is composed of four different chains. It is also observed in species, and therefore, the term "CD3" is used unless otherwise specified in the context, and refers to human CD3. It is not limited to this. In mammals, this complex is the CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No. P09693, or Cynomolgus macaque CD3γ (UniProtKB / Swiss-Prot No. Q95LI7) CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No. P04234 or cynomolgus monkey C) D3δKB / Swiss-Prot No. Q95LI8), two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No. P07766; Cryptomolgus macaque CD3ε-UniProt No. Q95LI5; or Rhesus macaque CD3 ε UniProtKB / Swiss-Prot No. G7NCB9), and CD3ζ (zeta)-chain (human CD3ζ UniProt KB / Swiss-Prot No. P20963, Crab-eating macaque CD3ζ UniProt KB / Swiss-Prot No. Q09TK0) These chains bind to molecules known as T cell receptors (TCRs) and are activated in T lymphocytes. It generates a chemical signal. TCR and CD3 molecules work together to form the TCR complex.

[0053] "CD3 antibodies" or "anti-CD3 antibodies" are specific to the antigen CD3, particularly human CD3ε (epsilon). It is an antibody that binds to [something].

[0054] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No. P11836). This refers to a gene or cDNA that is spontaneously expressed by cells, including tumor cells, or that contains the CD20 gene or cDNA. Any variant, isoform, and of CD20 expressed on transfected cells Includes species homologs. Species homologs include rhesus macaque CD20 (macaca mulatta; UniProtKB / Sw iss-Prot No. H9YXP1) and cynomolgus macaque CD20 (macaca fascicularis; UniProtKB No. G7P Q03) is included.

[0055] "CD20 antibody" or "anti-CD20 antibody" is an antibody that specifically binds to the antigen CD20, particularly human CD20. It is the body.

[0056] "CD3 x CD20 antibody", "anti-CD3 x CD20 antibody", "CD20 x CD3 antibody", or "anti-CD20 x CD3 antibody" This antibody is a bispecific antibody containing two different antigen-binding regions, one of which is the antigen CD20. It binds specifically to one of the molecules, and one of them specifically binds to CD3.

[0057] As used herein, the term "DuoBody-CD3×CD20" refers to IgG1 bispecific CD3×CD20. This refers to the antibody, where the CD3-binding Fab arm is VH as defined in SEQ ID NOs. 6 and 7, respectively. and VL sequence, constant light chain sequence as defined in SEQ ID NO: 22, and as defined in SEQ ID NO: 19 The CD20-binding Fab arms include stationary heavy chain sequences (FEALs) such as sequence numbers 13 and 14, respectively. The VH and VL sequences, the constant light chain sequence as defined in SEQ ID NO: 23, and the constant light chain sequence as defined in SEQ ID NO: 20 It contains a constant heavy chain sequence (FEAR) that is considered to be correct. This bispecific antibody is described in WO2016 / 110576. It can be prepared as described.

[0058] In a preferred embodiment, the bispecific antibody of the present invention is isolated. The term "isolated bispecific antibody" used in this book refers to an antibody that possesses different antigen specificity from another antibody. This refers to bispecific antibodies that do not qualitatively contain (for example, antibodies that specifically bind to CD20 and CD3). The isolated bispecific antibodies are monospecific antibodies that specifically bind to CD20 or CD3. (Essentially not included).

[0059] The present invention also provides functional variants of the VL region, VH region, or one or more CDRs of the antibodies in the examples. The report provides antibodies containing functional variants of VL, VH, or CDR used in the context of antibodies. The antibody still has low affinity and / or specificity / selectivity compared to the "reference" or "parent" antibody. Even if not, an effective percentage (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) (The above) can be maintained, and in some cases, such antibodies are higher in parental antibody levels than parental antibodies. This may be related to compatibility, selectivity, and / or specificity.

[0060] Such functional variants typically retain significant sequence identity with respect to the parent antibody. The percentage of identity between the two sequences is derived for optimal alignment of the two sequences. Considering the number of gaps that need to be filled and the length of each gap, the sequences to be aligned A function of the number of identical positions in (i.e., % homology = number of identical positions / total number of positions × 100) The percentage of identity between two nucleotides or amino acid sequences is, for example, PAM12. Using a 0-weight residue table, 12 gap length penalties, and 4 gap penalties, AL E. Meyers and W. Miller, Comput., incorporated into the IGN program (version 2.0). It can be determined using the algorithm in Appl. Biosci 4, 11-17 (1988). Furthermore, two The percentage of amino acid sequence identity is given by Needleman and Wunsch, J. Mol. Biol. 48, 444- This can be determined using the algorithm in 453 (1970).

[0061] Exemplary variants include VH and / or VL and / or parent antibody sequences, primarily through conservative substitutions. This includes elements different from the CDR region, for example, substitutions 9, 8, 7, 6, 5, 4, and 3 in the mutant. Ten of these, such as 2 or 1, are conservative amino acid residue substitutions.

[0062] In the context of the present invention, a conservative substitution is in the class of amino acids reflected in the following table. It may also be defined by substitution:

[0063] [ka] In the context of this invention, unless otherwise specified, the following notations refer to the following mutations. For use; i) an amino acid substitution at a certain position is denoted as, for example, K409R, 40 ii) A substitution of lysine to arginine at position 9; A specific three-letter or one-letter code is used, which includes the codes Xaa and X, indicating the amino acid residue. Therefore, the substitution of lysine to arginine at position 409 is shown as K409R, and at position 409 A substitution of lysine to any amino acid residue is denoted as K409X. A deletion of lysine at position 409 is In case K409 * This is shown.

[0064] In the context of the present invention, "competition" (or "blocking" or "cross-blocking") "G" is a term used when a particular molecule binds to a specific binding partner in the presence of another molecule that binds to that partner. This refers to a significant reduction in the tendency for toner to bind. The binding to CD20 by two or more anti-CD20 antibodies... Competition can be determined by any suitable technology.

[0065] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically derived from surface groups of molecules, such as amino acids or sugar side chains. Conformation And non-conformation epitopes lose their binding to the former in the presence of a denaturing solvent, but later It is distinguished in that the connection to the individual is not lost. Epitopes are the ami that are directly involved in the connection. anoic acid residues and other amino acid residues not directly involved in binding (e.g., specific antigen-binding peptides) It may also contain amino acid residues that are effectively blocked or covered by (in other words) (These amino acid residues are within the footprint of the specific antigen-binding peptide.)

[0066] As used herein, the term "chimeric antibody" refers to an antibody whose variable region originates from a non-human species (for example, a reticular mitochondrion). This refers to antibodies that originate from teeth, with their constant region derived from different species such as humans. Chimeric monoclonal antibodies are being developed to reduce antibody immunogenicity. The term "variable region" or "variable domain" used in the context of antibodies refers to the immunoglobulin This refers to a region that includes both the heavy chain and light chain CDR and framework regions. Chimeric antibodies are Sam brook et al., 1989, Molecular Cloning: A laboratory manual, New York: Cold Sprin Use standard DNA techniques as described in Harbor Laboratory Press, Ch. 15. Chimeric antibodies may be produced by genetically or enzymatically modified recombinant antibodies. It may be possible. Generating chimeric antibodies is within the scope of the knowledge of those skilled in the art, and therefore, The generation of chimeric antibodies according to the present invention may be carried out by methods other than those described herein. good.

[0067] As used herein, the term "humanized antibody" refers to a human antibody constant domain, as well as a human variable domain. Includes a non-human variable domain modified to have a high level of sequence homology to the domain. This refers to genetically modified non-human antibodies. These work together to form antigen-binding sites. Six non-human antibody complementarity-determining regions (CDRs) are used in homologous human acceptor framework regions. This can be achieved by grafting onto the FR (see WO92 / 22653 and EP0629240). To completely reconstitute the antibody binding affinity and specificity, the parent antibody (i.e., non-human antibody) ) Substitution of framework residues from the human framework region ( Post-mutation ) is necessary This can happen. Structural homology modeling is an important framework area for understanding antibody binding properties. It can be helpful in identifying amino acid residues within a non-human CDR. Therefore, humanized antibodies can help in identifying non-human CDRs. The sequence mainly contains one or more amino acid return mutations into non-human amino acid sequences, sometimes including them. It may include a framework region and a fully human steady-state region. In some cases, it is not necessarily required to include a fully human steady-state region. By applying additional amino acid modifications that are not transient mutations, preferred affinity and biochemical properties are achieved. Humanized antibodies with desirable properties may be obtained.

[0068] "Human antibodies" are derived from the variable and constant regions of human germline immunoglobulin sequences. This refers to the antibodies that a person possesses. Human antibodies are encoded by human germline immunoglobulin sequences. Absent amino acid residues (for example, by random or site-directed mutagenesis in vitro) This may include mutations introduced by somatic mutations in vivo. Furthermore, the term "human antibody" as used herein refers to antibodies derived from the germline of another mammalian species, such as mice. The resulting CDR sequence is intended to contain an antibody grafted onto a human framework sequence. This is not the case. The human monoclonal antibody of the present invention is not produced by conventional monoclonal antibody methods (for example, the standard somatic cell hybridization technique of Kohler and Milstein, Nature It can be produced by various techniques, including 256: 495 (1975). Somatic hybridises While the manual procedure is preferred, in principle, other techniques for producing monoclonal antibodies (e.g.) For example, viral or oncogenic transformation of B lymphocytes, or live human antibody genes Phage display technology using rallies can also be used. Human monoclonal antibodies The mouse is the appropriate animal strain for preparing secreting hybridomas. Hybridoma production is a very well-established procedure. Immune-treated splenocytes for fusion. Immunization protocols and techniques for the isolation of are publicly known in the art. Fusion partners (e.g.) For example, mouse myeloma cells and the fusion procedure are also known. Therefore, human monoclonal Nal antibodies, for example, possess a part of the human immune system rather than a mouse or rat immune system. It can be generated using genic or transchromosome mice or rats. Therefore, in one embodiment, a human antibody is used instead of an animal immunoglobulin sequence. Transgenic animals having germline immunoglobulin sequences (e.g., mice or It is obtained from rats. In such embodiments, the antibody is introduced into the animal human reproductive system. Although derived from germline immunoglobulin sequences, the final antibody sequence is derived from the above-mentioned human germline immunoglobulin sequence. The phosphorus sequence is further modified by somatic hypermutation and affinity maturation mediated by endogenous animal antibodies. This is the result of the study (see, for example, Mendez et al. 1997 Nat Genet. 15(2):146-56). The term "reduction conditions" or "reduction environment" refers to the hinge region of the substrate, or more specifically, the antibody. This refers to conditions or environments in which the cysteine ​​residue is more likely to be reduced than oxidized.

[0069] The term “recombinant host cell” (or simply “host cell”) is used herein, Cells into which an expression vector (for example, an expression vector encoding the antibody of the present invention) has been introduced This is the intention. Recombinant host cells include, for example, transfectomas (e.g., CHO (CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells), and lymphocyte cells Cells are mentioned.

[0070] The term "treatment" refers to alleviating, improving, stopping, or eradicating (curing) symptoms or disease conditions. This refers to administering an effective amount of the therapeutically active antibody of the present invention for the purpose of [details omitted].

[0071] The term "effective dose" or "therapeutic effective dose" refers to the dose required to achieve the desired therapeutic outcome. This refers to the effective amount in terms of quantity and duration. The therapeutically effective dose of an antibody depends on the individual's disease state, age, sex, and This varies depending on factors such as body weight and the ability of the antibody to induce the desired response in the individual. To obtain a therapeutically effective amount, also, any toxic or harmful effects of the antibody or antibody portion, are cured. The amount is below the level of therapeutically beneficial effects.

[0072] As used herein, the term "buffering agent" refers to a pharmaceutically acceptable buffering agent. The term "accelerating agent" includes, but is not limited to, any agent that maintains the pH value of a solution within an acceptable range. However, acetic acid, histidine, TRIS(registered trademark) (tris(hydroxymethyl)aminomethane) , including citrate, succinate, glycolate, etc. Generally used herein The "buffering agent" has a pKa and buffering capacity suitable for a pH range of approximately 5 to approximately 6, preferably approximately 5.5.

