Pharmaceutical Composition and Method of Use of Anti-CD20 / Anti-CD3 Bispecific Antibody

Abstract

The present invention relates to pharmaceutical compositions of anti-CD20 / anti-CD3 bispecific antibodies and methods of use thereof.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition of an anti-CD20 / anti-CD3 bispecific antibody and a method for using the same.

Background Art

[0002] One of the major challenges in the development of biotechnological therapies is the stability of proteins, and it is necessary to maintain the stability of proteins in multiple process steps until they are launched on the market. Furthermore, the stability of proteins must be maintained not only during storage but also during administration to patients. Therapeutic antibodies can be formulated in an aqueous carrier for administration to a subject, for example, by intravenous administration or subcutaneous administration. During the storage, handling, and administration of such pharmaceutical compositions, it is necessary to reduce the loss of therapeutic antibodies that can occur due to degradation and surface adsorption, such as the adsorption of proteins to the surfaces of filters, storage canisters, tubes, syringes, infusion bags, and other containers. Both low-concentration formulations and high-concentration formulations pose respective challenges during research and development and manufacturing. For example, low concentrations are strongly affected by surface adsorption, while high concentrations may exhibit high viscosities.

[0003] When a pharmaceutical composition contains a relatively low concentration of a therapeutic protein, the loss of the protein may increase dramatically due to these factors, and as a result, the therapeutic effect of the pharmaceutical composition decreases.

[0004] Therefore, in the art, it is necessary to develop a pharmaceutical formulation in which an anti-CD20 / anti-CD3 bispecific antibody (for example, a low-dose anti-CD20 / anti-CD3 bispecific antibody, for example, a low-dose anti-CD20 / anti-CD3 T cell-engaging bispecific antibody, for example, glofitamab) is stable and protected from loss due to adsorption.

Summary of the Invention

[0005] The present invention relates to pharmaceutical compositions of anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 T cell engaging bispecific antibodies (TCBs), e.g., glofitamab, RO7082859, or RG6026), and methods of using the same. The disclosed compositions and related methods address the problem of delivering anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 TCBs, e.g., glofitamab) formulated at low concentrations, enabling patients to receive the intended dose of anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) with little or no loss of protein during storage and administration.

[0006] In one aspect, the present invention is a liquid pharmaceutical composition comprising at a pH in the range of about 5.0 to about 6.0, from about 1 to 25 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody; from about 10 to 50 mM of a buffer; at least about 200 mM of an isotonicity agent; from about 0 to 15 mM of methionine; and at least about 0.2 mg / ml of a surfactant; wherein the anti-CD20 / anti-CD3 bispecific antibody comprises a) at least one antigen-binding domain that specifically binds to CD20, comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 in a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 in a light chain variable region, and at least one antigen-binding domain that specifically binds to CD20, b) at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising, at least one antigen-binding domain that specifically binds to CD3 comprising, a liquid pharmaceutical composition.

[0007] In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) ranges from about 1 to 5 mg / ml. In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) ranges from about 0.9 to 1.1 mg / ml. In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is about 1 mg / ml.

[0008] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) a) at least one antigen-binding domain that specifically binds to CD20, comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; b) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; comprising.

[0009] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises: a) a first Fab molecule that specifically binds to CD3, particularly CD3 epsilon, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are exchanged with each other; b) a second Fab molecule and a third Fab molecule that specifically bind to CD20, wherein in the constant domain CL of the second Fab molecule and the third Fab molecule, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted with lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second Fab molecule and the third Fab molecule, the amino acid at position 147 is substituted with glutamic acid (E) (EU numbering), the amino acid at position 213 is substituted with glutamic acid (E) (EU numbering); c) an Fc domain composed of a first subunit and a second subunit capable of stable association; and comprises.

[0010] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0011] In one embodiment, the buffer is a histidine buffer, optionally a histidine HCl buffer. In one embodiment, the buffer has a concentration of about 15 to 25 mM. In one embodiment, the buffer has a concentration of about 20 mM. In one embodiment, the buffer provides a pH of about 5.2 to about 5.8.

[0012] In one embodiment, the isotonic agent is selected from the group consisting of salts, sugars, and amino acids. In one embodiment, the isotonic agent is either sucrose or sodium chloride. In one embodiment, the isotonic agent is sucrose at a concentration of about 200 mM or more. In one embodiment, the isotonic agent is sucrose at a concentration of about 200 mM to 280 mM. In one embodiment, the isotonic agent is sucrose at a concentration of about 240 mM.

[0013] In one embodiment, methionine is at a concentration of about 5 to 15 mM.

[0014] In one embodiment, methionine is at a concentration of about 10 mM. In one embodiment, the surfactant is at a concentration of about 0.2 to 0.8 mg / ml. In one embodiment, the surfactant is polysorbate 20 or poloxamer 188. In one embodiment, the surfactant is polysorbate 20 at a concentration of 0.2 to 0.8 mg / ml. In one embodiment, the surfactant is polysorbate 20 at a concentration of about 0.5 mg / ml.

[0015] In one embodiment, the liquid pharmaceutical composition has a pH of from about 5 to about 6, and is from about 1 to 5 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), comprising a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region, At least one antigen-binding domain that specifically binds to CD20, b) At least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 A heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 A light chain variable region comprising, At least one antigen-binding domain that specifically binds to CD3, An anti-CD20 / anti-CD3 bispecific antibody at about 1 to 5 mg / ml; About 15-25 mM histidine buffer; About 200-280 mM sucrose; About 0-15 mM methionine; About 0.2-0.8 mg / ml PS20 Comprising.

[0016] In one embodiment, the liquid pharmaceutical composition is At a pH of about 5.5, About 1 mg / ml glofitamab; About 20 mM histidine buffer; About 240 mM sucrose; About 10 mM methionine; About 0.5 mg / ml PS20 Comprising.

[0017] In one embodiment, the present invention provides the use of a liquid pharmaceutical composition of any of the foregoing embodiments and aspects for the preparation of a medicament useful for the treatment of a cell proliferative disorder.

[0018] In another aspect, the invention features a pharmaceutical composition according to any of the foregoing aspects and embodiments for use in treating or delaying the progression of a cell proliferative disorder in a subject in need thereof.

[0019] In another aspect, the invention features a pharmaceutical composition according to any of the foregoing aspects and embodiments for use in treating or delaying the progression of a cell proliferative disorder in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition according to any of the foregoing aspects and embodiments.

[0020] In certain embodiments, the cell proliferative disorder is cancer.

[0021] A further aspect of the invention pertains to the invention described herein.

[0022] Each embodiment and any embodiment can be combined, unless the context clearly indicates otherwise. Each embodiment and any embodiment can be applied to each aspect and any aspect of the invention, unless the context clearly indicates otherwise.

[0023] Certain embodiments of the invention will become apparent from the following more detailed description of specific preferred embodiments and the claims.

[0024] The content of this application file contains at least one drawing created in color. A copy of this patent or this patent application with color drawings will be provided by the Patent Office upon request and payment of the necessary fees. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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Mode for Carrying Out the Invention

[0026] The present invention relates to a pharmaceutical composition of an anti-CD20 / anti-CD3 bispecific antibody and methods of using the same. The disclosed compositions and related methods address the problem of delivering an anti-CD20 / anti-CD3 bispecific antibody formulated at low concentrations such that a patient receives the intended dose of the anti-CD20 / anti-CD3 bispecific antibody with little or no loss of the bispecific antibody during storage and administration.

[0027] I. General Techniques The practice of the present disclosure, unless otherwise described, uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are well described in the literature, for example, in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001), and the like.

[0028] II. Definitions The terms used herein are used as generally used in the art, unless otherwise specifically defined herein.

[0029] As used herein, the term "cluster of differentiation 20" or "CD20" refers to any native CD20 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the human protein is characterized by UniProt database entry number P11836) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD that is expressed on pre-B and mature B lymphocytes (Valentine, M.A. et al., J. Biol. Chem. 264 (1989)11282-11287; Tedder, T.F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988)208-212; Stamenkovic, I., et al., J. Exp. Med. 167 (1988)1975-1980; Einfeld, D.A., et al., EMBO J. 7 (1988)711-717; Tedder, T.F., et al., J. Immunol. 142 (1989)2560-2568). The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes members of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splice boundaries and exhibit unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B-lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is located at 11q12 within a cluster of family members. This term encompasses "full-length", unprocessed CD20, as well as any form of CD20 resulting from intracellular processing. This term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. Alternative splicing of this gene results in two transcript variants that encode the same protein.In one embodiment, the CD20 is human CD20.

[0030] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that is capable of binding to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20. In one embodiment, the degree of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 From 10 -13 M, for example 10 -9 From 10 -13 Dissociation constant (K D In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 from different species.

[0031] "Type II anti-CD20 antibody" means an anti-CD20 antibody having the binding characteristics and biological activity of the type II anti-CD20 antibodies described in Cragg et al., Blood 103 (2004) 2738-2743; Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1 below. TIFF0007701982000001.tif83170

[0032] Examples of Type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumomab (B1), humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (disclosed in WO 2004 / 035607) and AT80 IgG1.

[0033] Examples of type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, belimumab, ocrelizumab, obinutuzumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in WO 2005 / 103081), 2F2 IgG1 (disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (disclosed in WO 2004 / 056312).

[0034] Unless otherwise indicated, "CD3" refers to any native CD3 derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). This term encompasses "full-length", unprocessed CD3, as well as any form of CD3 resulting from intracellular processing. This term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank number BAB71849.1.

[0035] The terms "anti-CD20 / anti-CD3 antibody", "anti-CD20 / anti-CD3 bispecific antibody", and "bispecific antibody that binds to CD20 and CD3" refer to bispecific antibodies that can bind to CD20 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CD20 and / or CD3. In one embodiment, the degree of binding of the bispecific antibody that binds to CD20 and CD3 to unrelated non-CD3 proteins and / or non-CD20 proteins is less than about 10% of the binding of the antibody to CD3 and / or CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody binds to each of CD20 and / or CD3 having a dissociation constant (K D ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the bispecific antibody that binds to CD20 and CD3 binds to an epitope of CD3 that is conserved among CD3s from different species and / or an epitope of CD20 that is conserved among CD20s from different species. An example of an anti-CD20 / anti-CD3 bispecific antibody is glofitamab (WHO Drug Information (International Nonproprietary Name), Recommended INN: List 83, 2020, vol. 34, no. 1, p. 39, anti-CD20 / anti-CD3 T cell engaging bispecific antibody (TCB), CD20-TCB, RO7082859, or RG6026; CAS #: 2229047-91-8).