[0073] The "surfactants" used herein prevent the adsorption and / or aggregation of drugs onto surfaces. It is a compound typically used in pharmaceutical formulations for this purpose. Furthermore, surfactants are, To reduce the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example... For example, exemplary surfactants are found in very low concentrations (e.g., 5% w / w or less, e.g., 3% w / w or less). For example, if present at a concentration of 1% w / w or less, the surface tension can be significantly reduced at the interface. Activators are amphiphilic (i.e., typically composed of both hydrophilic and hydrophobic or lipophilic groups). (and therefore, micelles or similar self-assembling structures are formed in aqueous solution.) This means that it is possible to do so. As a surfactant known for pharmaceutical use, glyceryl monooleate Roll, benzethonium chloride, sodium doxate, phospholipid, polyethylene alkyl Ether, sodium lauryl sulfate and tricaprylin (anionic surfactant); chloride Benzalkonium, citrimide, cetylpyridinium chloride, and phospholipids (cationic interface) Activators; as well as α-tocopherol, glycerol monooleate, myristyl alcohol Electrolytes, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene Castor oil derivative, polyoxyethylene sorbic acid ester, polyoxyethylene Stearate, polyoxylhydroxystearate, polyoxylglyceride, polys Rubate, propylene glycol dilaurate, propylene glycol monolaurate, Sorbitan ester, sucrose palmitate, sucrose stearate, tricaprylic acid Examples include nasal and TPGS (nonionic and zwitterionic surfactants).

[0074] The “diluents” referred to herein are pharmaceutically acceptable (for administration to humans). (Safe and non-toxic) and useful for preparing dilutions of pharmaceutical compositions. Preferably, such dilutions of the composition of the present invention dilute only the antibody concentration, but also allow for a slower dilution. The agitator and stabilizer are not diluted. Therefore, in a preferred embodiment, the diluent is the pharmaceutical agent of the present invention. Contains buffers and stabilizers at the same concentrations as those present in the composition. Further exemplary diluents. For example, sterile water, bacteriostatic water for injection (BWFI), preferably a pH buffer such as an acetate buffer, sterile water Examples include saline solution, Ringer's solution, or dextrose solution. In preferred embodiments, odor The diluent contains or is essentially composed of an acetate buffer and sorbitol.

[0075] The terms "pharmaceutical composition" and "pharmaceutical preparation" are used interchangeably in this specification.

[0076] Specific Embodiments of the Invention In its main embodiments, the present invention provides pharmaceutical compositions comprising: a. Bispecific antibodies that bind to human CD3 and human CD20 at concentrations of approximately 50-120 mg / mL. b. Approximately 20 to 40 mM acetic acid, c. Approximately 140-160 mM sorbitol Here, the pH of the composition is approximately 5 to 6, and the bispecific antibody contains human CD3 with the following CDR sequence. The first binding region to be joined: VH-CDR1: Sequence ID 1, VH-CDR2: Sequence ID 2, VH-CDR3: Sequence ID number No. 3, VL-CDR1:Sequence ID 4, VL-CDR2:GTN, and VL-CDR3:Sequence ID 5, and the following Second binding region that binds to human CD20 containing the CDR sequence: VH-CDR1: Sequence ID 8, VH-CDR2: Sequence ID Number 9, VH-CDR3: Sequence ID 10, VL-CDR1: Sequence ID 11, VL-CDR2: DAS, and VL-CDR3: Includes sequence number 12.

[0077] In another aspect, the present invention provides a pharmaceutical composition comprising: a. Bispecific antibodies that bind to human CD3 and human CD20 at concentrations of approximately 50-120 mg / mL. b. Approximately 20 to 40 mM acetic acid, c. Approximately 140-160 mM sorbitol Here, the pH of the composition is approximately 5 to 6, and the bispecific antibody contains the following human CD3 CDR sequence. First binding region that binds to: VH-CDR1: Sequence ID 1, VH-CDR2: Sequence ID 2, VH-CDR3: Sequence ID Number 3, VL-CDR1: Sequence ID 4, VL-CDR2: GTN, and VL-CDR3: Sequence ID 5, and the following Second binding region that binds to human CD20 containing the CDR sequence: VH-CDR1: SEQ ID NO: 8, VH-CDR2: Column number 9, VH-CDR3: Sequence ID 10, VL-CDR1: Sequence ID 11, VL-CDR2: DAS, and VL-CDR3 Includes Sequence ID No. 12. This provides a simple yet stable pharmaceutical composition. An advantage of the present invention is that the composition is suitable for both intravenous and subcutaneous administration. A further advantage is that the antibody concentration in the composition can range from a low concentration of about 4 μg / mL to about 120 mg / mL. Phase I dose grading in clinical trials where the concentration changes to high or even higher levels. To allow the same formulation to be used in further trials, and to allow the same composition to be used in later stages of clinical trials, Furthermore, the composition must be stable so that it can ultimately be used in commercially available formulations. In particular, the bispecific antibodies are stable across a wide range of antibody concentrations. Such a formulation can be used at temperatures ranging from 2°C to 25°C or even higher, and such a wide range of temperatures It is remarkable that the composition of the present invention is stable across antibody concentrations. If stored between these dates, at least 3 months, for example, at least 6 months, and even more, at least 9 months. It is stable for at least 12 months.

[0078] In one embodiment of the composition of the present invention, the first binding region of the bispecific antibody that binds to CD3 is sequence Includes VH and VL sequences numbered 6 and 7.

[0079] In a further embodiment of the composition of the present invention, the second binding region of a bispecific antibody that binds to CD20 This includes the VH and VL sequences of sequence numbers 13 and 14.

[0080] In a further embodiment of the pharmaceutical composition of the present invention, the bispecific antibody is DuoBody-CD3×CD20. ru.

[0081] In preferred embodiments of the composition of the present invention, the bispecific antibody is an IgG1 antibody. However, However, bispecific antibodies use either IgG2, IgG3, or IgG4 antibody isotypes or IgG1, IgG2 antibody isotypes instead. It may be a combination of IgG3 or IgG4. For example, the primary heavy chain is the IgG1 isotype. Furthermore, the double hemisphere may be an IgG4 isotype.

[0082] In further embodiments of the composition of the present invention, the bispecific antibody comprises Fc, which includes first and second heavy chains. The region includes the Fc region, and the Fc region exhibits effects compared to a bispecific antibody containing the wild-type IgG1 Fc region. The antibody has been modified to reduce its inhibitory function. Here, the bispecific antibody is a human Fc gamma receptor Due to its low ability to bind to the human complement component C1q, it is consequently less effective in antibody-dependent cell-mediated cells. cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). It has a low ability to induce Fc-mediated effector functions such as ). Therefore, effector function The bispecific antibody of the present invention, with reduced performance, activates only T cells in the presence of CD20-expressing cells. In other words, such bispecific antibodies are antibody-mediated FcR-dependent CD3 crosslinking and It does not induce subsequent target-independent T cell activation.

[0083] In another embodiment of the pharmaceutical composition of the present invention, a bispecific antibody is a C1q antibody against the antibody. Compared to a bispecific antibody containing the wild-type IgG1 Fc region, the combined antibody is at least 70% and at least 80%. Modify to be at least 90%, at least 95%, at least 97%, or 100% lower. It includes the Fc region, where binding of C1q is determined by ELISA.

[0084] The bispecific antibodies described herein include, for example, WO2011 / 131746 and Labrijn AF et al., (2 013) DuoBody® technology platform (Gen It can be produced according to mab A / S). DuoBody technology contains two heavy chains and two light chains. Half of the first monospecific antibody is converted into a second monospecific antibody containing two heavy chains and two light chains. It can be used to combine with half. In the resulting heterodimer, one is derived from the first antibody. The heavy chain and one light chain of the first antibody are paired with one heavy chain and one light chain from the second antibody. The second monospecific antibody recognizes different epitopes on different antigens such as CD3 and CD20. In this case, the resulting heterodimer is a bispecific antibody against CD3 and CD20.

[0085] DuoBody technology ensures that each monospecific antibody has a single point mutation in its CH3 domain. It must contain a heavy chain constant region. Point mutations can cause one of the monospecific antibodies to The relationship between CH3 domains in the resulting bispecific antibody is stronger than the relationship between CH3 domains in the object. This makes it possible for interactions to occur. A single point mutation in each monospecific antibody, even As stated in WO2011 / 131746, if you use the EU index for numbering... , in residues 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy chain constant region There is. Furthermore, a single point mutation is different in one monospecific antibody from other monospecific antibodies. It is located at different residues. For example, one monospecific antibody has mutation F405L (i.e., residue 40 It may also include a mutation from phenylalanine to leucine in 5, and other single-specific antibodies Even if the body contains the K409R mutation (i.e., a mutation from lysine to arginine at residue 409) Good. The heavy chain constant region of a single-specific antibody is IgG1, IgG2, IgG3, or IgG4 isotype ( For example, human IgG1 isotype, and dual generated by DuoBody technology. The specific antibody may retain Fc-mediated effector function, or the Fc region may be as described in this specification. It may be further modified to reduce the Fc-mediated effector function described in the book.

[0086] Therefore, in another embodiment of the pharmaceutical composition of the present invention, the bispecific antibodies are each small At the very least, it includes first and second heavy chains including the hinge region, CH2 and CH3 regions, where the first heavy chain In this context, the human IgG1 heavy chain consists of T366, L368, K370, D399, F405, Y407, and K409. At least one amino acid is substituted at the position corresponding to the position selected from the group. Furthermore, in the second heavy chain, T366, L368, K370, D399, F405, Y40 in the human IgG monoheavy chain Corresponds to a position selected from the group consisting of 7 and K409 (according to the EU numbering system). At least one amino acid at the position is substituted, and the first and second heavy chains It has not been replaced at the same position.

[0087] In yet another embodiment of the pharmaceutical composition of the present invention, (i) the first heavy chain is human IgG mono The amino acid at the position corresponding to F405 in the chain is substituted with L, and the second double chain In human IgG monohelic acid, the amino acid at the position corresponding to K409 is substituted with R. or, (ii) at the position in the first heavy chain corresponding to K409 in the human IgG monohelic acid The amino acid is R, and in the second heavy chain, the position corresponding to F405 in the human IgG single heavy chain. The amino acid in this position is L.

[0088] In one embodiment, the bispecific antibody of the pharmaceutical composition of the present invention is the first constant state of the bispecific antibody. Both the heavy chain and the second constant heavy chain are located at positions L234 and L235 (EU index number) in the human IgG1 heavy chain. Further substitution occurs at the position corresponding to the numbing, and L234 is replaced by F (L234F). Alternatively, L235 may be replaced with E (L235E). This allows for the Fc-mediated effect of the antibody. The function of the brain deteriorates.

[0089] In further embodiments, the bispecific antibody of the pharmaceutical composition has a first constant weight Both the chain and the second constant heavy chain are further positioned at the location corresponding to D265 in the human IgG monoheavy chain. It may also be possible to replace D265 with A (D265A).

[0090] In another embodiment, the bispecific antibody in the pharmaceutical composition comprises the first and second constant regions of the bispecific antibody comprising three substitutions L234F+L235E+D265A in both heavy chains.

[0091] In another embodiment, the bispecific antibody in the pharmaceutical composition comprises the first and second constant regions of the bispecific antibody comprising three substitutions L234F+L235E+D265A in both heavy chains, and the first constant heavy chain has F405L substitution and the second constant heavy chain further comprises K409R substitution, or vice versa. There fore, the first constant heavy chain has the substitutions L234F+L235E+D265A+F405L (referred to herein as the "FEAL" mutation also referred to as), and the second constant heavy chain has the substitutions L234F+L235E+D265A+K409R (herein also referred to as the "FEAR" mutation), or the first constant heavy chain has L234F+L235E+D26 5A+K409R substitutions and the second constant heavy chain comprises L234F+L235E+D265A+F405L substitutions. Preferr ed embodiment, the first and second constant heavy chains of the bispecific antibody are of an isotype and comprise L234F+L235E+D265A+F405L substitution and L234F+L235E+D265A+K409R substitution, respectivel y.

[0092] Therefore, in one embodiment of the present invention, the bispecific antibody of the pharmaceutical composition has the sequence of SEQ ID NO: 19 comprises a first heavy chain constant region and a second heavy chain constant region of SEQ ID NO: 20, or alternatively a first heavy chain constant region of SEQ ID NO: 20 comprises a heavy chain constant region and a second heavy chain constant region of SEQ ID NO: 19. In another embodiment, the bispecific antibody has at least 90% sequence identity to the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively, such as at least 91%, such as at least 92%, such as at least 93%, such as at least Also 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example It contains a heavy chain constant region with at least 98%, for example, at least 99%, sequence identity, but Contains the FEAR and FEAL amino acids described above.

[0093] The first and second light chains of the bispecific antibody in the composition preferably further extend the constant regions of the first and second light chains. Includes. The light chain steady region may be a lambda or kappa subtype. The preferred of the present invention In a more accurate embodiment, the steady-state region of the light chain of the CD3 coupling arm is of the lambda subtype, C The steady-state region of the light chain of the D20 binding arm is of the kappa subtype. In one embodiment, CD3 The light chain of the binding arm has the sequence of sequence number 24, and the light chain of the CD20 binding arm has the sequence of sequence number 25. It has an array.