[0036] As used herein, the term "amino acid mutation" is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made so long as the final construct reaches the final construct as long as the final construct has a decrease in a desired characteristic, such as binding to an Fc receptor. Deletions and insertions of amino acid sequences include amino-terminal and / or carboxy-terminal deletions, as well as insertions of amino acids. In particular, the amino acid mutation is an amino acid substitution. For the purpose of changing the binding properties of the Fc region, it is particularly preferred to perform a non-conservative amino acid substitution, that is, to replace one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids, or replacement with naturally occurring amino acid derivatives of the 20 common amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic methods or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering, such as methods for changing the side chain groups of amino acids by chemical modification, may also be considered useful. In this specification, various notations are used to indicate the same amino acid mutation. For example, the substitution of proline at position 329 in the Fc region with glycine can be indicated as 329G, G329, G 329 , P329G, or Pro329Gly.

[0037] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand) unless otherwise indicated. The affinity of molecule X for partner Y is usually expressed by the dissociation constant (K D ), which is the dissociation rate constant and the association rate constant (k off and k on) ratio. Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0038] The term "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to the parental antibody without such modifications, and such modifications improve the affinity of the antibody for the antigen.

[0039] As used herein, the term "antigen-binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding portion can direct the entity to which it binds (e.g., a cytokine or a second antigen-binding portion) to a target site, e.g., a particular type of tumor cell or a tumor stroma having an antigenic determinant. The antigen-binding portion includes antibodies and fragments thereof as further defined herein. Preferred antigen-binding portions include the antigen-binding domain of an antibody, which includes the variable region of the antibody heavy chain and the variable region of the antibody light chain. In some embodiments, the antigen-binding portion includes an antibody constant region known in the art, as further defined below. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include either of the two isotypes: κ and λ.

[0040] "Specific binding" means that the binding is antigen - selective and can be distinguished from unwanted or non - specific interactions. The ability of an antigen - binding moiety to bind to a specific antigenic determinant can be measured by enzyme - linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance technology (analysis with a BIACORE (registered trademark) instrument) (Liljeblad et al., Glyco J. 17, 323 - 329 (2000)), and classical binding assays (Heeley, Endocr Res. 28, 217 - 229 (2002)). In one embodiment, the degree of binding of the antigen - binding moiety to an irrelevant protein is less than about 10% of the binding of the antigen - binding moiety to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen - binding moiety that binds to an antigen, or an antigen - binding molecule comprising the antigen - binding moiety, has a dissociation constant (K -8 d) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 −9 -13 M to 10 -9 −8 -13 M, e.g., 10 D −9

[0041] "Reduction of binding", e.g., reduction of binding to an Fc receptor, refers to a decrease in affinity for each interaction, as measured, for example, by SPR. For clarity, the term also includes reduction to zero (or below the detection limit of the analytical method) of the affinity, i.e., complete termination of the interaction. Conversely, "increase of binding" refers to an increase in binding affinity for each interaction.

[0042] As used herein, the term "antigen - binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen - binding molecules are immunoglobulins and derivatives, such as fragments thereof.

[0043] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex (e.g., a conformational structure formed from a contiguous stretch of amino acids or different regions of non-contiguous amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., CD3) refers to any native form of a protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. This term, when referring to a specific protein herein, encompasses both the "full-length," unprocessed protein and any form of the protein obtained from intracellular processing. This term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants. Exemplary human proteins useful as antigens include CD3, particularly the epsilon subunit of CD3 (for the human sequence, see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1; for the cynomolgus monkey [Macaca fascicularis] sequence, see UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1). In certain embodiments, the T cell-activating bispecific antigen-binding molecules described herein bind to epitopes of CD3 or target cell antigens that are conserved among CD3s or target cell antigens from various species.

[0044] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly linked by amino bonds (also known as peptide bonds). The term "polypeptide" refers to a chain consisting of two or more amino acids, and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term referring to a chain of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modification of polypeptides, and such products include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, protein cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be obtained from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides can be produced by any method, including chemical synthesis. The polypeptides of the present invention are of a size consisting of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may or may not have a well-defined three-dimensional structure. A polypeptide having a well-defined three-dimensional structure is said to be "folded", and a polypeptide that can adopt a number of different conformations without having a well-defined three-dimensional structure is said to be "unfolded".

[0045] An "isolated" polypeptide or variant, or derivative thereof, is intended to be a polypeptide that is not in its natural environment. In particular, purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinant production polypeptides and proteins expressed in host cells are considered to be isolated for the purposes of the present invention, in the same manner as natural or recombinant polypeptides that have been separated, fractionated or partially or substantially purified by any suitable technique.

[0046] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps as necessary. Alignments for determining the percent amino acid sequence identity can be obtained using various methods within the scope of the art, such as commonly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR®) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment for the full lengths of the sequences being compared. However, for the purposes of this specification, the percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code was submitted to the U.S. Copyright Office, Washington, D.C. 20559, along with user documentation, and is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its source code. The ALIGN-2 program should be compiled for use on a UNIX® operating system, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In the situation where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to (or with respect to) a given amino acid sequence B (or, a given amino acid sequence A that has or contains a specific percent amino acid sequence identity to (or with respect to) a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y Here, X is the number of amino acid residues with scores that match identically in the alignment of the programs of A and B by the array alignment program ALIGN-2, and Y is the total number of amino acid residues of B. It will be understood that when the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0047] As used herein, the term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity.

[0048] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to an antibody that has a structure substantially similar to the native antibody structure or has a heavy chain containing an Fc region as defined herein.

[0049] “Antibody fragment” refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), and multispecific antibodies formed from antibody fragments. The term “antibody fragment” as used herein also encompasses single-domain antibodies.

[0050] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins can be assigned to one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains. An immunoglobulin consists essentially of two Fab molecules and an Fc domain linked via the hinge region of the immunoglobulin.

[0051] The term "antigen-binding domain" refers to the portion of an antibody that specifically binds to a part or all of an antigen and contains a region complementary to a part or all of the antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0052] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure, each containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). For example, see Kindt et al., Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007). In some cases, a single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0053] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire, or an amino acid sequence corresponding to a sequence encoding another human antibody. This definition of a human antibody clearly excludes humanized antibodies that contain non-human antigen-binding residues.

[0054] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially includes all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0055] The term "hypervariable region" or "HVR", as used herein, refers to each region of an antibody variable domain that is hypervariable in an array ( "complementary determining region" or "CDR"), and / or forms a predetermined loop structurally ( "hypervariable loop"), and / or contains residues that contact an antigen ( "antigen contact"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs of the invention include the following: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) Combinations of (a), (b) and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0056] Unless otherwise indicated, HVR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. as described above.

[0057] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of the variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0058] "Human consensus framework" is a framework that represents the amino acid residues that occur most commonly in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is the subgroup of Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. supra.

[0059] For the purposes of this specification, an "acceptor human framework" is a framework that includes the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0060] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0061] As used herein, the terms "IgG isotype" or "subclass" mean any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0062] The terms "Fc domain" or "Fc region" are used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an IgG heavy chain can vary slightly, but the Fc region of a human IgG heavy chain is typically defined as extending from Cys226 or Pro230 of the heavy chain to the carboxyl terminus. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, expression of a particular nucleic acid molecule encoding a full-length heavy chain may result in antibodies produced by a host cell that include either the full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Thus, the C-terminal lysine (Lys447) of the Fc region, or glycine (Gly446) and lysine (K447) at the C-terminus may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). The "subunit" of an Fc domain as used herein refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain and has the ability to stably self-associate. For example, the subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0063] "Modifications that facilitate the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the formation of homodimers by the association of a polypeptide containing the Fc domain subunits with an identical polypeptide. The modifications that facilitate association as used herein specifically include separate modifications made to each of the two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and these modifications are complementary to each other in order to facilitate the association of the two Fc domain subunits. For example, the modifications that facilitate association change the structure or charge of one or both of these Fc domain subunits such that their association is sterically or electrostatically desirable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first Fc domain subunit and a polypeptide containing the second Fc domain subunit, which can be non-identical in the sense that additional components (e.g., antigen-binding portions) fused to each of the subunits are not the same. In some embodiments, the modifications that facilitate association include amino acid mutations within the Fc domain, specifically amino acid substitutions. In certain embodiments, the modifications that facilitate association include separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0064] "Activating Fc receptor" is an Fc receptor that, following engagement by the Fc region of an antibody, induces a signaling event that stimulates receptor-bearing cells to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0065] As used herein, the term "effector function," when used in reference to an antibody, refers to a biological activity attributable to the Fc region of the antibody that varies by the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0066] As used herein, the term "effector cell" refers to a population of lymphocytes that express on their surface an effector moiety receptor, such as a cytokine receptor, and / or an Fc receptor, thereby binding to an effector moiety, such as a cytokine, and / or the Fc region of an antibody, and contributing to the destruction of target cells, such as tumor cells. Effector cells mediate, for example, cytotoxic or phagocytic effects. Effector cells include, but are not limited to, effector T cells, such as CD8 + cytotoxic T cells, CD4 + helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages / monocytes.

[0067] As used herein, the terms "engineer(ed)(ing)" and "engineering" with respect to a naturally occurring polypeptide, a recombinant polypeptide, or a fragment thereof are considered to include any manipulation of the peptide backbone or post-translational modification. Engineering manipulations include modification of the amino acid sequence, modification of the glycosylation pattern, or modification of the side chain groups of individual amino acids, as well as combinations of these techniques. In particular, "engineering" with the prefix "glyco-", and the term "glycosylation engineering" include metabolic engineering of the glycosylation machinery of a cell, including genetic manipulation of the oligosaccharide synthesis pathway to effect changes in the glycosylation of glycoproteins expressed intracellularly. Further, glycosylation engineering includes mutations to glycosylation and the effects of the cellular environment. In one embodiment, glycosylation engineering is a change in glycosyltransferase activity. In certain embodiments, the engineering results in a change in N-acetylglucosaminyltransferase activity and / or fucosyltransferase activity. Glycosylation engineering can be used to obtain a "host cell having increased GnTIII activity" (e.g., a host cell engineered to express one or more polypeptides at elevated levels having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), a "host cell having increased ManII activity" (e.g., a host cell engineered to express one or more polypeptides at elevated levels having α-mannosidase II (ManII) activity), or a "host cell having reduced α(1,6) fucosyltransferase activity" (e.g., a host cell engineered to express α(1,6) fucosyltransferase at reduced levels).