[0094] The concentration of the bispecific antibody in the pharmaceutical composition may be approximately 1 mg / mL to approximately 200 mg / mL. In one embodiment of the present invention, the concentration of the bispecific antibody is about 50 to about 120 mg / mL. In the application method, the concentration of the bispecific antibody is approximately 50 to approximately 110 mg / mL. Other embodiments of the present invention In this case, the concentration of the bispecific antibody is approximately 50 to approximately 100 mg / mL. In another embodiment of the present invention, The concentration of the bispecific antibody is approximately 50 to approximately 90 mg / mL. In other embodiments of the present invention, bispecific The concentration of the heterozygous antibody is approximately 50 to approximately 80 mg / mL. In other embodiments of the present invention, bispecific antibodies The concentration in the body is approximately 50 to approximately 70 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody The concentration is approximately 60 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody is approximately 70 mg / mL. This is mL. In other embodiments of the present invention, the concentration of the bispecific antibody is approximately 80 mg / mL. In another embodiment of the invention, the concentration of the bispecific antibody is approximately 90 mg / mL. In the application method, the concentration of the bispecific antibody is approximately 100 mg / mL. In another embodiment of the present invention, The concentration of the bispecific antibody is approximately 110 mg / mL. In other embodiments of the present invention, the bispecific antibody The concentration in the body is approximately 120 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody is It is approximately 130 mg / mL. In another embodiment of the present invention, the concentration of the bispecific antibody is approximately 140 mg / mL. Yes. In another embodiment of the present invention, the concentration of the bispecific antibody is approximately 150 mg / mL.

[0095] The concentration of the bispecific antibody in the pharmaceutical composition may be 1 mg / mL to 200 mg / mL. In this embodiment, the concentration of the bispecific antibody in the pharmaceutical composition is 50 to 120 mg / mL. In this embodiment, the concentration of the bispecific antibody is 50-110 mg / mL. Other embodiments of the present invention In this case, the concentration of the bispecific antibody is 50-100 mg / mL. In another embodiment of the present invention, bispecific antibodies are used. The concentration of the specific antibody is 50-90 mg / mL. In other embodiments of the present invention, a bispecific antibody is used. The concentration is 50-80 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody is 5 The concentration is 0 to 70 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody in the pharmaceutical composition is The concentration is 60 mg / mL. In another embodiment of the present invention, a bispecific antibody in a pharmaceutical composition. The concentration is 70 mg / mL. In another embodiment of the present invention, the pharmaceutical composition has dual specificity. The antibody concentration is 80 mg / mL. In other embodiments of the present invention, a dual feature in the pharmaceutical composition is used. The concentration of the heterozygous antibody is 90 mg / mL. In another embodiment of the present invention, two antibodies in the pharmaceutical composition The concentration of the bispecific antibody is 100 mg / mL. In other embodiments of the present invention, in the pharmaceutical composition thereof, the concentration of the bispecific antibody is 110 mg / mL. In other embodiments of the present invention, in the pharmaceutical composition thereof, the concentration of the bispecific antibody is 120 mg / mL. In other embodiments of the present invention, in the pharmaceutical composit ion thereof, the concentration of the bispecific antibody is 130 mg / mL. In other embodiments of the present invention, in the pharmaceuti cal composition thereof, the concentration of the bispecific antibody is 140 mg / mL. In other embodiments of the present invention, the concentration of the bispecific antibody in the pharmaceutical composition is 150 mg / mL.

[0096] The pharmaceutical composition of the present invention controls pH within a range that optimizes the therapeutic effect and stability of the bispecific antibody, and comprises an acetate buffer used for said pH control. The acetate buffer can be prepared by mixing sodium acetate trihydrate and aceti c acid in water for injection. The pH can be adjusted by adding sodium hydroxide . In one embodiment of the present invention, the acetate buffer is present at a concentration between 20 mM and 40 mM . In one embodiment of the present invention, the concentration of the acetate buffer in the composition is 20 mM. In other embodiments of the present invention, the concentration of the acetate buffer in the composition is 25 mM. In other embodiment s of the present invention, the concentration of the acetate buffer in the composition is 30 mM. In other embodiment s of the present invention, the concentration of the acetate buffer in the composition is 35 mM. In other embodiments of the present invention, the concentration of the acetate buffer in the composition is 40 mM. In one embodiment of the present invention, the pharmaceutical compositio n may further comprise an additional buffer. In another embodiment, the pharmaceutical composition does not contain an additional buffe r.

[0097] In one embodiment of the present invention, the pH of the pharmaceutical composition is within the range of 5 to 6. In other embodiments of the present In its morphology, the pH of the pharmaceutical composition is in the range of 5.2 to 5.8. In another embodiment of the present invention, The pH of the pharmaceutical composition is in the range of 5.4 to 5.6. In other embodiments of the present invention, the pH of the pharmaceutical composition It is approximately 5.5, for example, 5.5.

[0098] The pharmaceutical composition of the present invention is a "stabilizer" that can interact with the charged group of the amino acid side chain. It further contains sorbitol, thereby reducing the possibility of intermolecular and intramolecular interactions. To lower the blood pressure. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 100 mM to 250 mM. In another embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 130 mM to 200 mM. In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 140 mM. In the application form, sorbitol is present in the pharmaceutical composition at a concentration of 150 mM. In one embodiment... In one embodiment, sorbitol is present in the pharmaceutical composition at a concentration of 180 mM. Bitol is present in the pharmaceutical composition at a concentration of 200 mM. In one embodiment, sorbitol It is present in the pharmaceutical composition at a concentration of 220 mM. In one embodiment, sorbitol is a pharmaceutical It is present in the composition at a concentration of 230 mM. In one embodiment, sorbitol is present in the pharmaceutical composition. It is present at a concentration of 240 mM in one embodiment. It exists at a concentration of 0 mM.

[0099] In one embodiment, the osmotic pressure (mOsm / kg) of the pharmaceutical composition is 200 mOsm / kg. Another embodiment In this embodiment, the osmotic pressure of the pharmaceutical composition is 210 mOsm / kg. The pressure is 220 mOsm / kg. In another embodiment, the osmotic pressure of the pharmaceutical composition is 230 mOsm / kg. In another embodiment, the osmotic pressure of the pharmaceutical composition is 240 mOsm / kg. The osmotic pressure of the drug composition is 250 mOsm / kg.

[0100] In one embodiment of the present invention, the concentration ratio of the acetic acid buffer to sorbitol in the pharmaceutical composition is 1 The ratio is between 5 and 1:10. In one embodiment of the present invention, the concentration ratio of acetic acid buffer to sorbitol is 1 :5. In another embodiment of the present invention, the concentration ratio of the acetic acid buffer to sorbitol is 1:6. Yes. In another embodiment of the present invention, the concentration ratio of the acetic acid buffer to sorbitol is 1:7. In another embodiment of the present invention, the concentration ratio of the acetic acid buffer to sorbitol is 1:8. In other embodiments of the present invention, the concentration ratio of acetic acid buffer to sorbitol is 1:9. In this embodiment, the concentration ratio of the acetate buffer to the sorbitol is 1:10.

[0101] In one embodiment of the present invention, the pharmaceutical composition has a pH of about 5.5 and essentially consists of the following: a. Bispecific antibodies in concentrations of 50-120 mg / mL b. 20-40 mM acetate buffer c. 140-160 mM sorbitol.

[0102] In certain embodiments of the present invention, the pharmaceutical composition has a pH of about 5.5 and is essentially one of the following: ru: a. 60 mg / mL bispecific antibody b. 30 mM acetate buffer c. 150 mM sorbitol Here, the CD3-binding Fab arms of the bispecific antibody are defined by SEQ ID NOs: 6 and 7, respectively. It contains VH and VL sequences and a constant heavy chain sequence (FEAL) as defined by SEQ ID NO: 19, and is CD20-bound. The Fab arms are defined by the VH and VL sequences of sequence numbers 13 and 14, respectively, and sequence number 20. It contains a constant heavy chain sequence (FEAR).

[0103] In another embodiment of the present invention, the pharmaceutical composition has a pH of about 5.5 and is essentially comprised of the following: : a. 60 mg / mL bispecific antibody b. 30 mM acetate buffer c. 250 mM sorbitol, Here, the CD3-binding Fab arms of the bispecific antibody are defined by SEQ ID NOs: 6 and 7, respectively. It contains VH and VL sequences and a constant heavy chain sequence (FEAL) as defined by SEQ ID NO: 19, and is CD20-bound. The Fab arms are defined by the VH and VL sequences of sequence numbers 13 and 14, respectively, and sequence number 20. It contains a constant heavy chain sequence (FEAR).

[0104] In one embodiment, the pharmaceutical composition is formulated with 30 mM acetic acid, 150 mM sorbitol, and pH 5.5. This is a concentrated formulation (DuoBody CD3×CD20). This concentrated formulation is diluted with a diluent immediately before administration. The concentration of the bispecific antibody may be set to 2 μg / mL to 5 mg / mL. In one embodiment, the diluent is 30 The solution consists of mM acetic acid, 150 mM sorbitol, and a pH of 5.5.

[0105] In one embodiment of the present invention, the pharmaceutical composition does not contain a surfactant. In another embodiment, The pharmaceutical composition does not contain hyaluronidase. In further embodiments, the pharmaceutical composition is a surfactant. It does not contain hyaluronidase.

[0106] In a preferred embodiment, the pharmaceutical composition is a subcutaneous composition, or the pharmaceutical composition is subcutaneously administered. It is intended for use in administration. However, the pharmaceutical composition of the present invention is administered intravenously. This may also be done. Therefore, one embodiment of the pharmaceutical composition is an intravenous composition or an intravenous administration composition. This is a pharmaceutical composition for use. The pharmaceutical composition is suitable for both subcutaneous and intravenous administration. This is an advantage of the present invention.

[0107] In one embodiment of the present invention, the pharmaceutical composition is for use in the treatment of cancer. In one embodiment of the present invention, the pharmaceutical composition is for use in the treatment of B-cell malignancies. be.

[0108] In another embodiment, the pharmaceutical composition of the present invention relates to T cell-mediated immune response, inflammation, and the microenvironment. It can be used to induce remodeling.

[0109] In certain embodiments, the pharmaceutical composition treats, prevents, or diagnoses various CD20-related diseases. Therefore, it is intended for in vivo use. Examples of CD20-related diseases include, in particular, B Cellular lymphomas, for example, non-Hodgkin lymphoma (NHL), B-cell leukemia and immune diseases, for example These include autoimmune diseases such as those listed below.

[0110] In one embodiment, the pharmaceutical composition according to the present invention is used in the treatment of NHL or B-cell leukemia. It is for practical use.

[0111] In one embodiment, the pharmaceutical composition according to the present invention is rituximab or ofatumumab resistant N HL or B-cell leukemia, such as rituximab-resistant non-invasive B-cell lymphoma, which is resistant to CD20 antibodies. It is intended for use in the treatment of sexual NHL or B-cell leukemia.

[0112] In one embodiment, the pharmaceutical composition according to the present invention is used for acute lymphoblastosis such as relapsed or refractory ALL. This is intended for use in the treatment of leukemia cysticum (ALL).

[0113] In one embodiment, the pharmaceutical composition according to the present invention is used for the treatment of CLL such as relapsed or refractory CLL. It is intended for use in [location / situation].

[0114] In one embodiment, the pharmaceutical composition according to the present invention is used for the treatment of FL such as relapsed or refractory FL. It is intended for use in this way.

[0115] The present invention further provides the above-mentioned pharmaceutical composition to a target that requires it, when sufficient to treat cancer. The present invention provides a method for treating cancer in a subject, including intermittent administration.

[0116] The present invention further provides the above-mentioned pharmaceutical composition to a target that requires it, when sufficient to treat cancer. The present invention provides a method for treating cancer in a subject, which includes subcutaneous administration to the subject.

[0117] The present invention further provides the above-mentioned pharmaceutical composition to a target that requires it, when sufficient to treat cancer. The present invention provides a method for treating cancer in a subject, which includes intravenous administration to the subject. In one embodiment, the cancer treated by this method is a B-cell malignancy, such as NHL, CLL, ALL, or FL. Or CD20 antibody-resistant NHL or B-cell leukemia, e.g., rituximab- or ofatumumab-resistant. NHL or B-cell leukemia, such as rituximab-resistant non-aggressive B-cell lymphoma.