[0068] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell and may include mutations. Mutant progeny having the same function or biological activity as selected or screened in the original transformed cell are included herein. A host cell is any type of cell line that can be used to produce a protein of the invention. In one embodiment, the host cell is engineered to enable the production of an antibody having modified oligosaccharides. In certain embodiments, the host cell is engineered to increase the expression level of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cell is further engineered to increase the expression level of one or more polypeptides having α-mannosidase II (ManII) activity. A host cell includes cultured cells, such as mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloid cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0069] As used herein, the term "polypeptide having GnTIII activity" refers to a polypeptide capable of catalyzing the addition of an N-acetylglucosamine (GlcNAc) residue to the β-linked mannoside of the trimannosyl core of an N-linked oligosaccharide. This includes fusion polypeptides that exhibit enzymatic activity similar to, but not necessarily identical to, the activity of β(1,4)-N-acetylglucosaminyltransferase III, also known as β-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.144) according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), with or without dose-dependence when measured in a specific biological assay. When dose-dependence exists, it need not be the same as that of GnTIII; rather, it should be substantially similar to the dose-dependence at a given activity when compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or an activity of less than about 1 / 25, preferably less than about 1 / 10, and most preferably less than about 1 / 3 of the activity of GnTIII). In certain embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi-resident polypeptide. In particular, the Golgi localization domain is the localization domain of mannosidase II or GnTI, and most specifically the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of α1,6 core fucosyltransferase. Methods for producing such fusion polypeptides and using them to produce antibodies with enhanced effector functions are disclosed in International Publication No. WO 2004 / 065540, U.S. Provisional Patent Application No. 60 / 495142, and U.S. Patent Publication No. 60 / 495142, the entire contents of which are hereby expressly incorporated by reference.

[0070] As used herein, the term "Golgi localization domain" refers to the amino acid sequence of a Golgi-resident polypeptide that serves to anchor a polypeptide at a location within the Golgi complex. Generally, the localization domain constitutes the amino-terminal "tail" of the enzyme.

[0071] As used herein, the term "polypeptide having ManII activity" refers to a polypeptide that is capable of catalyzing the hydrolysis of the terminal 1,3- and 1,6-linked α-D-mannose residues of the branched GlcNAcMan5GlcNAc2 mannose intermediate of N-linked oligosaccharides. This includes polypeptides that exhibit activity similar to, but not necessarily identical to, that of Golgi α-mannosidase II, also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC 3.2.1.114) according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).

[0072] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of target cells coated with antibodies by immune effector cells. The target cells are cells to which an antibody or a fragment thereof containing an Fc region specifically binds, usually via a protein moiety that is the N-terminus relative to the Fc region. As used herein, the term "increased / decreased ADCC" refers to an increase / decrease in the number of target cells lysed within a predetermined time by the ADCC mechanism at a predetermined antibody concentration in the medium surrounding the target cells, and / or a decrease / increase in the antibody concentration in the medium surrounding the target cells required to lyse a predetermined number of target cells within a predetermined time by the ADCC mechanism. Increased / decreased ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell, which is not manipulated, using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, an increase in ADCC mediated by an antibody produced by a host cell engineered to have a glycosylation pattern (e.g., glycosyltransferase, GnTIII, or other glycosyltransferases) engineered by the methods described herein is relative to ADCC mediated by the same antibody produced by the same type of non-engineered host cell.

[0073] An "antibody having increased / decreased antibody-dependent cell cytotoxicity (ADCC)" means an antibody having an increase / decrease in ADCC determined by any suitable method known to those skilled in the art. One acceptable in vitro ADCC assay is as follows: 1) The assay uses target cells known to express a target antigen recognized by the antigen-binding region of the antibody; 2) The assay uses, as effector cells, human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors; 3) The assay is performed according to the following protocol. i) Isolate PBMCs using standard density centrifugation procedures and resuspend them in RPMI cell culture medium at 5×10 6 cells / ml; ii) Grow the target cells by standard tissue culture methods, harvest them from the exponential growth phase with a viability higher than 90%, wash them with RPMI cell culture medium, and label them with 100 microcuries of 51 Cr, wash them twice with cell culture medium, and resuspend them in cell culture medium at a density of 10 5 cells / ml; iii) Transfer 100 microliters of the final target cell suspension above to each well of a 96-well microtiter plate; iv) Serial dilute the antibody in cell culture medium from 4000 ng / ml to 0.04 ng / ml, add 50 microliters of the resulting antibody solution to the target cells in the 96-well microtiter plate, and test various antibody concentrations covering the above full concentration range in triplicate; v) For the maximum release (MR) control, add 50 microliters of a 2% (v / v) aqueous solution of a non-ionic detergent (Nonidet, Sigma, St. Louis) to three additional wells in the plate containing the labeled target cells, instead of the antibody solution (in iv) above; vi) As a spontaneous release (SR) control, add 50 microliters of RPMI cell culture medium to three additional wells in the plate containing the labeled target cells, instead of the antibody solution (in iv) above; vii) Then, centrifuge the 96-well microtiter plate at 50 × g for 1 minute and incubate it at 4°C for 1 hour; viii) Add 50 microliters of the PBMC suspension (in i) above to each well to obtain an effector:target cell ratio of 25:1, and place the plate in an incubator at 37°C for 4 hours in a 5% CO2 atmosphere; ix) Recover the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) Calculate the percentage of specific lysis for each antibody concentration according to the formula (ER - MR) / (MR - SR)×100 (where ER is the average radioactivity quantified for that antibody concentration (see above ix), MR is the average radioactivity quantified for the MR control (see above v) (see above ix), and SR is the average radioactivity quantified for the SR control (see above vi) (see above ix)); 4) "Increased / decreased ADCC" is defined as either an increase in the maximum percentage of specific lysis observed within the tested antibody concentration range and / or a decrease / increase in the antibody concentration required to achieve one-half of the maximum percentage of specific lysis observed within the tested antibody concentration range. The increased / decreased ADCC measured in the above assay is relative to the ADCC mediated by the same antibody produced by the same type of host cell, produced using the same standard production, purification, formulation, and storage methods, but not manipulated, known to those of ordinary skill in the art.

[0074] As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (which may include, for example, naturally occurring variants or those that arise during the production of a monoclonal antibody preparation, and such variants are usually present in minor amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0075] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in a pharmaceutical formulation.

[0076] "Natural antibodies" refer to naturally occurring immunoglobulin molecules having various structures. For example, natural IgG antibodies are approximately 150,000 Dalton heterotetrameric glycoproteins composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by one constant light (CL) domain. The light chains of an antibody may be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains.

[0077] As used herein, terms such as "first," "second," "third," etc. with respect to antigen-binding portions or domains are used for convenience of distinction when more than one of each type of portion or domain is present. The use of these terms is not intended to give a particular order or orientation unless so expressly indicated.

[0078] The terms "multispecificity" and "bispecificity" mean that an antigen-binding molecule is capable of specifically binding to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule is capable of binding simultaneously to two antigenic determinants (particularly, two antigenic determinants expressed on two distinct cells).

[0079] As used herein, the term "valent" ("valent" or "valency") means the presence of a specific number of antigen-binding sites in an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" means the presence of one (and only one) antigen-binding site specific for the antigen in the antigen-binding molecule.

[0080] "Antigen-binding site" refers to the site of an antigen-binding molecule that confers interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody includes the amino acid residues of the complementarity-determining region (CDR). A natural immunoglobulin molecule typically has two antigen-binding sites, and a Fab molecule typically has a single antigen-binding site.

[0081] As used herein, "activated T cell antigen" refers to an antigen determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, and capable of inducing or enhancing T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with an activated T cell antigen can induce T cell activation by triggering the signaling cascade of the T cell receptor complex. An exemplary activated T cell antigen is CD3. In a particular embodiment, the T cell activation antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1 for the human sequence; UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1 for the cynomolgus macaque [Macaca fascicularis] sequence).

[0082] As used herein, "T cell activation" refers to one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of T lymphocytes, particularly cytotoxic T lymphocytes. The T cell activation therapeutic agents used in the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0083] As used herein, "target cell antigen" refers to an antigen determinant presented on the surface of target cells, such as cells within a tumor, for example, cancer cells or cells of the tumor stroma. In a particular embodiment, the target cell antigen is CD20, particularly human CD20 (see UniProt number P11836).

[0084] As used herein, "B cell antigen" refers to an antigenic determinant present on the surface of B lymphocytes, particularly malignant B lymphocytes (in this case, the antigen is also referred to as "malignant B cell antigen").

[0085] As used herein, "T cell antigen" refers to an antigenic determinant presented on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0086] "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").

[0087] "Fused" means that components (e.g., Fab molecule and Fc domain subunit) are joined by peptide bonds, either directly or via one or more peptide linkers.

[0088] "Effective amount" of a drug refers to the amount necessary to cause a physiological change in the cell or tissue to which the drug is administered.

[0089] "Therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an effective amount for achieving a desired therapeutic or prophylactic result, in the required dosage of the drug and for the required time. The therapeutically effective amount of a drug, for example, eliminates, reduces, delays, minimizes, or prevents side effects of a disease.

[0090] "Therapeutic agent" means, for example, the active ingredient of a pharmaceutical composition, and the drug is administered to a subject for the purpose of altering the natural course of a disease in the subject being treated, for prophylaxis, or during the course of a clinical pathology. "Immunotherapeutic agent" refers to a therapeutic agent that is administered to a subject, for example, for the purpose of restoring or enhancing the subject's immune response against a tumor.

[0091] The term "pharmaceutical composition" refers to a formulation in a form that enables the biological activity of the active ingredient contained therein and does not contain additional components that are so toxic as to be unacceptable to the subject to which the composition is administered.

[0092] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0093] The term "package insert" or "instructions for use" is used to refer to the instructions customarily included in the commercial packaging of a therapeutic product and includes information about indications, usage, dosage, administration, combination therapy, contraindications and / or precautions regarding the use of the therapeutic product.

[0094] As used herein, the term "combination therapy" encompasses co-administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administrations, in which case the administration of the antibodies reported herein can occur before, simultaneously with, and / or after the administration of one or more additional therapeutic agents, preferably one or more antibodies.

[0095] The "crossover" Fab molecule (also referred to as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced by each other), that is, the crossover Fab molecule includes a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (in the direction from the N-terminus to the C-terminus), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (in the direction from the N-terminus to the C-terminus). Briefly, in a crossover Fab molecule in which the variable domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0096] In contrast, the "conventional" Fab molecule means a Fab molecule in the native format, that is, a Fab molecule containing a heavy chain composed of the variable domain and constant domain of the heavy chain (in the direction from the N-terminus to the C-terminus, VH-CH1), and a light chain composed of the variable domain and constant region of the light chain (in the direction from the N-terminus to the C-terminus, VL-CL).

[0097] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides can include common phosphodiester bonds or non-common bonds (e.g., amide bonds as found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments.

[0098] An "isolated" nucleic acid molecule or polynucleotide is intended to mean a nucleic acid molecule, DNA or RNA, removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or (partially or substantially) purified polynucleotides in solution. An isolated polynucleotide contains polynucleotide molecules that are normally contained in a cell that contains polynucleotide molecules, but the polynucleotide molecules are present extrachromosomally or at a chromosomal location different from their original chromosomal location. Isolated RNA molecules include RNA transcripts of the present invention in vivo or in vitro, and positive and negative strand forms, double-stranded forms. The isolated polynucleotides or nucleic acids of the present invention further include such molecules produced by synthesis. Also, the polynucleotide or nucleic acid may or may not contain regulatory elements such as promoters, ribosome binding sites or transcription terminators.

[0099] The reference nucleotide sequence of the present invention and a nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" means that the nucleotide sequence of the polynucleotide is identical to the reference nucleotide sequence except that it may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or at any position between these terminal positions, and may be scattered individually among the residues within the reference sequence or scattered in one or more continuous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the present invention can be determined using a computer program (e.g., ALIGN-2) such as that described above for polypeptides.

[0100] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically using a series of specific nucleic acid elements that enable the transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0101] The terms "vector" or "expression vector" are synonymous with "expression construct" and refer to a DNA molecule that is used to introduce and direct the expression of a specific gene that operably binds within a target cell. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of the introduced host cell. The expression vectors of the present invention include an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. When the expression vector enters the interior of the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell transcription and / or translation machinery. In certain embodiments, the expression vectors of the present invention include an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0102] As used herein, the term "about" refers to the normal error range of each value, which would be readily understood by one of ordinary skill in the art. References to "about" values or parameters herein include (describe) embodiments that are directed to the value or parameter itself.

[0103] "B cell proliferative disorder" means a disease in which the number of a patient's B cells is increased compared to the number of B cells in a healthy individual, particularly a disease in which the increase in the number of B cells is the cause or evidence of the disease. "CD20-positive B cell proliferative disease" is a B cell proliferative disease in which B cells, particularly malignant B cells (in addition to normal B cells), express CD20.

[0104] Exemplary B cell proliferative disorders include non-Hodgkin lymphoma (NHL), diffuse large B cell lymphoma (DLBCL; e.g., recurrent or refractory DLBCL not otherwise specified (NOS)), high-grade B cell lymphoma (HGBCL; e.g., HGBCL NOS, double-hit HGBCL, and triple-hit HGBCL), primary mediastinal large B cell lymphoma (PMBCL), and DLBCL transformation from FL; trFL)); follicular lymphoma (FL) (including Grade 1-3b FL); mantle cell lymphoma (MCL); and marginal zone lymphoma (MZL) (including splenic, nodal, or extranodal MZL). In one embodiment, the CD20-positive B cell proliferative disorder is a recurrent or refractory NHL (e.g., recurrent or refractory DLBCL, recurrent or refractory FL, or recurrent or refractory MCL).

[0105] A "refractory disease" is defined as no complete remission to primary therapy. In one embodiment, a refractory disease is defined as no response to prior therapy or recurrence within 6 months of prior therapy. In one embodiment, a refractory disease is characterized by one or more of the following: progressive disease (PD) as the best response to primary therapy, stable disease (SD) as the best response after at least 4 cycles of primary therapy (e.g., 4 cycles of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone (also abbreviated as R-CHOP)), or partial response (PR) as the best response after at least 6 cycles, and residual disease or disease progression demonstrated by biopsy after partial response. A "recurrent disease" is defined as a complete remission to primary therapy. In one embodiment, recurrence of the disease is demonstrated by biopsy. In one embodiment, the patient has recurred or not responded after at least 2 prior systemic treatment regimens (including at least 1 prior regimen containing an anthracycline and at least 1 prior regimen containing anti-CD20-directed therapy).

[0106] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human. In one example, each subject in the subject population is a human. In one example, each subject in the reference subject population is a human.

[0107] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated and can be implemented for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating a disease state, and remission or improvement of prognosis. In some embodiments, the methods of the invention are used to delay the onset or slow the progression of a disease.

[0108] As used herein, "slowing the progression of" a disorder or disease means delaying, preventing, decelerating, retarding, stabilizing, and / or delaying the onset of a disease or disorder (e.g., a CD20-positive B cell proliferative disorder, e.g., NHL, e.g., DLBCL). Such delays can be of various durations depending on the disease being treated and / or the medical history of the individual. As will be apparent to those skilled in the art, a sufficient or significant delay can in effect encompass prevention in that the individual does not develop the disease. For example, in advanced cancer, the onset of central nervous system (CNS) metastasis can be delayed.

[0109] As used herein, "reduce" or "inhibit" means, for example, the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. For clarity, this term also includes reduction to 0 (or below the detection limit of the assay method), i.e., complete disappearance or elimination. In certain embodiments, reduction or inhibition refers to the reduction or inhibition of cytokine-mediated toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevation of liver enzymes, and / or central nervous system (CNS) toxicity, etc., after treatment with an anti-CD20 / anti-CD3 bispecific antibody using the step-up dosing regimen of the present invention, as compared to when the target dose of the bispecific antibody is administered pre-set without changing the target dose of the bispecific antibody. In other embodiments, "reduce" or "inhibit" can refer to the effector function of the antibody mediated by the Fc region of the antibody, and such effector functions specifically include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). In other embodiments, reduction or inhibition can refer to the symptoms, presence or size of metastases, or size of the primary tumor of a CD20-positive B cell proliferative disorder to be treated (e.g., NHL (e.g., DLBCL), FL (e.g., relapsed and / or refractory FL, or transformed FL), MCL, high-grade B cell lymphoma, or PMLBCL).

[0110] As used herein, "administer" means a method of giving a dosage of a pharmaceutical composition of an anti-CD20 / anti-CD3 bispecific antibody to a subject. The pharmaceutical compositions described herein can be administered intravenously (e.g., by intravenous infusion).

[0111] As used herein, "buffer" refers to a buffer solution (also referred to herein as "buffering agent") that resists changes in pH due to the action of its acid-base conjugate components. In some embodiments, the buffer of the present invention has a pH in the range of about 5 to about 6. Exemplary buffering agents for use in the present invention include, but are not limited to, histidine (e.g., histidine HCl), acetate, phosphate, succinate, or combinations thereof. In some embodiments, histidine is histidine hydrochloride (histidine HCl), histidine acetate, monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or mixtures thereof.

[0112] The pharmaceutical compositions according to the present invention may also contain one or more tonicity modifiers. The term "tonicity modifier" refers to a pharmaceutically acceptable additive used to adjust the tonicity of a formulation. The formulation may be hypotonic, isotonic or hypertonic. Generally, isotonicity relates to the osmotic pressure of a solution relative to the osmotic pressure of normal human serum (about 250 - 350 mOsmol / kg). The formulations of the present invention may be hypotonic, isotonic or hypertonic, but are preferably isotonic. Hypertonic formulations are liquids or liquids reconstituted from a solid form (e.g., lyophilized form) and exhibit the same tonicity as several other solutions being compared, e.g., physiological saline solutions and sera. Suitable tonicity modifiers include, but are not limited to, salts such as sodium chloride or potassium chloride, glycerin and any component derived from amino acids or saccharides, particularly glucose. Tonicity modifiers are generally used in an amount of ≧200 mM.

[0113] Among stabilizers and tonicity modifiers, there is a group of compounds that can function as both, i.e., can simultaneously act as stabilizers and tonicity modifiers. Examples thereof can be found in the group of saccharides, amino acids, polyols, cyclodextrins, polyethylene glycols and salts. An example of a sugar that can be both a stabilizer and a tonicity modifier is trehalose.

[0114] As used herein, "surfactant" refers to a surface active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85); poloxamers (e.g., poloxamer 188); TRITON®; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauroamidopropyl betaine, cocoamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyldimethylamine, palmidopropyldimethylamine, or isostearamidopropyldimethylamine; sodium methyl cocoyl taurate, or disodium methyl oleoyl taurate; and MONAQUAT TM series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONIC® type block copolymers, e.g., PLURONIC® F-68); and the like. In one embodiment, the surfactant herein is polysorbate 20 (PS20). In yet another embodiment, the surfactant herein is poloxamer 188 (P188).

[0115] An "antiseptic" is a compound that can optionally be included in a formulation to substantially reduce the bacterial action in the formulation and thus, for example, facilitate the production of multi-purpose formulations. Examples of possible preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of antiseptics include phenol, aromatic alcohols such as butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol; 3-pentanol, and m-cresol. In one embodiment, the antiseptic of the present specification is benzyl alcohol. In some embodiments, the formulation does not contain a preservative.