[0118] In one embodiment, the pharmaceutical composition according to the present invention is in the form of a unit dose dosage. The unit dose of Ming refers to the unit dose of liquid. In one embodiment of the present invention, the unit dose dosage form includes the following: a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5, Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 Contains approximately 5 μg to 50 mg of bispecific antibodies. b. Acetate buffer and sorbitol, Here, the pH is approximately 5.5.

[0119] In one embodiment of the unit dose formulation, the acetate buffer and sorbitol are in a ratio of 1:5 to 1:10. For example, they are included in concentration ratios of 1:6, 1:7, 1:8, or 1:9.

[0120] In further embodiments, the osmotic pressure of the unit dose formulation is approximately 210 to approximately 250, for example, 220, 230, 240 or It is 250 mOsm / kg.

[0121] In further embodiments, the present invention relates to a unit dose formulation including the following: a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5, Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 Contains approximately 5 μg to 50 mg of bispecific antibodies. b. Acetate buffer solution at a concentration of approximately 30 mM, c. Sorbitol at a concentration of approximately 150 mM, Here, the pH is approximately 5.5.

[0122] In one embodiment of the above-described unit dose formulation, the amount of bispecific antibody is approximately 50 μg to approximately 40 mg, for example. The dosage is 50 μg to 40 mg.

[0123] In one embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 100 μg to approximately 30 mg. In another embodiment of the dose-type formulation, the amount of bispecific antibody is approximately 150 μg. In another embodiment, the amount of bispecific antibody is approximately 200 μg. Another embodiment of the unit dose formulation. In this configuration, the amount of bispecific antibody is approximately 250 μg. In another embodiment of the unit dose formulation, two The amount of the bispecific antibody is approximately 300 μg. In another embodiment of the unit dose formulation, the bispecific antibody The amount of the body is approximately 350 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 4 The amount is 00 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 450 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 500 μg. In another embodiment of the dosage form, the amount of bispecific antibody is approximately 600 μg. In this embodiment, the amount of bispecific antibody is approximately 700 μg. In another embodiment of the unit dose formulation... In this case, the amount of bispecific antibody is approximately 800 μg. In another embodiment of the unit dose formulation, bispecific The amount of heterozygous antibody is approximately 900 μg. In another embodiment of the unit dose formulation, the bispecific antibody is The amount is approximately 1 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 2 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 3 mg. In another embodiment of the type, the amount of bispecific antibody is approximately 4 mg. In terms of form, the amount of bispecific antibody is approximately 5 mg. In another embodiment of the unit dose formulation, bispecific antibody is used. The amount of specific antibody is approximately 6 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody The amount is approximately 7 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 8 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 9 mg. In another embodiment, the amount of bispecific antibody is approximately 10 mg. Another embodiment of the unit dose formulation In this configuration, the amount of bispecific antibody is approximately 11 mg. In another embodiment of the unit dose formulation, bispecific antibodies are used. The amount of specific antibody is approximately 12 mg. In another embodiment of the unit dose formulation, the bispecific antibody is The amount is approximately 13 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 14 mg. Yes. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 15 mg. In another embodiment of the dosage form, the amount of bispecific antibody is approximately 16 mg. In this embodiment, the amount of bispecific antibody is approximately 17 mg. In another embodiment of the unit dose formulation, The amount of bispecific antibody is approximately 18 mg. In another embodiment of the unit dose formulation, bispecific The amount of antibody is approximately 19 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 2 It is 0 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 21 mg. In another embodiment of the tuplicate dose formulation, the amount of bispecific antibody is approximately 22 mg. In another embodiment, the amount of bispecific antibody is approximately 23 mg. Another embodiment of the unit dose formulation. In this case, the amount of bispecific antibody is approximately 24 mg. In another embodiment of the unit dose formulation, bispecific The amount of heterozygous antibody is approximately 25 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody The amount is approximately 26 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 27 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is approximately 28 mg. In another embodiment of the dosage form, the amount of bispecific antibody is approximately 29 mg, for example, approximately 30 mg.

[0124] In one embodiment of the unit dose formulation, the amount of bispecific antibody is 100 μg to 30 mg. Unit dose In another embodiment of the dosage form, the amount of bispecific antibody is 150 μg. In this formulation, the amount of bispecific antibody is 200 μg. In another embodiment of the unit dose formulation, The amount of bispecific antibody is 250 μg. In another embodiment of the unit dose formulation, the bispecific antibody The amount of the body is 300 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 350 μg. The amount is g. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 400 μg. In another embodiment of the dosage formulation, the amount of bispecific antibody is 450 μg. In one embodiment, the amount of bispecific antibody is 500 μg. In another embodiment of the unit dose formulation, In this case, the amount of bispecific antibody is 600 μg. In another embodiment of the unit dose formulation, bispecific The amount of sex antibody is 700 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 8 The amount is 00 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 900 μg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 1 mg. In one embodiment, the amount of bispecific antibody is 2 mg. In another embodiment of the unit dose formulation, The amount of bispecific antibody is 3 mg. In another embodiment of the unit dose formulation, the bispecific antibody The amount is 4 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 5 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 6 mg. In another embodiment, the amount of bispecific antibody is 7 mg. In another embodiment of the unit dose formulation... In this case, the amount of bispecific antibody is 8 mg. In another embodiment of the unit dose formulation, bispecific antibody The amount of the body is 9 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 10 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 11 mg. In another embodiment of the type, the amount of bispecific antibody is 12 mg. Another embodiment of the unit dose type In this configuration, the amount of bispecific antibody is 13 mg. In another embodiment of the unit dose formulation, bispecific The amount of heterozygous antibody is 14 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 1 The amount is 5 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 16 mg. In another embodiment of the dosage formulation, the amount of bispecific antibody is 17 mg. In this embodiment, the amount of bispecific antibody is 18 mg. In another embodiment of the unit dose formulation, The amount of bispecific antibody is 19 mg. In another embodiment of the unit dose formulation, the bispecific antibody The amount is 20 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 21 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 22 mg. In another embodiment of the type, the amount of bispecific antibody is 23 mg. Another embodiment of the unit dose type In this configuration, the amount of bispecific antibody is 24 mg. In another embodiment of the unit dose formulation, bispecific The amount of heterozygous antibody is 25 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 2 The amount is 6 mg. In another embodiment of the unit dose formulation, the amount of bispecific antibody is 27 mg. In another embodiment of the dosage formulation, the amount of bispecific antibody is 28 mg. In this embodiment, the amount of bispecific antibody is 29 mg, for example, 30 mg.

[0125] In another embodiment of the unit dose formulation of the present invention, the first bispecific antibody that binds to human CD3 The binding region includes the VH and VL sequences of SEQ ID NOs: 6 and 7, and is a bispecific antibody that binds to human CD20. The second binding region includes the VH and VL sequences of SEQ ID NOs: 13 and 14. The preferred unit dose formulation of the present invention In this embodiment, the bispecific antibody is DuoBody-CD3×CD20 as described above.

[0126] In another embodiment, the total volume of the unit dose dosage form of the present invention is approximately 0.3 mL to approximately 3 mL, for example, 0.3 mL to 3 mL. It is mL. In another embodiment, the total volume of the unit dose formulation of the present invention is 0.5 mL. Another embodiment In its morphology, the total volume of the unit dose formulation of the present invention is 0.8 mL. In another embodiment, the present invention The total volume of the unit dose dosage form is 1 mL. In another embodiment, the total volume of the unit dose dosage form of the present invention It is 1.2 mL. In another embodiment, the total volume of the unit dose dosage form of the present invention is 1.5 mL. In one embodiment, the total volume of the unit dose formulation of the present invention is 1.7 mL. In another embodiment, The total volume of the unit dose dosage form of the present invention is 2 mL. In another embodiment, the total volume of the unit dose dosage form of the present invention The volume is 2.5 mL. This type of unit dose formulation is suitable for subcutaneous administration.

[0127] In embodiments where the unit dose dosage form is for intravenous administration, the volume is typically large. For example, between 10 mL and 500 mL. In one embodiment, the volume of the unit dose dosage form is 20 mL. In the application form, the volume of the unit dose formulation is 50 mL. In one embodiment, the volume of the unit dose formulation is 8 It is 0 mL. In one embodiment, the volume of the unit dose dosage form is 100 mL. In one embodiment, The volume of the dosage form is 150 mL. In one embodiment, the volume of the unit dose dosage form is 200 mL. In terms of form, the volume of the unit dose dosage form is 250 mL. In one embodiment, the volume of the unit dose dosage form is 30 It is 0 mL. In one embodiment, the volume of the unit dose dosage form is 350 mL. In one embodiment, The volume of the dosage form is 400 mL. In one embodiment, the volume of the unit dose dosage form is 450 mL. In terms of form, the volume of the unit dose formulation is 500 mL.

[0128] The unit dose formulation is a suitable diluent for the pharmaceutical composition of the present invention, such as an acetate buffer and sorbitol. It can be prepared by diluting it with a diluent that consists of a liquid and has a pH of 5.5. The diluent consists of a buffer and sorbitol so that the concentration of the bispecific antibody is affected only by the dilution. It is preferable that the concentration of Thor is the same as that of the pharmaceutical composition.

[0129] In another embodiment, the present invention further includes a container or a unit dose dosage form as described herein. Provide a container.

[0130] Furthermore, the present invention provides the above-mentioned unit dose formulation to subjects who require it, sufficient to treat cancer. The present invention provides a method for treating cancer in a subject, which involves administering a drug for a certain period of time. In one embodiment, The present invention provides a unit dose formulation to a target that requires it for a sufficient amount of time to treat cancer. The present invention provides a method for treating cancer in a subject, which includes subcutaneous administration. In one embodiment, The present invention provides a unit dose formulation to a target that requires it for a sufficient amount of time to treat cancer. The present invention provides a method for treating cancer in a subject, including intravenous administration.

[0131] In another embodiment, the present invention provides the unit dose described herein for use in the treatment of cancer. Regarding dosage form. In another embodiment, the unit dose dosage form is for subcutaneous administration. In terms of administration methods, the unit dose formulation is intended for intravenous administration.

[0132] The present invention a. Pharmaceutical compositions described herein b. Diluent containing acetic acid and sorbitol c. Container for unit dose formulations d. Instructions for dilution and / or use This also applies to parts kits that include this.

[0133] It is preferable that the concentration ratio of acetic acid to sorbitol in the diluent and the pharmaceutical composition be equal.

[0134] In one embodiment of the present invention, the parts kit is a. Pharmaceutical compositions containing the following: i. A 60 mg / mL bispecific antibody, e.g., DuoBody-CD3×CD20 ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. The pH is 5.5. b. Diluents containing the following: v. 30 mM acetate buffer vi. 150 mM sorbitol c. Containers for unit dose formulations, and d. Instructions for dilution and / or use Includes.

[0135] In one embodiment, the present invention further relates to a method for preparing a pharmaceutical composition as described herein, a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2:GTN, and VL-CDR3: Sequence ID 5, Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: A bispecific antibody containing 60-120 mg / mL VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 b. 3.53 mg / mL sodium acetate trihydrate c. 0.32 mg / mL acetic acid d. 27.3 mg / mL sorbitol The process of mixing in water for injection; and A step to adjust the pH to 5.5 by adding sodium hydroxide; Regarding methods including

[0136] In one embodiment of the method for preparing the pharmaceutical composition of the present invention, a is 60 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition, a is 70 mg / mL. In another embodiment of the preparation method, a is 80 mg / mL. Method for preparing the pharmaceutical composition of the present invention In another embodiment of the method, a is 90 mg / mL. Another embodiment of the method for preparing the pharmaceutical composition of the present invention In the application form, a is 100 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention In this case, a is 110 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a is 12 It is 0 mg / mL. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a is 150 mg / mL. Yes. In another embodiment of the method for preparing the pharmaceutical composition of the present invention, a is 200 mg / mL.

[0137] The present invention further relates to a method for preparing the unit dose dosage forms described herein, a. a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence 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: Sequence ID 5, and, The second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8, VH -CDR2:Sequence ID 9, VH-CDR3:Sequence ID 10, VL-CDR1:Sequence ID 11, VL-CDR2:DAS, and VL-CDR3: Sequence ID 12 A bispecific antibody containing 60-120 mg / mL, for example, 60 mg or 120 mg. b. 3.53 mg / mL sodium acetate trihydrate c. 0.32 mg / mL acetic acid d. Mix 27.3 mg / mL of sorbitol in water for injection and add sodium hydroxide. A step in preparing a pharmaceutical composition by adjusting the pH to 5.5; b. i. 3.53 mg / mL sodium acetate trihydrate ii. 0.32 mg / mL acetic acid iii. Mix sorbitol at a concentration of 27.3 mg / mL or less in water for injection, and add sodium hydroxide. A process of preparing a diluent by adjusting the pH to 5.5 by adding [a certain substance]; c. Mix the pharmaceutical composition and the diluent to obtain a bispecific antibody at a desired concentration in a unit dose dosage form. The process to be achieved; Regarding methods including

[0138] In further embodiments of the present invention, pharmaceutical compositions or unit dose agents obtainable by the method described above. Regarding types.