[0116] A "stable" pharmaceutical composition is a pharmaceutical formulation in which the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity, upon storage (e.g., upon frozen storage). The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability is measured at a selected amount of light exposure and / or temperature over a selected period. Stability can be evaluated by assessment of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection); assessment of ROS formation (e.g., by using a light stress assay or a 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues of the anti-CD20 / anti-CD3 bispecific antibody (e.g., Met residues of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab)); assessment of charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing chromatography, or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis for comparing reduced intact anti-CD20 / anti-CD3 bispecific antibody; peptide mapping (e.g., trypsin or LYS-C) analysis; assessment of the biological activity or target binding function of the anti-CD20 / anti-CD3 bispecific antibody (e.g., binding to T cells and / or B cells), etc., and can be qualitatively and / or quantitatively evaluated by various different methods.Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and / or Trp oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, and glycosylation differences.

[0117] As used herein in the context of formulations according to the invention, the term "liquid" means a formulation that is liquid at a temperature of at least about 2 to about 8 °C under atmospheric pressure.

[0118] In this application, unless otherwise specified, the techniques utilized can be found in any of several well-known references as follows: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), and Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0119] Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer, unless otherwise specified. Accordingly, prior to describing the methods and uses, it should be understood that the invention is not limited to a particular methodology, protocol, cell line, animal species or genus, construct, and reagents so described, and can of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0120] III. Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) at a low concentration for the treatment of, for example, B cell proliferative disorders (e.g., non-Hodgkin lymphoma, NHL), and its use. The pharmaceutical composition of the present invention can be formulated to carry a low concentration of anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) and is stable against protein loss due to adsorption during storage and clinical administration. Adsorption is a serious problem for low-concentration antibodies that require further dilution and handling before clinical administration, and can result in low titers. Glofitamab is administered at doses of 2.5 mg and 10 mg (fractionated dosing) and a maintenance dose of 30 mg (target dose, uniform dosing). Glofitamab is intended for intravenous administration by intravenous bag infusion after dilution with 0.9% or 0.45% sodium chloride. The dose can be achieved in the intravenous bag with an administration solution concentration of 0.05 mg / ml to 0.6 mg / ml.

[0121] In one embodiment, a liquid pharmaceutical composition is provided comprising: at a pH in the range of about 5.0 to about 6.0, about 1 to 25 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab); about 10 to 50 mM of a buffer; about ≧200 mM of an isotonic agent; about 0 to 15 mM of methionine; and about ≧0.2 mg / ml of a surfactant.

[0122] In one embodiment, a liquid pharmaceutical composition is provided comprising: at a pH in the range of about 5.0 to about 6.0, about 5 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab); about 10 to 50 mM of a buffer; An isotonic agent of about ≧200 mM; Methionine of about 0 - 15 mM; and A surfactant of about ≧0.2 mg / ml.

[0123] In one embodiment, a liquid pharmaceutical composition is provided that includes: At a pH in the range of about 5.0 to about 6.0, An anti-CD20 / anti-CD3 bispecific antibody of about 0.9 to 1.1 mg / ml (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab); A buffer of about 10 - 50 mM; An isotonic agent of about ≧200 mM; Methionine of about 0 - 15 mM; and A surfactant of about ≧0.2 mg / ml.

[0124] In one embodiment, a liquid pharmaceutical composition is provided that includes: At a pH in the range of about 5.0 to about 6.0, An anti-CD20 / anti-CD3 bispecific antibody of about 1 mg / ml (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab); A buffer of about 10 - 50 mM; An isotonic agent of about ≧200 mM; Methionine of about 0 - 15 mM; and A surfactant of about ≧0.2 mg / ml.

[0125] In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody ranges from about 1 to 5 mg / ml. In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 mg / ml, about 1.1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, or about 5 mg / ml. In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody is about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, or about 30 mg / ml.

[0126] In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody ranges from about 0.9 - 1.1 mg / ml. In one embodiment, the concentration of the anti-CD20 / anti-CD3 bispecific antibody is about 1 mg / ml.

[0127] In one embodiment, the liquid pharmaceutical composition is an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprising at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 comprising a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 comprising a light chain variable region, and Comprising an anti-CD20 / anti-CD3 bispecific antibody.

[0128] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20, the heavy-chain variable region sequence being at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7, and the light-chain variable region sequence being at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8. In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20, the heavy-chain variable region sequence of SEQ ID NO: 7 and the light-chain variable region sequence of SEQ ID NO: 8.

[0129] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 Comprising a heavy-chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 Comprising a light-chain variable region, and Comprising at least one antigen-binding domain that specifically binds to CD3.

[0130] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD3, the heavy-chain variable region sequence of which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 15, and the light-chain variable region sequence of which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16, and comprises at least one antigen-binding domain. In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD3, which comprises the heavy-chain variable region sequence of SEQ ID NO: 15 and the light-chain variable region sequence of SEQ ID NO: 16.

[0131] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition a) at least one antigen-binding domain that specifically binds to CD20, comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 comprising a heavy-chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 comprising a light-chain variable region, and comprising at least one antigen-binding domain that specifically binds to CD20, and b) at least one antigen-binding domain that specifically binds to CD3, comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region comprising the same, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the same, and at least one antigen-binding domain that specifically binds to CD3 and comprises the same. It comprises the same.

[0132] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises: (i) at least one antigen-binding domain that specifically binds to CD20 and comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; (ii) at least one antigen-binding domain that specifically binds to CD3 and comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16. It comprises the same.

[0133] In one embodiment, the antigen-binding domain that specifically binds to CD3 of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is an antibody fragment, particularly a Fab molecule or an scFv molecule, more specifically a Fab molecule. In certain embodiments, the antigen-binding domain that specifically binds to CD3 of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced with each other).

[0134] In one embodiment, the antigen-binding domain that specifically binds to CD20 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is an antibody fragment, particularly a Fab molecule or an scFv molecule, more specifically a Fab molecule. In certain embodiments, the antigen-binding domain that specifically binds to CD20 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a conventional Fab molecule.

[0135] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition comprises at least one antigen-binding domain that specifically binds to CD20 and at least one antigen-binding domain that specifically binds to CD3. In one embodiment, the anti-CD20 / anti-CD3 antibody of the liquid pharmaceutical composition comprises a first antigen-binding domain that specifically binds to CD3 and second and third antigen-binding domains that specifically bind to CD20. In one embodiment, the first antigen-binding domain is a crossover Fab molecule and the second and third antigen-binding domains are each a conventional Fab molecule. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) further comprises an Fc domain. The anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition may comprise modifications in the Fc region and / or antigen-binding domain as described herein. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU numbering).

[0136] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition is (i) an antigen-binding domain that specifically binds to CD3 and is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain; (ii) a first antigen-binding domain that specifically binds to CD20 and is fused to the N-terminus of the Fab heavy chain of the antigen-binding domain that specifically binds to CD3 at the C-terminus of the Fab heavy chain; (iii) a second antigen-binding domain that specifically binds to CD20 and is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain; and comprises.

[0137] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of the liquid pharmaceutical composition is a) a first Fab molecule that specifically binds to CD3, particularly CD3 epsilon, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are exchanged with each other; (b) a second Fab molecule and a third Fab molecule that specifically bind to CD20, wherein in the constant domain CL of the second Fab molecule and the third Fab molecule, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted with lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second Fab molecule and the third Fab molecule, the amino acid at position 147 is substituted with glutamic acid (E) (EU numbering), the amino acid at position 213 is substituted with glutamic acid (E) (EU numbering), the second Fab molecule and the third Fab molecule; c) an Fc domain composed of a first subunit and a second subunit capable of stable association and comprises.

[0138] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody of a liquid pharmaceutical composition (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0139] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is bivalent with respect to CD20 and monovalent with respect to CD3.

[0140] In one embodiment, the first Fab molecule of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c), the second Fab molecule of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the heavy chain of the first Fab molecule of a), and the third Fab molecule of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other subunit of the Fc domain of c). In one embodiment, the first Fab molecule of a) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 15 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16.

[0141] In yet a further embodiment, the first Fab molecule of a) comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

[0142] In one embodiment, the second and third Fab molecules of b) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8.

[0143] In one embodiment, the second and third Fab molecules of b) each comprise the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

[0144] In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 17, a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 18, a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 19, and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 20. In further certain embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 17, the polypeptide sequence of SEQ ID NO: 18, the polypeptide sequence of SEQ ID NO: 19, and the polypeptide sequence of SEQ ID NO: 20. In yet further certain embodiments, the bispecific antibody comprises one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19, and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 20.

[0145] Certain anti-CD20 / anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 and European Patent Application Nos. EP15188093 and EP16169160, each of which is incorporated herein by reference in its entirety.

[0146] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) specifically binds to CDε.

[0147] In one embodiment, the anti-CD20 / anti-CD3 CD3 antibody of the liquid pharmaceutical composition can compete for binding with antibody H2C (International Publication No. WO2008 / 119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and US Patent No. 6054297), antibody FN18 (Nooij et al., Eur J Immunol 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J 4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31 (Spits et al., J Immunol.135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol.129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., Eur J Immunol.21-1709 (1991)), or antibody Leu-4. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition may also include an antigen-binding portion that specifically binds to CD3 as described in International Publication No. WO2005 / 040220, WO2005 / 118635, WO2007 / 042261, WO2008 / 119567, WO2008 / 119565, WO2012 / 162067, WO2013 / 158856, WO2013 / 188693, WO2013 / 186613, WO2014 / 110601, WO2014 / 145806, WO2014 / 191113, WO2014 / 047231, WO2015 / 095392, WO2015 / 181098, WO2015 / 001085, WO2015 / 104346, WO2015 / 172800, WO2016 / 020444, or WO2016 / 014974.

[0148] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition may include an antibody or antigen-binding portion from rituximab, ofatumumab, ocrelizumab, ofatumumab, ocaratuzumab, belzutifan, and blinatumomab.

[0149] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0150] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody can be composed of generic, biosimilar, or non-comparable biological versions of the antibodies named herein.