[0139] [Table 1-1]

[0140] [Table 1-2]

[0141] [Table 1-3] [Examples]

[0142] Examples The pharmaceutical composition of the present invention can be prepared by mixing the components listed in Table 2. ru.

[0143] [Table 2]

[0144] Example 1: Stability of Duobody-CD3×CD20 in various formulations

[0145] [ka]

[0146] material Unless otherwise specified, Duobody-CD3×CD20 was formulated at 2 mg / mL. method Thermal stability due to fluorescence / static light scattering Conformation stability and colloidal stability are determined in the unit device (Unchained Labs). This was determined by combining fluorescence / static light scattering (SLS) measurements. This measurement is thermal stress By utilizing the increase in protein, protein unfolding and aggregation are induced to achieve conformation. Evaluate the stability of the ion and colloid. Amphoteric ion caused by increased thermal stress. The folding state transition is caused by changes in the local environment during protein unfolding. It is detected by changes in the intrinsic fluorescence of Trp (and Tyr) residues in the protein. When a putophane residue is exposed, the maximum emission wavelength shifts to a longer wavelength. (Central-average (BCM)) ), that is, the wavelengths at which the fluorescence emission spectrum is equally divided are plotted, and the relationship between temperature and wavelength This shows the conformational changes of the protein. Fluorescence analysis reveals the opening of unfolding. Starting temperature (T onset ) and melting temperature (T m ) All values ​​are generated from the BCM curve. onset is, This is the calculated temperature at which the protein begins to unravel. mThe value represents the state in which the protein is folded. This is an intermediate point in the transition from the expanded state.

[0147] Unit measurements also provide SLS measurements that determine the stability of the protein colloid. It was irradiated with laser light scattered by molecules in the solution. Static light scattering intensity This is proportional to the average molecular weight of the species in the solution. Therefore, this analysis is useful for protein growth over a temperature gradient. It is susceptible to aggregates. Static light scattering was measured at 266 nm to detect smaller aggregates, and similarly... Larger aggregate species were detected at 473 nm. Aggregation temperature (T agg The protein begins to coagulate at the start of the protein This was determined from the data regarding the temperature at which counting begins. These data are based on the count intensity. The greater the change, the better the analysis; in other words, the higher the count, the more protein aggregation is observed. This indicates that more light was scattered due to body formation. Over the increasing temperature gradient, 10 3 vinegar Changes in SLS count data in kale are typically due to significant protein aggregation. This is because the SLS count only changes slightly, and the aggregation is only partial. Therefore, the fact that the SLS count does not change across the temperature gradient indicates that protein aggregation is negligible. To demonstrate that one is capable of doing so.

[0148] exterior The exterior appearance was determined by visual inspection.

[0149] pH pH was measured using a Mettler Toledo SevenMulti pH meter.

[0150] viscosity Viscosity was measured using a Wells-Brookfield Cone / Plate Rheometer.

[0151] Osmotic pressure Osmotic pressure was measured using an osmometer.

[0152] Absorbance A 280 Protein concentration Protein concentration is measured using UV / Vis spectroscopy (Agilent UV / Vis spectrophotometer (model 8453)). The value was determined by absorbance measurement at 280 nm (A280). Size exclusion chromatography (SEC) Size exclusion chromatography was performed using a TOSOH, TSK-gel G3000SWxL (7.8×300mm) column (Si The tests were performed using Agilent 1100 and 1200 HPLC systems (gma, catalog number 08541).

[0153] Image capillary isoelectric focusing (icIEF) Image capillary isoelectric focusing electrophoresis was performed using the iCE 3 Analyzer equipped with a PrinCE Autosampler. I went.

[0154] Reducing and non-reducing microchip capillary electrophoresis - sodium dodecyl sulfate Microchip capillary electrophoresis (both reducing and non-reducing) is performed using the Labchip GXII instrument. I used it and followed the manufacturer's instructions.

[0155] Dynamic light scattering (DLS) Dynamic light scattering analysis was performed using a Wyatt DynaPro Plate Reader. DLS analysis was performed at room temperature. Protein size and aggregation were evaluated. For DLS analysis, the time autocorrelation function of scattered light was determined. The average size of molecules in the solution is determined based on single-exponential cumulative fitting of the data. It is calculated. The reportable values ​​are polydispersity and hydrodynamic radius. In a constituent protein sample, the sample is considered monodisperse, but it contains a collection of multiple particle sizes. In a sample, the sample is considered polydispersible. The Percentage Polydispersity Index (%Pd) is a measure of the particle size distribution. This is a measure of width; the higher the %Pd, the wider the particle distribution. Therefore, a test with a high %Pd... The material typically contains (large) aggregates. Fluid of non-spherical protein particles. The mechanical radius is the radius of a sphere that has the same translational diffusion velocity as the particle. The diffusion velocity is the fraction of the particle. It depends on the amount of particles, surface structure, and the concentration and type of counterions in the formulation. Monodisperse size A larger hydrodynamic radius in the distribution indicates that the oligomers in the solution are of a higher order (for example) This may be due to the fact that it is a tetramer, but not due to the large size of the aggregate.

[0156] Solubility screening To determine the solubility of Duobody-CD3×CD20, first, the material was concentrated using a centrifugal concentrator. The formulation was prepared in a selected buffer at a low starting concentration. Subsequently, the solution was divided into 20, 50, 60, and 90 minutes. The protein was concentrated to a target concentration of >120 mg / mL by time-controlled spinning. The concentration was measured.

[0157] result 1. Baseline biophysical screening and excipient screening Perform an initial biophysical screening to select a buffer / pH / excipient combination. We then proceeded to a more detailed screening. Baseline biophysical screening And excipient screening is performed across a wide range of buffer / pH / excipient combinations, using DuoBod. This includes screening the thermal stability of y-CD3×CD20 (2 mg / mL) by fluorescence / SLS and DLS. Yes. Table 3 lists the buffers used in the initial screening and their pH values. ru.

[0158] Table 1 shows the data obtained from the initial buffering agent screening, including 30 mM acetic acid and 30 mM hydroxypropyl alcohol. Stidine buffer, excipient (150 mM NaCl, 150 mM arginine, 150 mM sorbitol or 1 Tests were conducted with and without 50 mM sucrose. Thermal stability was evaluated using fluorescence / SLS measurement and DLS. The aggregation of Duobody-CD3×CD20 (2 mg / mL) at room temperature was determined using [method / tool]. Fluorescence / SLS analysis was performed. Therefore, the melting temperature (T m ), Start of deployment (T onset ) and T agg DLS analysis revealed that Tan Information regarding the polydispersity and hydrodynamic radius of the protein was obtained.

[0159] In thermal stability analysis, T onset and T m These are the corresponding histidine preparations (each at 53-55°C) Compared to acetic acid preparations (in the ranges of 53-58°C and 60-62.5°C, respectively), It is slightly higher in the surrounding area. onset and T m The higher the value, the better the thermal stability of the protein. To indicate something. T between multiple excipients onset and T m Although the difference is small, the presence of arginine The stability is slightly lower under the given conditions, and slightly higher under sorbitol or sucrose. This may indicate that T by SLS agg Based on this decision, acetic acid and histidine preparations In either case, the addition of NaCl or arginine results in a mixture containing sorbitol or sucrose. Compared to formulations that do not contain excipients, T aggIt was shown that the temperature would decrease (59-60°C). Partial aggregation was observed at 66°C in acetic acid preparations containing sorbitol or sucrose, but No aggregation was observed with histidine buffers containing the excipients.

[0160] In DLS at room temperature, the greater the mean radius and multimode consistency, the greater the sucrose It showed a negative effect on the aggregation behavior of molecules in the presence of sorbitol. Furthermore, sorbitol affected the average radius and It appears to induce a slight increase in %Pd.

[0161] Based on data obtained from initial screening, acetic acid pH 5.5 without excipients, DuoBody-CD3×CD20 is stable and monodisperse in stidine pH 6.0 and histidine pH 6.5 buffers. It was concluded that it exists. Sorbitol and sucrose slightly increased thermal stability. Na Cl and arginine reduced thermal stability. Based on DLS results in initial screening. Therefore, sucrose was excluded as an excipient to be used in the next solubility screening. Excipient ( With or without using 150 mM NaCl, 150 mM arginine, or 150 mM sorbitol, acetate p H5.5, histidine pH6.0, and histidine pH6.5 formulations were selected for further solubility testing.

[0162] [Table 3]

[0163] [ka]

[0164] 2. Solubility screening The second stage of the baseline biophysical screening test is the initial baseline biophysical A solution is created by combining a pH / buffer combination selected from physical screening with an excipient. It included resolution screening. Buffer used in the second screening test. The list is shown in Table 4.

[0165] To measure the solubility in the presence of excipients, the materials are formulated in a selected buffer, The samples were continuously concentrated using a centrifugal concentrator at a set spin interval. Figure 1 shows the results for 20 minutes, 50 minutes, and 6 minutes. The concentrations of each formulation after spin intervals of 0 and 90 minutes are shown. Vinegar with or without sorbitol. The acid formulation was concentrated to 150 mg / mL at the fastest possible rate (50 minutes). Histamine containing and without sorbitol. The thidine preparation was concentrated most quickly (50 minutes), but was concentrated to a lower concentration (120 mg / mL). While other formulations could be concentrated to 120 mg / mL, it took time to reach this concentration. It took longer (60-90 minutes) compared to acetic acid preparations.

[0166] The concentrated sample (final concentration 120-150 mg / mL) was analyzed using fluorescence / SLS and DLS (Table 4) and viscosity. Further analysis was conducted on this (Figure 2).

[0167] Table 4 shows that formulations containing sorbitol but no excipients have the highest T m To indicate that one possesses. T in formulations containing sorbitol but not a binder agg The values ​​are for preparations containing NaCl and Arg. The metastasis was unclear compared to previous measurements, making interpretation difficult.

[0168] DLS data at room temperature is higher at DuoBody-CD3×CD20 concentrations compared to the low concentrations shown in Table 3. This shows the general increase in %Pd (>15% is interpreted as multivariance). Average hydrodynamic radius The amount of change may be influenced by the viscosity of the concentrated material, which affects the appropriateness of the formulation. It hinders the rankings.

[0169] Figure 2 shows the viscosity (cP) of various formulations with and without sorbitol. Acetate formulations are It exhibited a viscosity in the range of 7.9 to 12.1 cP. In contrast, the histidine-based solution without sorbitol... The drug had a higher cP than the acetic acid preparation (ranging from 28.4 to 79.9 cP). The addition of sorbitol was... While it reduced the viscosity of stidine preparations (in the range of 18-30 cP), sorbitol reduced the viscosity of acetic acid preparations. It did not affect that.

[0170] [Table 4]

[0171] [ka]

[0172] Furthermore, acetic acid (pH 5.5) with or without sorbitol, and acetic acid containing sorbitol. Permeation of concentrated sample in histidine (pH 6.5) containing thidine buffer (pH 6.0) and sorbitol. The pressure was measured using an osmometer. The results are shown in Table 5.

[0173] The osmotic pressure of acetic acid pH 5.5 without sorbitol was in the range of 70-80 mOsm / kg. The osmotic pressure of acetic acid and histidine preparations containing ethanol is in the range of 220-230 mOsm / kg, which is correct. It is close to the osmolality of normal plasma (275-295 mOsm / kg; Rasouli 2016 Clin Biochem 49 (12):9 36-41).

[0174] [Table 5]

[0175] Based on the above results, 30 mM acetic acid pH 5.5 containing 150 mM sorbitol is equivalent to Duobody-CD3 It was found that ×CD20 is a preferred formulation because acetic acid containing sorbitol It exhibits thermal stability equivalent to histidine preparations, is most efficient at high concentrations (150 mg / mL), and is the most effective. This was because it was a low-viscosity formulation.

[0176] 3. Real-time and acceleration stability Real-time aging of Duobody-CD3×CD20 at 30 mM acetic acid, 150 mM sorbitol, and pH 5.5 Qualitative analysis was performed using the assay described above (appearance, pH, UV[A]). 280 ], SEC, icIEF, CE-SDS [reduced and non-reduced] We used archetypes to evaluate at various points in time ranging from 0 to 12 months.

[0177] Table 6 shows the stability of Duobody-CD3×CD20 (5 mg / mL) samples stored at 5±3°C for 0, 2, 3, or 6 months. The test results are shown.