[0151] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the liquid pharmaceutical composition provided herein is glofitamab. Glofitamab (WHO Pharmaceutical Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN: List 83, 2020, Volume 34, Issue 1, p. 39, also known as: CD20-TCB, RO7082859, or RG6026; CAS number: 2229047-91-8) is a novel T cell-engaging bispecific (TCB) full-length antibody with a 2:1 molecular composition that binds bivalently to CD20 on B cells and monovalently to CD3 on T cells, particularly the CD3 epsilon chain (CD3e). Its CD3 binding region is fused head-to-tail to one of the CD20 binding regions via a flexible linker. This structure gives glofitamab superior in vitro potency compared to other CD20-CD3 bispecific antibodies with a 1:1 composition and results in a large antitumor effect in preclinical DLBCL models. The bivalency of CD20 preserves this potency in the presence of competing anti-CD20 antibodies, providing opportunities for pretreatment or co-treatment with these agents. Glofitamab contains a engineered heterodimeric Fc region with complete loss of binding to FcgR and C1q. By simultaneously binding to CD3e of the T cell receptor (TCR) complex on T cells and human CD20-expressing tumor cells, it induces lysis of tumor cells in addition to activation, proliferation, and cytokine release of T cells. Lysis of B cells by glofitamab is CD20-specific and does not occur when CD20 is not expressed or when binding (bridging) of T cells to CD20-expressing cells does not occur simultaneously. In addition to death, T cells are activated by CD3 bridging detected by increased T cell activation markers (CD25 and CD69), cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), cytotoxic granule release (granzyme B), and T cell proliferation. A schematic diagram of the molecular structure of glofitamab is shown in Figure 2. The sequence of glofitamab is summarized in Table 2. TIFF0007701982000002.tif85170

[0152] In some embodiments, the buffer is histidine, acetate, phosphate, succinate, citrate, or a combination thereof. In some embodiments, the histidine is histidine acetate. Alternative buffers include histidine hydrochloride (histidine HCl), histidine acetate, monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or mixtures thereof. In certain embodiments, the liquid pharmaceutical composition comprises a histidine buffer, i.e., a buffer having histidine, generally L-histidine, as the buffer. In certain embodiments, the buffer comprises L-histidine, i.e., L-histidine, or a mixture of L-histidine and L-histidine HCl, and the pH adjustment is achieved using hydrochloric acid. The L-histidine HCl buffer can be prepared by dissolving an appropriate amount of L-histidine and L-histidine hydrochloride in water, or by dissolving an appropriate amount of L-histidine in water and adjusting the pH to the desired value by addition of hydrochloric acid.

[0153] In certain embodiments, the buffer (e.g., histidine, e.g., L-histidine HCl) is at a concentration of 10 mM to 50 mM. For example, the buffer can be from 10 mM to 15 mM, or from 15 mM to 20 mM, e.g., from 6 mM to 18 mM, from 7 mM to 16 mM, from 8 mM to 15 mM, or from 9 mM to 12 mM, e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In particular, the concentration of the buffer (e.g., histidine, e.g., L-histidine HCl) is about 15 to 25 mM. In one embodiment, the buffer (e.g., histidine, e.g., L-histidine HCl) is at a concentration of about 20 mM.

[0154] Regardless of the buffer used, the pH can be adjusted to a value in the range of about 5.0 to about 6.0, preferably about 5.2 to about 5.8, using an acid or base known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide.

[0155] By the inventors of the present invention, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab), a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising, at least one antigen-binding domain that specifically binds to CD20, comprising, b) at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising, at least one antigen-binding domain that specifically binds to CD3, comprising, It has been discovered that anti-CD20 / anti-CD3 bispecific antibodies, including , are particularly stable in compositions with a pH of from about 5.2 to about 5.8. In one embodiment, the buffer provides a pH of from about 5.2 to about 5.8, particularly a pH of about 5.5.

[0156] In some embodiments, the pharmaceutical composition comprises an isotonic agent such as a sugar, an amino acid, a salt, etc. In embodiments where the isotonic agent is a sugar, the sugar can be, for example, sucrose, glucose, glycerol or trehalose. In certain embodiments, the sugar is sucrose, optionally D-sucrose. In some embodiments, the isotonic agent is either sucrose or sodium chloride. The isotonic agent (e.g., a sugar, e.g., sucrose) can be at a concentration of at least about ≧200 mM. For example, the isotonic agent (e.g., a sugar, e.g., sucrose) can be, for example, from 200 mM to 220 mM, from 220 mM to 240 mM, from 240 mM to 260 mM, from 260 mM to 280 mM, from 280 mM to 300 mM, from 300 mM to 320 mM, from 320 mM to 340 mM, from 340 mM to 360 mM, from 360 mM to 380 mM, from 380 mM to 400 mM, from 400 mM to 420 mM, from 420 mM to 440 mM, from 440 mM to 460 mM, from 460 mM to 480 mM, or from 480 mM to 500 mM, for example, from 200 mM to 300 mM, for example, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM. In some embodiments, the concentration of the isotonic agent is from about 200 mM to 280 mM. In some embodiments, the concentration of the isotonic agent is about 240 mM. In a particular embodiment, the isotonic agent is sucrose and is present at a concentration of at least about 200 mM, i.e., ≧ about 200 mM. In other particular embodiments, the isotonic agent is sucrose (e.g., D-sucrose) and is present at a concentration of from about 200 mM to 280 mM. In a particular embodiment, the isotonic agent is sucrose (e.g., D-sucrose) and is present at a concentration of about 240 mM.

[0157] In some embodiments, the liquid pharmaceutical composition contains methionine as a stabilizer.

[0158] A stabilizer methionine at any suitable concentration can be used. For example, in some embodiments of any of the aforementioned pharmaceutical compositions, the concentration of the stabilizer (e.g., methionine) is from about 0.01 mM to about 15 mM, such as about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM.

[0159] In certain embodiments, the concentration of methionine is from about 5 mM to 15 mM. In certain embodiments, the concentration of methionine is about 10 mM.

[0160] Any of the pharmaceutical compositions described herein may contain a surfactant. Any suitable surfactant may be used. In some embodiments, the surfactant is a nonionic surfactant (e.g., polysorbate (polyoxyethylene (n) sorbitan monolaurate), poloxamer, polyoxyethylene alkyl ether, alkylphenyl polyoxyethylene ether, or combinations thereof). In some embodiments, the nonionic surfactant is a polysorbate (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate (PS20), TWEEN 20 (registered trademark); e.g., particularly purified PS20 (PS20 with enhanced purity by an exclusive flash chromatography process and available from Avantor Performance Materials, LLC (Center Valley, PA, US))) or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate (PS80), e.g., TWEEN 80 (registered trademark); e.g., particularly purified PS80 (Avantor)). In certain embodiments, the polysorbate is polysorbate 20. In other embodiments, the nonionic surfactant is a poloxamer (e.g., poloxamer 188, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)).

[0161] The pharmaceutical surfactant can be at a concentration of at least about ≧0.2 mg / ml, i.e., at least ≧ about 0.02% (w / v).

[0162] In some embodiments of any of the pharmaceutical compositions described herein, the concentration of the surfactant (e.g., PS20 or P188) is from about 0.01% (w / v) to about 2% (w / v), such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% (w / v).

[0163] In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is from about 0.1 to 1 mg / ml, i.e., from 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is from about 0.2 to 1 mg / ml, i.e., from 0.02% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is from about 0.2 to 0.8 mg / ml, i.e., from 0.02% (w / v) to about 0.08% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is about 0.5 mg / ml, i.e., about 0.05% (w / v).

[0164] In certain embodiments, the surfactant is P188 and the concentration of P188 is about 0.05% (w / v), 0.07% (w / v), or 0.1% (w / v).

[0165] In certain embodiments, the surfactant is PS20 and the concentration of PS20 is at least about ≧0.2 mg / ml, i.e., at least about ≧0.02% (w / v) of the concentration of PS20.

[0166] In certain embodiments, the surfactant is PS20, and the concentration of PS20 is about 0.2 - 0.8 mg / ml, i.e., from about 0.02% (w / v) to about 0.08% (w / v). In certain embodiments, the surfactant is PS20, and the concentration of PS20 is about 0.5 mg / ml, i.e., about 0.05% (w / v). In certain embodiments, the surfactant is PS20, and the concentration of PS20 is at least about ≧0.02% (w / v) PS20. In certain embodiments, the surfactant is PS20, and the concentration of PS20 is from about 0.02% (w / v) to about 0.08% (w / v). In certain embodiments, the surfactant is PS20, and the concentration of PS20 is about 0.05% (w / v).

[0167] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5 to about 6, from about 1 to 5 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region, and at least one antigen-binding domain that specifically binds to CD20, b) at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region containing (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 14 and a light chain variable region containing at least one antigen-binding domain that specifically binds to CD3 containing an anti-CD20 / anti-CD3 bispecific antibody of about 1 to 5 mg / ml containing about 15 - 25 mM histidine buffer; about 200 - 280 mM sucrose; about 0 - 15 mM methionine; about 0.2 - 0.8 mg / ml of PS20 and containing.

[0168] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8, an anti-CD20 / anti-CD3 bispecific antibody of about 1 to 5 mg / ml (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), which a) at least one antigen-binding domain that specifically binds to CD20, which (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region containing (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 6 and a light chain variable region containing at least one antigen-binding domain that specifically binds to CD20 containing b) At least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising, at least one antigen-binding domain that specifically binds to CD3 comprising, an anti-CD20 / anti-CD3 bispecific antibody at about 1 to 5 mg / ml comprising; a histidine buffer at about 15 - 25 mM; sucrose at about 200 - 280 mM; methionine at about 0 - 15 mM; PS20 at about 0.2 - 0.8 mg / ml comprising.

[0169] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8, an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) at about 0.9 to 1.1 mg / ml, a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 6 a light chain variable region containing the same, and at least one antigen-binding domain that specifically binds to CD20 containing the same, b) at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region containing the same, and (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 14 a light chain variable region containing the same, and at least one antigen-binding domain that specifically binds to CD3 containing the same, an anti-CD20 / anti-CD3 bispecific antibody of about 1 mg / ml containing the same; a histidine buffer of about 15 - 25 mM; sucrose of about 200 - 280 mM; methionine of about 0 - 15 mM; PS20 of about 0.2 - 0.8 mg / ml containing the same.