[0178] Table 7 shows the stability of Duobody-CD3×CD20 (5 mg / mL) samples stored at 25±℃ for 0, 1, 2, 3, or 6 months. The results of the sex test are shown.

[0179] Table 8 shows Duobody-CD3×CD20 (60 mg / mL) samples stored at 5±3°C for 0, 2, 3, 6, 9, or 12 months. The results of the stability test are shown below.

[0180] Table 9 shows the results of storing Duobody-CD3×CD20 (60 mg / mL) samples at 25±3℃ for 0, 1, 2, 3, or 6 months. The results of the qualitative test are shown below.

[0181] After storage at 5±3℃ and 25±3℃ for 12 months and 6 months, respectively, all samples were found to contain 5 mg / mL and 60 mg At a concentration of / mL, the sample remained stable under all test methods. Samples stored at 5±3℃ were stable. Compared to the start of the trial, no significant change was observed in either the June or December timeframe using either testing method. No chemical changes were observed. The purity profile in the accelerated stability test at 25±3℃ was expected. Minor changes were observed by UV spectroscopy, icIEF, CE-SDS depletion, and SEC testing.

[0182] [Table 6]

[0183] [Table 7]

[0184] [Table 8]

[0185] [Table 9]

[0186] 4. Conclusion Based on the results obtained from analytical tests of Duobody-CD3×CD20 in various formulations, 30 mM acetate A 150 mM sorbitol solution with a pH of 5.5 was found to be the optimal formulation for this molecule. This formulation is available in doses of 2-150 mg. Supports concentrations in the range of / mL. The inventors have found that DuoBody-CD3×CD20 is 5 and 60 mg / mL (30m The product was shown to be stable until December at 5±3℃ in acetic acid, 150 mM sorbitol, and pH 5.5. Furthermore, in accelerated stability tests at 25±3℃, 5 and 60 mg / mL (30 mM acetic acid, 150 mM sorbitol) were found. Regarding DuoBody-CD3×CD20 (Tall, pH 5.5), slight changes within the predicted range were expected until June. It was observed.

[0187] Example 2: Canine administered DuoBody-CD3×CD20 via intravenous (IV) and subcutaneous (SC) routes. Cytokine analysis of monkey blood

[0188] A dose-range finding (DRF) study of DuoBody-CD3×CD20 using female cynomolgus monkeys, and a study of male and female cynomolgus monkeys. In a GLP toxicity study of DuoBody-CD3×CD20 using nymph monkeys, t=0 (before administration), 2, 4, Blood samples were collected from animals at 6, 12, and 24 hours. The sample (0.25 ml) was treated with a reagent containing K2EDTA. Transfer to a test tube and, in order to obtain plasma, centrifuge at 3000 rpm (approximately 1500 g) for 10 minutes at 4°C. The plasma was processed. It was transferred to a clear 0.5 mL polypropylene tube and stored at -80°C until analysis. Ta.

[0189] BioPlex200 Reader (BioRad) along with Milliplex MAP NHP Cytokine Magnetic Bead Pan Using el (Millipore Cat.No.PRCYTOMAG-40K), test according to the manufacturer's protocol. IL-1β, IL-2, IL-6, IL-4, IL-8, IL-10, IL-12p40, IL-15, IF The concentrations of Nγ, TNFα, and MCP-1 were measured.

[0190] Figure 3 shows the results of a single intravenous injection of DuoBody-CD3×CD20 with the pharmaceutical composition of the present invention in a GLP toxicity test. The patient received either oral administration (0.1 or 1 mg / kg) or a single subcutaneous dose (0.1 or 1 mg / kg). This shows the average cytokine levels for each group in blood samples obtained from animals.

[0191] When DuoBody-CD3×CD20 was administered, cytokines IL-1β, IL-4, IL-12p40 and IL The level of -15 was slightly reduced (less than 150 pg / mL) (Figure 3A).

[0192] Cytokines IL-2, IL-6, IL-8, IL-10, IFNγ, TNFα, and MCP-1 are found in DuoBody-CD3 Intravenous administration of ×CD20 clearly induced a peak, which reached its peak within 2 to 12 hours after administration (Figure 3B). ). Subsequently, cytokine levels returned to baseline. Each of these cytokines Regarding this, the peak level was higher in the blood of animals that received subcutaneous administration (0.1 or 1 mg / kg). The levels were lower compared to the corresponding intravenous administration levels. Regarding the peak levels of IL-8 and IFNγ... In all cases, subcutaneous administration resulted in lower delays compared to intravenous administration.

[0193] Similar findings were observed in the DRF trial, with the exception of this (smaller) trial. No difference in IFNγ concentrations was observed between animals administered intravenously and those administered subcutaneously.

[0194] Example 3: Four repeated intravenous administrations of DuoBody-CD3×CD20 to cynomolgus monkeys, with the initial dose Evaluation of B cell depletion after a single intravenous or subcutaneous administration (dose-range setting study). In accordance with this overview, female cynomolgus monkeys were given the formulation of the present invention (30 mM acetic acid, 150 mM sorbitol). DuoBody-CD3×CD20 at pH 5.5 is administered intravenously four times a week (0.01, 0.1, or 1 mg / kg). ), followed by an initial intravenous dose (0.01 mg / kg) and then an intravenous target dose of 1 mg / kg, or a single subcutaneous injection. DuoBody-CD3×CD20 was administered by injection at one of the following doses: 0.01, 0.1, 1, 10, or 20 mg / kg:

[0195] [ka]

[0196] This research will be conducted under the supervision of the UK Home Office and will be used for experimental and other scientific purposes. In accordance with the European Convention for the Protection of Vertebrates (Council of Europe), Charles River Laboratories The experiment was conducted in Leeds (Charles River Laboratories) (Tranent, UK).

[0197] The Mauritian-origin crab-eating macaque, Macaca fascicularis, is a species of macaque bred for specific purposes. ius)Limited(Riviere de Anguilles, Mauritius) or Noveprim(Mahebourg, Maurit Obtained from ius. The animals were socially raised in communal barns while being provided with a rich environment. .

[0198] Sample collection A whole blood sample (approximately 0.5 mL) is collected from the femoral vein using a sterile subcutaneous needle and sterile syringe. Collected. For immunophenotyping by flow cytometry, heparin sodium was included. The blood was transferred to a tube and stored at room temperature until analysis was performed within 48 hours.

[0199] The biopsy (approximately 20 mg) was cut into the lymph nodes using standard surgical sterile techniques and then extracted into the superficial lymph nodes. The biopsy was taken from the animal, which was under general anesthesia during the procedure. The biopsy was taken at Roswell Park Memorial Institute. Single-cell suspensions were collected using itute (RPMI) and stored on moist ice until processed within 24 hours. The solution was prepared using the Medimachine System for automated mechanical deaggregation of tissue (Becton Dickinson; see the full CRL research report for details). The obtained cells were placed in 2 mL of d The sample was resuspended in Rubecco's phosphate-buffered saline (PBS; Gibco, catalog number 14190).

[0200] During the autopsy, samples from the lymph nodes and spleen were oriented on a cork disc and then placed in aluminum. Each sample is individually wrapped in foil, uniquely labeled, rapidly frozen in liquid nitrogen, and then evaluated by immunohistochemistry. It was stored in a freezer set to maintain a temperature of -80°C.

[0201] Flow cytometry A mixture of directly labeled antibodies (see below) was prepared in a round-bottom test tube (Falcon, catalog number 352052). The antibody mixture was prepared and selected to allow for the analysis of CD19+ B cells as well.

[0202] For immunophenotyping of peripheral blood, 50 μL of anticoagulated whole blood was added to the antibody mixture and heated at room temperature for 2 hours. Red blood cells (RBCs) were incubated for 0 minutes, protected from light. Red blood cells (RBCs) were thawed with 1x RBC lysis buffer (eBio Using science (catalog number 00-4300-54), RT for 10 minutes (or until RBC dissolution is complete). Dissolve the cells. Centrifuge the tube at 300-500g for 5 minutes using RT. Discard the supernatant and remove the cell pellet. The mixture was resuspended in 0.5 mL of PBS (Gibco, catalog number 10010). 50 μL of Flow Count beads (B Eckman Coulter (catalog number 7546053) was added to each tube before analysis.

[0203] To determine the immunophenotype of lymph node cells, 50 μL of cell suspension was added to the antibody mixture. Incubate on ice for 15 minutes and protect from light. After incubation, add 0.5 mL of Dulbecco's PBS to each cell. It was added to the tube.

[0204] The sample is scanned using a 2-laser 5-color Beckman Coulter FC500 or BD LSR Fortessa X-20 flow scanner. Analysis was performed using a tometer. CD4 - CD8 - CD15 - CD19 + The events were classified as B cells.

[0205] The following antibody mixture was used:

[0206] [ka]

[0207] immunohistochemistry Frozen lymph nodes and spleen collected at autopsy were sectioned and subjected to standard immunohistochemistry procedures for CD19 The sample was stained with an antibody against (Abcam, catalog number ab134114).

[0208] result Repeated intravenous and single subcutaneous administration, as well as intravenous administration at the initial dose, all affect peripheral blood flow. This resulted in dose-dependent depletion of B cells from lymph nodes (Figures 4-9). At 0.01 mg / kg, the most The first two intravenous administrations induced B-cell depletion to a (nearly) undetectable level. 3 In the first and fourth doses, B cells were not partially or completely depleted. This lack of effect after repeated administration. This may be due to the formation of anti-drug antibodies. Repeated intravenous administration at 0.1 or 1 mg / kg is Induce complete B cell depletion, then administer (partially) at 21 days (0.1 mg / kg) or 42-119 days (1 mg / kg). Recovery has begun. The pharmaceutical composition of the present invention (30 mM acetic acid, 150 mM sorbitol, pH 5.5) A single subcutaneous injection of DuoBody-CD3×CD20 is effective at all dose levels for the cardiovascular and lymph nodes. This resulted in B cell depletion down to undetectable levels. B cell recovery was observed in all groups. With low doses, baseline return occurred in a few weeks, and with high doses, it took approximately 70 days after administration. If the target dose of 1 mg / kg is administered one day after intravenous administration (0.01 mg / kg), peripheral blood and liver cells will be affected. B cells were completely depleted from the lymph nodes and this continued until the scheduled autopsy day (day 29). In this study, Immunohistochemistry confirmed the depletion of B cells from the lymph nodes and spleen (Figure 10).

[0209] Example 4: CaniQuiza after 5 repeated intravenous infusions or a single subcutaneous injection of DuoBody-CD3×CD20 B cell depletion in the phlebotomy (GLP toxicity test) Male and female cynomolgus monkeys were given DuoBody-CD3×CD20 from the pharmaceutical composition of the present invention via intravenous infusion five times a week. (0.01, 0.1 or 1 mg / kg), single intravenous infusion (0.1 or 1 mg / kg), or subcutaneous injection (0. The patient was administered 1, 1, or 10 mg / kg; a control group also received intravenous infusion of physiological saline five times a week. Included:

[0210] [ka]

[0211] This research is for experimental and other scientific purposes under the jurisdiction of the UK Home Office. In accordance with the European Convention on the Protection of Vertebrate Animals (Council of Europe), Charles River Laboratories ( It was held in Tranent, UK.

[0212] The crab-eating macaque, Macaca fascicularis, bred for the purpose of originating from Mauritius, is LCL-C Obtained from ynologics (Port Louis, Mauritius). Animals are socially rich in their environment. It was raised in a jungle pen.

[0213] Whole blood samples and lymph node biopsies were collected as described above.

[0214] The B cells were quantified by flow cytometry in the same manner as above, except for the following: For peripheral blood immunophenotyping, 50 μL of anticoagulated whole blood was added to the antibody mixture and incubated at +4°C for 30 minutes. Incubated for minutes, protected from light. The TruCount introduction tube (BD Biosiences) was used during data acquisition. CD45 + CD4 - CD8 - CD15 - CD19 + The event was classified as a B cell. The following antibody mixture was used:

[0215] [ka]

[0216] result The results are shown in Figures 11-16. Intravenous infusion of physiological saline partially reduced the number of B cells in peripheral blood. However, the B cell count returned to baseline within 3 weeks after the final infusion. DuoBody-CD3×CD20 Intravenous infusion five times a week induced dose-dependent B cell depletion, and in the low-dose group, partial depletion occurred. Depletion was observed, and in the high-dose group, complete depletion over a long period was seen. Single intravenous administration of 0.1 or 1 mg / kg. Internal infusion completely depletes B cells from peripheral blood, and at the highest dose tested, depletion occurred at autopsy. The effect lasted until day 36. Subcutaneous administration of DuoBody-CD3×CD20 was the most effective dose in all the trials. Bell caused complete B cell depletion from peripheral blood. At the lowest dose, B cell levels were partially reduced. Although recovery occurred, complete B-cell depletion persisted until the autopsy day (day 33) in the 1 mg / kg and 10 mg / kg groups.