[0170] In one embodiment, the liquid pharmaceutical composition is at a pH of about 5.2 to about 5.8, an anti-CD20 / anti-CD3 bispecific antibody of about 1 mg / ml (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region containing (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 6 a light chain variable region containing at least one antigen-binding domain that specifically binds to CD20 and contains b) at least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region containing (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 14 a light chain variable region containing at least one antigen-binding domain that specifically binds to CD3 and contains an anti-CD20 / anti-CD3 bispecific antibody at about 1 mg / ml containing about 15 - 25 mM histidine buffer; about 200 - 280 mM sucrose; about 0 - 15 mM methionine; about 0.2 - 0.8 mg / ml PS20 and containing.

[0171] In one embodiment, the liquid pharmaceutical composition is at a pH of about 5.5, an anti-CD20 / anti-CD3 bispecific antibody at about 1 mg / ml (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), a) At least one antigen-binding domain that specifically binds to CD20, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region, and at least one antigen-binding domain that specifically binds to CD20, b) At least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region, and at least one antigen-binding domain that specifically binds to CD3, an anti-CD20 / anti-CD3 bispecific antibody of about 1 mg / ml, and; about 20 mM histidine buffer; about 240 mM sucrose; about 10 mM methionine; about 0.5 mg / ml PS20 and.

[0172] In one embodiment, the liquid pharmaceutical composition according to the present invention is At a pH of about 5.2 to about 5.8, An anti-CD20 / anti-CD3 bispecific antibody at about 1 to 5 mg / ml (for example, anti-CD20 / anti-CD3 TCB, for example, glofitamab), (i) At least one antigen-binding domain that specifically binds to CD20, comprising the heavy-chain variable region sequence of SEQ ID NO: 7 and the light-chain variable region sequence of SEQ ID NO: 8, (ii) At least one antigen-binding domain that specifically binds to CD3, comprising the heavy-chain variable sequence of SEQ ID NO: 15 and the light-chain variable region sequence of SEQ ID NO: 16, An anti-CD20 / anti-CD3 bispecific antibody at about 1 to 5 mg / ml comprising; A histidine buffer at about 15 - 25 mM; Sucrose at about 200 - 280 mM; Methionine at about 0 - 15 mM; PS20 at about 0.2 - 0.8 mg / ml and comprising.

[0173] In one embodiment, the liquid pharmaceutical composition according to the present invention is At a pH of about 5.2 to about 5.8, An anti-CD20 / anti-CD3 bispecific antibody at about 0.9 to about 1.1 mg / ml, (i) At least one antigen-binding domain that specifically binds to CD20, comprising the heavy-chain variable region sequence of SEQ ID NO: 7 and the light-chain variable region sequence of SEQ ID NO: 8, (ii) At least one antigen-binding domain that specifically binds to CD3, comprising the heavy-chain variable region sequence of SEQ ID NO: 15 and the light-chain variable region sequence of SEQ ID NO: 16, An anti-CD20 / anti-CD3 bispecific antibody at about 0.9 to about 1.1 mg / ml comprising; A histidine buffer at about 15 - 25 mM; Sucrose at about 200 - 280 mM; Methionine at about 0 - 15 mM; PS20 at about 0.2 - 0.8 mg / ml and comprising.

[0174] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8, about 1 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), comprising (i) at least one antigen-binding domain that specifically binds to CD20, comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; about 1 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody comprising the above; about 15-25 mM of histidine buffer; about 200-280 mM of sucrose; about 0-15 mM of methionine; about 0.2-0.8 mg / ml of PS20 and comprising the above.

[0175] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.5, about 1 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab), comprising (i) at least one antigen-binding domain that specifically binds to CD20, comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16; about 1 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody comprising the above; about 20 mM of histidine buffer; about 240 mM of sucrose; about 10 mM of methionine; about 0.5 mg / ml of PS20 and comprising the above.

[0176] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8, about 1 to 5 mg / ml of golimumab; about 15 - 25 mM of histidine buffer; about 200 - 280 mM of sucrose; about 0 - 15 mM of methionine; and about 0.2 - 0.8 mg / ml of PS20 and comprises.

[0177] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8, about 0.9 to about 1.1 mg / ml of golimumab; about 15 - 25 mM of histidine buffer; about 200 - 280 mM of sucrose; about 0 - 15 mM of methionine; and about 0.2 - 0.8 mg / ml of PS20 and comprises.

[0178] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.2 to about 5.8; about 1 mg / ml of golimumab; about 15 - 25 mM of histidine buffer; about 200 - 280 mM of sucrose; about 0 - 15 mM of methionine; and about 0.2 - 0.8 mg / ml of PS20 and comprises.

[0179] In one embodiment, the liquid pharmaceutical composition according to the present invention is at a pH of about 5.5, about 1 mg / ml of golimumab; about 20 mM of histidine buffer; about 240 mM of sucrose; about 10 mM of methionine; and about 0.5 mg / ml of PS20 comprises.

[0180] The formulation may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The presence of microorganisms can be reliably prevented by both sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Preservatives are generally used in an amount of about 0.001 to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl parabens, and benzalkonium chloride.

[0181] IV. Therapeutic Agents for Use in the Pharmaceutical Compositions of the Present Invention A. Anti-CD20 / Anti-CD3 Bispecific Antibodies The present invention relates to novel pharmaceutical compositions of anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 T cell engaging bispecific antibodies (TCBs), e.g., glofitamab). In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a polyclonal antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a human antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a humanized antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a chimeric antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a full-length antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is an IgG class antibody, particularly an IgG1 subclass antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a recombinant antibody.

[0182] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises a human antibody. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. WO 93 / 16185 and U.S. Patents Nos. 5,571,894 and 5,587,458. For descriptions of Fab and F(ab’)2 fragments that include salvage receptor binding epitope residues and have an increased in vivo half-life, see U.S. Patent No. 5,869,046. In one embodiment, the antibody fragment is a Fab fragment or an scFv fragment.

[0183] A diabody is an antibody fragment that has two antigen-binding sites and can be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. WO 93 / 01161, Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0184] A single-domain antibody is an antibody fragment that comprises all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516).

[0185] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolysis and production of intact antibodies by recombinant host cells (e.g., E. coli or phage) as described herein.

[0186] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In a further example, a chimeric antibody is a “class switch” antibody in which the class or subclass has been changed from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0187] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a humanized antibody. Typically, a non-human antibody is humanized while retaining the specificity and affinity of the parent non-human antibody in order to reduce its immunogenicity in humans. Generally, a humanized antibody comprises one or more variable domains in which the HVRs (e.g., CDRs) (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody also optionally includes at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) in order to, for example, restore or improve antibody specificity or affinity.

[0188] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989), U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing grafting of specificity-determining regions (SDRs)), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"), Dall’Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" approach to FR shuffling).

[0189] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0190] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a human antibody. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 36874 (2001), and Lonberg, Curr. Opin. Immunol. 20:450459 (2008).

[0191] Human antibodies may be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies having human variable regions in response to an antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is present episomally, or is randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent No. 6,075,181 and U.S. Patent No. 6,150,584, which describe XENOMOUSE™ technology, U.S. Patent No. 5,770,429, which describes HuMab® technology, U.S. Patent No. 7,041,870, which describes K-M MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0192] Human antibodies can also be made by methods based on hybridomas. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0193] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0194] The binding domains contained in an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) can be isolated by screening a combinatorial library for binding sites having the desired activity. For example, phage display libraries can be created and various methods for screening such libraries for antibodies having the desired binding properties are known in the art. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0195] In certain phage display methods, the repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. A library from an immunogen provides high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and also self antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993), without immunization. Finally, a naive library can also be synthetically generated by cloning unrearranged V gene segments from stem cells and achieving rearrangement in vitro using PCR primers containing random sequences to encode a highly variable CDR3 region, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0196] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or fragments of human antibodies.

[0197] Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (Milstein and Cuello, Nature 305: 537 (1983)); WO 93 / 08829; and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-into-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); making bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and may also be made by preparing trispecific antibodies such as, for example, described in Tutt et al. J. Immunol. 147: 60 (1991).

[0198] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576A1).

[0199] The anti-CD20 / anti-CD3 bispecific antibodies herein also include "dual action Fab" or "DAF" which contain antigen-binding sites that bind two different antigens (see, e.g., US 2008 / 0069820).

[0200] "Crossmab" antibodies are also included herein (see, e.g., International Publication Nos. 2009080251, O2009080252, 2009080253, and 2009080254).

[0201] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain contains two single-chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and since these epitopes may be specific for different cell types, when each scFv engages its cognate epitope, cells of two different cell types are brought into proximity or tethered. One particular embodiment of this approach includes an scFv that recognizes a cell surface antigen expressed by an immune cell, e.g., the CD3 polypeptide on a T cell, which is bound to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0202] Since it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, such as a CHO cell line. However, certain purification techniques (see, e.g., EP1691833) may require separating the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a gradient of sodium chloride concentration. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from multimeric species.

[0203] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Sites suitable for derivatization of the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof (but not limited thereto). Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers bound to the antibody may vary, and when multiple polymers are bound, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under the defined conditions, etc.

[0204] The anti-CD20 / anti-CD3 bispecific antibody can be conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, or enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants or animals), or radioisotopes.

[0205] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an antibody-drug conjugate (ADC), where the antibody is conjugated to one or more agents including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0425235B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin and doxorubicin (e.g., see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065.

[0206] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is conjugated to an enzymatic active toxin or a fragment thereof including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crocin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0207] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of the radioactive conjugate. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When used for detection, the radioactive conjugate may contain a radioactive atom for scintigraphy, such as Tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as, again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0208] Conjugates of anti-CD20 / anti-CD3 bispecific antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radio nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a "cleavable linker" that promotes the release of the cytotoxic drug intracellularly. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131(1992); U.S. Patent No. 5,208,020) may be used.

[0209] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is adapted for the treatment of a cell proliferative disorder (e.g., cancer). In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cancer is a B cell proliferative disorder. In one embodiment, the cancer is a CD20-positive B cell proliferative disorder. In one embodiment, the cancer is non-Hodgkin lymphoma (NHL). In one embodiment, the NHL is diffuse large B cell lymphoma (DLBCL), high-grade B cell lymphoma (HGBCL), follicular lymphoma (FL) [transformed FL; trFL]-derived DLBCL, primary mediastinal large B cell lymphoma (PMBCL), or marginal zone lymphoma (MZL). MZL is classified into splenic MZL, nodular MZL, and extranodal MZL. In one embodiment, the NHL is mantle cell lymphoma (MCL). In one embodiment, the NHL is grade 1-3a follicular lymphoma (FL). In some embodiments, the CD20-positive B cell proliferative disorder is a relapsed or refractory B cell proliferative disorder. In one embodiment, the relapsed or refractory B cell proliferative disorder is relapsed or refractory NHL (e.g., relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL).