[0217] Example 5: Pharmacokinetics of DuoBody-CD3×CD20 in cynomolgus monkeys: intravenous (IV) and subcutaneous (SC) Route of administration Materials and methods DuoBody-CD3×CD20 formulated in 30 mM acetate buffer, 150 mM sorbitol, and pH 5.5. To evaluate the pharmacokinetic (PK) properties of the drug, both via intravenous (IV) and subcutaneous (SC) administration routes, in toxicity tests. The determination was made in cynomolgus monkeys through experimentation. Blood samples were taken from female cynomolgus monkeys using DuoBody-CD3×CD2 Dose-Range-Finding (DRF) study of 0, and GLP toxicity of DuoBody-CD3×CD20 in cynomolgus monkeys. The drugs were obtained from animals through sex testing. The design and details of these studies are described in Example 2. Dynamic evaluation was performed on animals that received a single intravenous or subcutaneous injection. DuoBody-CD To measure the plasma concentration of 3×CD20, blood samples (approximately 0.5 mL each) were collected from all animals. The following was collected: The DuoBody-CD3×CD20 concentration in cynomolgus monkey plasma obtained from the DRF test was measured using Imperace. The results were obtained from GLP toxicity testing of cynomolgus. The DuoBody-CD3×CD20 concentration in plasma was measured using the monomolecule counting (SMC) method. These evaluations Details of the pricing are shown in the following section.

[0218] DuoBody-CD3×CD20-specific PK Imperacer(registered trademark) Immuno-PCR The concentration of DuoBody-CD3×CD20 in cynomolgus monkey plasma obtained from DRF studies was compared with the antibody-DNA complex. Advanced ultra-sensitive immunoassay utilizing exponential amplification of DNA markers for subsequent protein detection. This was determined using the Imperacer® method, a type of immunopolymerase chain reaction (PCR) method. In short, the 8-level calibration curve of DuoBody-CD3×CD20 prepared in 100% cynomolgus monkey plasma. Quality control (QC) and (diluted) cynomolgus monkey test samples are processed using Imperacer®. Contains combined CHI-SAB1 A1 (Chimera Biotec GmbH, Dortmund, Germany, Cat no. 11-272). The sample was diluted with the dilution buffer SDB6000. The sample was then treated with H of CD3, which can be bound to DuoBody-CD3×CD20. Coated with an is-tagged extracellular domain (CD3ECDHis; Genmab, Utrecht, Netherlands). It was added to a prepared 96-well ELISA plate. The plate was washed and PCR master mix (Mo lzym (catalog number C-022) was added, and the sample was processed using an Imperacer® RT-PCR reader (Ag I moved to the Enabled RT Cycler (MX 3000P / MX 3005P) obtained from ilent Technologies / Chimera. The immobilized DuoBody-CD3×CD20 is included in the Imperacer® detection conjugate. It can be detected in PCR amplification of the A marker. Sequence-specific fluorescence in PCR-Mastermix The treatment directly relates to the initial amount of DNA marker present, and is shown as a ΔCt signal. This results in an increase in the fluorescence signal. After the completion of real-time PCR signal generation, the measured fluorescence signal The data is processed by instrument software (MXPro; Chimera Biotec GmbH) and mathematical software ( Analysis was performed using Microsoft Excel (XLfit analysis plugin). The density of the combined DuoBody-CD3×CD20 was analyzed. The degree is measured using a nonlinear S-shaped 4-parameter regression, and the ΔCt signal is converted to the logarithmic spike concentration DuoBody-CD. This was determined from the standard curve created by plotting against 3×CD20. The procedure was established and implemented at Chimera Biotec GmbH, Dortmund. LLOQ was 1.0 pg / mL clarified. It was plasma.

[0219] DuoBody-CD3×CD20 PK Monomolecule Counting (SMC) Method DuoBody-CD3×CD20 concentrations in cynomolgus monkey plasma obtained from GLP toxicity tests were analyzed using the single-molecule counting (SMC) method. The determination was made using the following method: SMC immunoassay measures DuoBody-CD3×CD20 molecules in cynomolgus monkey plasma. This is a reliable fluorescence sandwich immunoassay method.

[0220] In short, the calibrator, QC, and research samples are filtered before use, and magnetic beads are added. According to the manufacturer's protocol (Merck Millipore, catalog number 03-0077-02), DuoBody-C On a D3×CD20 CD3 tonearm (UM-IgG1mm-3005-101-3-1-MP; Genmab, Utrecht, Netherlands) The sample was labeled with an anti-idiotype antibody. The filtered sample was then coated with magnetic particles w and the anti-idiotie of DuoBody-CD3×CD20 bonded to fluorochrome against the CD20 arm Along with the antibody (UM-IgG1mm-3001-2F2-Sab1.1(-FL); Genmab, Utrecht, Netherlands) Incubated. After incubation, the particles were washed and the unbound conjugates were removed. The tetraparticle was removed. Next, the magnetic particles to which the bonded analyte and conjugate were attached were cleaned. The analyte and conjugate were transferred to a plate, and the remaining buffer was aspirated. According to the protocol of the company (Merck Millipore), using an elution buffer The sample was dissociated from the magnetic particles, and the eluate was transferred to a 384-well plate containing a neutralizing buffer. Capillaries are counted using the Erenna® single-molecule counting system (Merck / Millipore). The molecules were drawn in and irradiated with a laser. The fluorescently labeled molecules emitted light, and a signal exceeding a threshold was detected. It is counted as a detected event. Furthermore, the amount of light from each event (event photon) and the total amount of light (total) are counted. The number of photons was measured.

[0221] This method was developed by PRA Health Sciences Bioanalytical Laboratory (PRA), ASEN, O The test was validated and performed in Randa. In the validation, LLOQ was measured and quantified in 0.100 ng / mL clear plasma. The upper limit (ULOQ) was measured in clear plasma at 50 ng / mL.

[0222] result Dose range setting study: Single intravenous administration with the initial dose. The plasma concentration profile of DuoBody-CD3×CD20 was determined on day 1 when DuoBody-CD3×CD20 was administered at 0.01 mg / kg. The initial dose was administered on the first day, and on the second day, the target dose of DuoBody-CD3×CD20 at 1 mg / kg was administered to cynomolgus monkeys. Measurements were taken in two females (n=2). Both the initial dose and the target dose were administered at a dose of 10 mL / kg over 30 minutes. It was administered by intravenous infusion. DuoBody-CD3×CD20 was administered as an initial intravenous dose of 0.01 mg / kg (day 1), followed by 1 mg / kg. After intravenous administration of the target dose of g (on day 2), C max The infusion of 1 mg / kg dose was completed very quickly. CL value (10.7~13.7 mL / day / kg) and V D The values ​​(56.1-64.9 mL / kg) were for the repeated intravenous infusion group. The same level was observed after the first dose.

[0223] Individual plasma concentration profiles generated from the Imperacer Immuno-PCR method are shown in Figure 17A. The group-average PK parameters are shown in Table 10. PK parameters were calculated only for the 1 mg / kg dose.

[0224] [Table 10]

[0225] Dose-finding study: Single subcutaneous dose The plasma concentration profiles of DuoBody-CD3×CD20 are as follows: DuoBody-CD3×CD20 at 0.01, 0.1, 1, and 10 Alternatively, measurements were taken after a single subcutaneous injection at a dose level of 20 mg / kg (n=2 females / group). Subcutaneous injection was 1 mL / The dose was administered in kg. After subcutaneous administration, C max This was reached 0.5 to 7 days after administration. nC max or AUC 0- Based on any of the infinity factors, a hyperproportional increase in exposure was observed up to a dose of 1 mg / kg. Between ~20 mg / kg, a proportional increase in exposure was observed with increasing dose. (Imperator) Figure 17B shows the individual blood cell concentration profiles generated from the (registered trademark) Immuno-PCR method, by Gunpei. The average PK parameters are shown in Table 11.

[0226] Absolute subcutaneous bioavailability (F) is measured as AUC after subcutaneous administration of 1 mg / kg. inf and 1 mg / kg AUC after initial intravenous administration 0- Using infinity, we can express the percentage of intravenous bioavailability and Calculations show that this dose provides 111% complete (100%) subcutaneous bioavailability. The result is shown.

[0227] [Table 11]

[0228] GLP toxicity test: Single intravenous administration The plasma concentration profile of DuoBody-CD3×CD20 is as follows: DuoBody-CD3×CD20 at 0.1 or 1 mg / kg Measurements were taken after a single intravenous infusion at the specified dose level (3 males and 3 females per group). Generated by the SMC method. The group-mean plasma concentration profiles are shown in Figure 17C, and the group-mean pharmacokinetic parameters are shown in Table 12. uoBody-CD3×CD20 (Average C max and AUC (0-t) Systemic exposure to (based on) is associated with increased doses in males and females. It increased along with the dose-normalized estimates of whole-body exposure to DuoBody-CD3×CD20. As a result, in both males and females, the levels increased generally in a manner exceeding dose-proportionality within the dose range of 0.1 to 1 mg / kg. maxThe median time was consistently 0.5 hours (end of infusion time). CL, VD, and VSS were administered in increasing doses. A tendency for it to decrease with increasing activity was observed. 1 / 2 For the desorption phase, the time required is 168 or 336 hours at a desorption phase of 0.1 mg / kg. On the other hand, in both sexes, a high value of 1 mg / kg resulted in a maximum incubation period of 840 hours (98 hours for men and 1 for women). The average value over 25 hours was likely to be appropriately derived. Whole-body exposure was 0.1 and 1 mg / kg. While there were nearly identical results between males and females, the average AUC for males was 0.1 mg / kg. (0-t) The female that was administered the drug was larger This was because one animal showed a total exposure approximately seven times greater than all other animals combined. It is highly likely that this is the case. Considering the variation caused by this animal, C max and AUC (0-t)) female / The male-to-female ratio is in the range of 0.8 to 1.3 (AUC of 0.1 mg / kg). (0-t) The result was 0.3).

[0229] [Table 12]

[0230] Single subcutaneous administration The plasma concentration profiles of DuoBody-CD3×CD20 are as follows: DuoBody-CD3×CD20 at 0.1, 1, and 10 mg / kg Measurements were taken after a single subcutaneous injection at the specified dose level (3 males and 3 females per group). The subcutaneous injection was administered at a dose of 0.2 mL / kg. The dose was administered. The group-average plasma concentration profiles generated from the SMC method are shown in Figure 17C. Pharmacokinetic parameters are shown in Table 13. DuoBody-CD3×CD20(mean C) max and AUC (0-t) Based on Systemic exposure to ) increased in both males and females with increasing subcutaneous doses. Based on this, C maxIn both males and females, the dose-proportional increase is between 0.1 and 1 mg / kg overall. Furthermore, although it was greater than the dose-proportional ratio between 1 and 10 mg / kg, the dose-normalized AUC (0-t) is 0.1-10 mg / kg The increase was greater than dose-proportional. Overall, this increase was 0.1-10 mg / kg in males and females after SC administration. The dose-proportional relationship was exceeded. max The median time was consistently 72 hours for males, but individually for females. T max Because there is a large variation between values, T max No consistency was observed in the trends. 1 / 2 is high The longest discharge period occurs at the appropriate dose, and at this time, the characteristics of the efflux phase appear most appropriately in both males and females. Generally, whole-body exposure is higher in males than females at 0.1 mg / kg, and between females and females in the 1 and 10 mg / kg groups. They were equivalent; C max and AUC (0-t) The male / female ratio was 0.5 and 0.4, respectively, at 0.1 mg / kg, and 1 mg The values ​​were 0.8 per kg for all cases, and 1.0 for all cases at 10 mg / kg.

[0231] [Table 13]

[0232] In summary, after intravenous infusion of DuoBody-CD3×CD20, plasma concentrations were elevated at the end of the 30-minute administration period. The levels rose until [time], and then generally decreased in a biphasic pattern. The peak level was reached approximately 72 hours after subcutaneous administration. An increase was observed until the indicated level, and it remained at a relatively steady level until 168 hours after administration. Subsequently, the concentration decreased monophasically until the end of the 4-week sampling period. The maximum plasma concentration after intravenous administration was significantly higher than the maximum plasma concentration after subcutaneous administration.

Claims

1. Pharmaceutical compositions containing or essentially consisting of the following: a. Bispecific antibodies that bind to human CD3 and human CD20 at concentrations of 50–120 mg / mL. b. 20–40 mM acetic acid c. 140–160 mM sorbitol Here, the pH of the composition is 5 to 6, and the bispecific antibody is The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2: GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID No. 12 A pharmaceutical composition containing the following:

2. The first binding region of the bispecific antibody that binds to CD3 is relative to the VH and VL sequences of SEQ ID NOs. 6 and 7. At least 90% sequence identity, for example, at least 95% for the VH and VL sequences of sequence numbers 6 and 7. Includes VH and VL sequences having %, 96%, 97%, 98%, 99%, or 100% sequence identity, The pharmaceutical composition according to claim 1.

3. The second binding region of the bispecific antibody that binds to CD20 is located in the VH and VL sequences of SEQ ID NOs. 13 and 14. In contrast, at least 90% of the VH and VL sequences of sequence identity sequence numbers 13 and 14 are It also includes VH and VL sequences having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The pharmaceutical composition according to claim 1 or 2.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the bispecific antibody is an IgG1 antibody.

5. Bispecific antibodies include the constant region of the lambda light chain and the constant region of the kappa light chain, for example, SEQ ID NO: 22 and The first and second light chains include first and second light chain steady regions selected from 23 light chain steady regions. or the pharmaceutical composition according to any one of claims 1 to 4.

6. The bispecific antibody comprises an Fc region containing the first and second heavy chains, and the Fc region is wild-type IgG1 Fc It has been modified to have lower effector function compared to bispecific antibodies that include the region. ru or the pharmaceutical composition according to any one of claims 1 to 5.

7. A bispecific antibody is one in which the binding of C1q to the antibody has a wild-type IgG1 Fc region. Compared to the body, at least 70%, at least 80%, at least 90%, at least 95%, less It includes an Fc region that has been modified to be at least 97% or 100% lower, where the C1q bond The pharmaceutical composition according to any one of claims 1 to 6, wherein is determined by ELISA.

8. The bispecific antibodies each contain at least the hinge region, CH2, and CH3 regions, respectively, as first and second It includes a heavy chain, where the first heavy chain contains T366, L368, K370, and D399 of the human IgG1 heavy chain. amino acids at positions selected from the group consisting of F405, Y407, and K409 At least one of the following is substituted, and in the second heavy chain, T366 in the human IgG monohelic acid Positions corresponding to positions selected from the group consisting of L368, K370, D399, F405, Y407, and K409. At least one amino acid in the position is substituted, and the first and second heavy chains are the same A pharmaceutical composition according to any one of claims 1 to 7, which is not substituted in position.

9. (i) The amino acid in the first heavy chain at the position corresponding to F405 in the human IgG monoheavy chain. is L, and in the second heavy chain, at the position corresponding to K409 in the human IgG single heavy chain The amino acid is R, or (ii) corresponds to K409 in the human IgG monoheavy chain in the first heavy chain. The amino acid at that position is R, and in the second heavy chain, F4 is present in the human IgG monoheavy chain. The amino acid at the position corresponding to 05 is L, according to any one of claims 1 to 8. Finished product.

10. In the bispecific antibody, positions L234 and L235 in both the primary and secondary heavy chains of the human IgG monoheavy chain The pharmaceutical composition according to any one of claims 1 to 9, wherein the corresponding positions are F and E, respectively.

11. The positions L234, L235, and in the human IgG monohelic acid The positions corresponding to D265 are F, E, and A, respectively, as described in any one of claims 1 to 10. A pharmaceutical composition.

12. Position L234 in the human IgG monoheavy chain of both the primary and secondary constant heavy chains of the bispecific antibody. The positions corresponding to L235 and D265 are F, E, and A, respectively, and are the first constant heavy chain of human IgG1 The position corresponding to F405 in the heavy chain is L, and K4 in the human IgG monoheavy chain of the second constant heavy chain. The pharmaceutical composition according to any one of claims 1 to 11, wherein the position corresponding to 09 is R.

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

16. A pharmaceutical composition according to any one of claims 1 to 12, comprising the amino acid sequence.

14. The first and second constant heavy chains each comprise the amino acid sequences of SEQ ID NOs: 19 and 20, respectively, according to claims 1 to 1. A pharmaceutical composition according to any one of item 3.

15. a is 50-120 mg / mL, for example 50-110 mg / mL, or for example 50-100 mg / mL, for example 50-90 mg / mL, for example 50-80 mg / mL, for example 50-70 mg / mL, for example 55-65 mg / mL, for example 58-62 mg / mL, For example, 60 mg / mL, or a is approximately 120 mg / mL, as described in any one of claims 1 to 14. A pharmaceutical composition.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein b is 28 to 32 mM, for example, 30 mM.

17. c is 145–155 mM, for example 148–152 mM, for example 150 mM, according to any one of claims 1 to 16. The pharmaceutical composition described.

18. The pH is 5.3 to 5.6, for example 5.4 to 5.6, for example about 5.5, according to any one of claims 1 to 17. The pharmaceutical composition described.

19. The composition has a pH of 5.4 to 5.6, for example, 5.

5. a. Bispecific antibodies in concentrations of 50–120 mg / mL b. 20–40 mM acetic acid c. 140–160 mM sorbitol, A pharmaceutical composition according to any one of claims 1 to 18, comprising essentially the above.

20. The composition has a pH of 5.4 to 5.

6. a. Bispecific antibody at 58–62 mg / mL b. 28–32 mM acetic acid c. 145–155 mM sorbitol, A pharmaceutical composition according to any one of claims 1 to 19, which is essentially derived from the above.

21. The composition has a pH of 5.

5. a. 60 mg / mL bispecific antibody b. 30 mM acetic acid c. 150 mM sorbitol, A pharmaceutical composition according to any one of claims 1 to 20, which is essentially derived from the above.

22. The composition has a pH of 5.4 to 5.

6. a. Bispecific antibody at 110–130 mg / mL b. 28–32 mM acetic acid c. 145–155 mM sorbitol, A pharmaceutical composition according to any one of claims 1 to 19, which is essentially derived from the above.

23. The composition has a pH of 5.

5. a. 120 mg / mL bispecific antibody b. 30 mM acetic acid c. 150 mM sorbitol, The pharmaceutical composition according to claim 22, which is essentially derived from the above.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the composition does not contain a surfactant.

25. The composition is a pharmaceutical combination according to any one of claims 1 to 24, wherein the composition does not contain hyaluronidase. Finished product.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the composition is a subcutaneous composition.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein the composition is a venous composition.

28. The composition is for use in the treatment of cancer, according to any one of claims 1 to 27. The pharmaceutical composition described.

29. The composition is for use in subcutaneous administration, according to any one of claims 1 to 28. The pharmaceutical composition described.

30. The composition is for use in intravenous administration, any one of claims 1 to 24. The pharmaceutical composition described above.

31. A pharmaceutical composition according to any one of claims 1 to 30, available in a dose-unit dosage form.

32. Storage temperature of 2-8°C, for example 5°C, for at least 6 months, for example at least 9 months or less. It remains stable for pharmaceutical use for at least 12 months. or the pharmaceutical composition according to any one of claims 1 to 31.

33. Use of the pharmaceutical composition according to any one of claims 1 to 25 for subcutaneous administration.

34. Use of the pharmaceutical composition according to any one of claims 1 to 25 for intravenous administration.

35. The use of the pharmaceutical composition according to claim 33 or 34, wherein the use is for the treatment of cancer.

36. A method for treating cancer in a subject, wherein the subject in need of the treatment is provided according to claims 1 to 32. A method comprising administering any of the pharmaceutical compositions described in item 1 for a period of time sufficient to treat cancer. 。

37. The method according to claim 36, wherein the composition is administered subcutaneously or intravenously.

38. The method according to claim 36 or 37, wherein the cancer is a B-cell malignant tumor.

39. Unit dose formulations that include or are essentially derived from the following: a The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2: GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID No. 12 It contains a bispecific antibody in an amount of 5 μg to 50 mg. b. Acetate buffer and sorbitol in a ratio between 1:5 and 1:10, where the unit dose The osmotic pressure of the dosage form is approximately 210 to 250, and the pH is approximately 5.4 to 5.

6.

40. Unit dose formulations that include or are essentially derived from the following: a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2: GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID No. 12 It contains a bispecific antibody in an amount of 5 μg to 50 mg. b. 30 mM acetic acid c. Sorbitol at a concentration of 150 mM, The pH here is 5.

5.

41. The first binding region of the bispecific antibody that binds to human CD3 contains the VH and VL sequences of SEQ ID NOs: 6 and 7. Furthermore, the second binding region of the bispecific antibody that binds to human CD20 is VH and V of SEQ ID NOs. 13 and 14. A unit dose dosage form according to claim 39 or 40, comprising an L sequence.

42. The bispecific antibodies include the first and second heavy chain constant regions of SEQ ID NOs. 19 and 20, respectively, as requested. The unit dose dosage form as described in item 41.

43. The amount of bispecific antibody is 50 μg to 40 mg, as per any one of claims 39 to 42. Dosage-based formulation.

44. The amount of bispecific antibody ranges from 100 μg to 30 mg, for example, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg. , 400μg, 450μg, 500μg, 600μg, 700μg, 800μg, 900μg, 1mg, 2mg, 3mg, 4mg, 5m g, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19m g, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, for example, 30mg. or the unit dose dosage form according to any one of claims 39 to 43.

45. The total volume is 0.5 mL to 2 mL, for example, 1 mL, as per any one of claims 39 to 44. Dosage form.

46. The unit dose dosage form according to claim 45, wherein the unit dose dosage form is for subcutaneous administration.

47. The total volume is 20 mL to 200 mL, and the dosage form is for intravenous administration, claim 3. The unit dose dosage form described in any one of items 9 to 44.

48. A method for treating cancer in the subject, To treat cancer, the unit dose formulation described in any one of claims 39 to 47 is administered to the target who requires it. A method that includes administering the dose for a sufficient amount of time.

49. A unit dose formulation according to any one of claims 39 to 47 for use in the treatment of cancer.

50. A container comprising a unit dose dosage form according to any one of claims 39 to 45.

51. a. The pharmaceutical composition according to any one of claims 1 to 25 b. Diluent containing acetic acid and sorbitol c. Container for unit dose formulations d. Instructions for dilution and / or use A parts kit that includes this.

52. The diluent and the pharmaceutical composition have the same concentration ratio of acetic acid to sorbitol, as described in claim 51. Parts kit.

53. a. Pharmaceutical compositions containing the following: i. 60 mg / mL bispecific antibody ii. 30 mM acetate buffer iii. 150 mM sorbitol iv. The pH is 5.

5. b. Diluents containing the following: i. 30 mM acetate buffer ii. 150 mM sorbitol c. Containers for unit dose formulations, and, d. Instructions for dilution and / or use, A parts kit according to claim 51 or 52, including the following:

54. A method for preparing a pharmaceutical composition according to any one of claims 1 to 32, a. The first binding region that binds to human CD3 containing the following CDR sequence: VH-CDR1: Sequence ID 1 VH-CDR2: Sequence ID 2 VH-CDR3: Sequence ID 3 VL-CDR1: Sequence ID 4 VL-CDR2: GTN, and VL-CDR3: Sequence ID 5 Furthermore, a second binding region that binds to human CD20 containing the following CDR sequence: VH-CDR1: Sequence ID 8 VH-CDR2: Sequence ID 9 VH-CDR3: Sequence ID 10 VL-CDR1: Sequence ID 11 VL-CDR2:DAS, and VL-CDR3: Sequence ID No. 12 A bispecific antibody containing 60-120 mg / mL, b. 3.53 mg / mL sodium acetate trihydrate c. 0.32 mg / mL acetic acid d. 27.3 mg / mL sorbitol The process of mixing in water for injection; and A step to adjust the pH to 5.5 by adding sodium hydroxide; A method that includes this.

55. The method according to claim 54, wherein a is 60 mg / mL.

56. The method according to claim 54, wherein a is 120 mg / mL.

57. A method for preparing a unit dose dosage form as defined in any one of claims 39 to 45, 、 a. A step of preparing a pharmaceutical composition by the method described in any one of claims 54 to 56; b. Diluents containing the following i. 3.53 mg / mL sodium acetate trihydrate ii. 0.32 mg / mL acetic acid iii. 27.3 mg / mL sorbitol iv. Sodium hydroxide to adjust the pH to 5.5 A step of preparing a diluent containing the above in water for injection; and c. A step of mixing the pharmaceutical composition and diluent to obtain the desired bispecific antibody concentration; Methods that include...

58. Pharmaceutical compositions or unit dose agents obtainable by the method described in any one of claims 54 to 57. Type.

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