[0210] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) specifically binds to CD3ε.

[0211] In one embodiment, the anti-CD20 / anti-CD3 CD3 antibody can compete for binding with antibody H2C (International Publication No. WO 2008 / 119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and US Patent No. 6,054,297), antibody FN18 (Nooij et al., Eur J Immunol 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J 4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31 (Spits et al., J Immunol. 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol. 129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., Eur J Immunol. 21-1709 (1991)), or antibody Leu-4. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody can also include an antigen-binding portion that specifically binds to CD3 as described in International Publication Nos. WO 2005 / 040220, WO 2005 / 118635, WO 2007 / 042261, WO 2008 / 119567, WO 2008 / 119565, WO 2012 / 162067, WO 2013 / 158856, WO 2013 / 188693, WO 2013 / 186613, WO 2014 / 110601, WO 2014 / 145806, WO 2014 / 191113, WO 2014 / 047231, WO 2015 / 095392, WO 2015 / 181098, WO 2015 / 001085, WO 2015 / 104346, WO 2015 / 172800, WO 2016 / 020444, or WO 2016 / 014974.

[0212] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody can include an antibody or antigen-binding portion from rituximab, ofatumumab, ocrelizumab, obinutuzumab, ocaratuzumab, belatacept, and blinatumomab.

[0213] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0214] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody can be composed of a generic, biosimilar, or non-comparable biological version of the antibodies named herein.

[0215] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is at least one antigen-binding domain that specifically binds to CD20 and (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising, and at least one antigen-binding domain comprising.

[0216] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20, the at least one antigen-binding domain comprising a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8. In a further embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20, the at least one antigen-binding domain comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

[0217] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is at least one antigen-binding domain that specifically binds to CD3, the (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 comprising a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 comprising a light chain variable region, and comprises at least one antigen-binding domain.

[0218] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD3, the heavy chain variable region sequence being at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 15, and the light chain variable region sequence being at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16. In a further embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD3, the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

[0219] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) a) at least one antigen-binding domain that specifically binds to CD20, comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region, and at least one antigen-binding domain that specifically binds to CD20, b) at least one antigen-binding domain that specifically binds to CD3, comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region containing the same, (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 14 and a light chain variable region containing the same, and at least one antigen-binding domain that specifically binds to CD3.

[0220] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises (i) at least one antigen-binding domain that specifically binds to CD20, comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16, and the same.

[0221] In one embodiment, the antigen-binding domain of the anti-CD20 / anti-CD3 bispecific antibody that specifically binds to CD3 is an antibody fragment, particularly a Fab molecule or an scFv molecule, more specifically a Fab molecule. In certain embodiments, the antigen-binding domain of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) that specifically binds to CD3 is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced with each other).

[0222] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20 and at least one antigen-binding domain that specifically binds to CD3. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises a first antigen-binding domain that specifically binds to CD3 and second and third antigen-binding domains that specifically bind to CD20. In one embodiment, the first antigen-binding domain is a crossover Fab molecule and the second and third antigen-binding domains are each a conventional Fab molecule. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) further comprises an Fc domain. The anti-CD20 / anti-CD3 bispecific antibody may comprise modifications of the Fc region and / or antigen-binding domains as described herein. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU numbering).

[0223] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is (i) an antigen-binding domain that specifically binds to CD3, which is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (ii) a first antigen-binding domain that specifically binds to CD20, which is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the antigen-binding domain that specifically binds to CD3; (iii) At the C-terminus of the Fab heavy chain, a second antigen-binding domain that specifically binds to CD20 and is fused to the N-terminus of the second subunit of the Fc domain; comprising.

[0224] In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises: a) A first Fab molecule that specifically binds to CD3, particularly CD3 epsilon, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are exchanged with each other; b) A second Fab molecule and a third Fab molecule that specifically bind to CD20, wherein in the constant domain CL of the second Fab molecule and the third Fab molecule, the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second Fab molecule and the third Fab molecule, the amino acid at position 147 is substituted by glutamic acid (E) (EU numbering), the amino acid at position 213 is substituted by glutamic acid (E) (EU numbering), the second Fab molecule and the third Fab molecule; c) An Fc domain composed of a first subunit and a second subunit capable of stable association comprising.

[0225] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0226] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is bivalent with respect to CD20 and monovalent with respect to CD3.

[0227] In one embodiment, the first Fab molecule of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c), the second Fab molecule of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the heavy chain of the first Fab molecule of a), and the third Fab molecule of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other subunit of the Fc domain of c). In one embodiment, the first Fab molecule of a) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 15 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16.

[0228] In yet a further embodiment, the first Fab molecule of a) comprises the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

[0229] In one embodiment, the second and third Fab molecules of b) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8.

[0230] In one embodiment, the second and third Fab molecules of b) each comprise the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

[0231] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 17, a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 18, a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 19, and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 20. In further certain embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 17, the polypeptide sequence of SEQ ID NO: 18, the polypeptide sequence of SEQ ID NO: 19, and the polypeptide sequence of SEQ ID NO: 20. In yet further certain embodiments, the bispecific antibody comprises one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 19, and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 20.

[0232] Certain anti-CD20 / anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 and European Patent Application Nos. EP15188093 and EP16169160, each of which is incorporated herein by reference in its entirety. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the pharmaceutical composition of the present invention is glofitamab.

[0233] B. Antibody Format 1. Structure of the Anti-CD20 / anti-CD3 Bispecific Antibody The components of the anti-CD20 / anti-CD3 bispecific antibody can be fused to each other in various structures. Exemplary structures are shown in Figure 1.

[0234] In certain embodiments, the antigen-binding portions included in the anti-CD20 / anti-CD3 bispecific antibody are Fab molecules. In such embodiments, the first, second, third, etc. antigen-binding portions may be referred to herein as the first, second, third, etc. Fab molecules, respectively. Further, in certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody includes an Fc domain composed of a first subunit and a second subunit that can stably associate.

[0235] In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0236] In such an embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific such embodiment, the anti-CD20 / anti-CD3 bispecific antibody consists essentially of the first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a structure is schematically shown in FIGS. 1G and 1K. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

[0237] In another embodiment, the second Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In a specific such embodiment, the antibody consists essentially of the first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first and second Fab molecules are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain. Such a structure is schematically shown in FIGS. 1A and 1D. The first and second Fab molecules can be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the first and second Fab molecules are fused to the Fc domain by an immunoglobulin hinge region, respectively. In a specific embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, and in particular, the Fc domain is the IgG1 Fc domain.

[0238] In other embodiments, the second Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In one such embodiment, the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In a specific such embodiment, the antibody consists essentially of the first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such a structure is schematically shown in FIGS. 1H and 1L. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be further fused to each other.

[0239] The Fab molecule can be fused to the Fc domain or to each other directly or through a peptide linker containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SEQ ID NO: 21), (SG4) n (SEQ ID NO: 22), or G4(SG4) n (SEQ ID NO: 23) peptide linkers. "n" is typically an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment from 5 to 100, and in a further embodiment from 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS) n or (GxS) n G m [wherein, G = glycine, S = serine, (x = 3, n = 3, 4, 5 or 6, m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5, m = 0, 1, 2 or 3)], and in one embodiment, x = 4, n = 2 or 3, and in a further embodiment, x = 4, n = 2. In one embodiment, the peptide linker is (G4S)2 (SEQ ID NO: 24). A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2 (SEQ ID NO: 24). Exemplary peptide linkers suitable for connecting the Fab heavy chains of the first and second Fab molecules include the sequences (D)-(G4S)2 (SEQ ID NOs: 24 and 25). Another suitable such linker includes the sequence (G4S)4 (SEQ ID NO: 26). In addition, the linker may include (a part of) an immunoglobulin hinge region. Particularly when the Fab molecule is fused to the N-terminus of the Fc domain subunit, it can be fused via an immunoglobulin hinge region or a part thereof, regardless of the presence or absence of an additional peptide linker.

[0240] An antibody having a single antigen-binding portion (e.g., a Fab molecule) that can specifically bind to a target cell antigen (e.g., such as those shown in FIGS. 1A, 1D, 1G, 1H, 1K, or 1L) is useful particularly when internalization of the target cell antigen is expected following binding of a high-affinity antigen-binding portion. In such cases, the presence of more than one antigen-binding portion specific for the target cell antigen can promote internalization of the target cell antigen, thereby reducing its availability.

[0241] However, in many other cases, it would be advantageous to have an antibody that includes two or more antigen-binding portions (e.g., Fab molecules) specific for a target cell antigen (see examples shown in FIGS. 1B, 1C, 1E, 1F, 1I, 13J, 1M or 1N), for example, to optimize targeting to a target site or to enable cross-linking of the target cell antigen.

[0242] Thus, in certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody includes two anti-CD20 binding portions, e.g., two Fab molecules that target CD20. In one embodiment, the two Fab molecules that target CD20 are conventional Fab molecules. In one embodiment, the two Fab molecules that target CD20 include the same heavy and light chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover).

[0243] In an alternative embodiment, the anti-CD20 / anti-CD3 bispecific antibody includes two anti-CD3 binding portions, e.g., two Fab molecules that target CD3. In such an embodiment, both of the two Fab molecules that target CD3 are crossover Fab molecules (Fab molecules in which the variable domains VH and VL of the Fab heavy and light chains or the constant domains CL and CH1 are exchanged / replaced by each other). In such an embodiment, the two Fab molecules that target CD3 include the same heavy and light chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover).

[0244] In one embodiment, the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0245] In certain embodiments, the second and third Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, the antibody consists essentially of the first, second, and third Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are shown schematically in FIGS. 1B and 1E (embodiment where the third Fab molecule is a conventional Fab molecule and is identical to the second Fab molecule) and FIGS. 1I and 1M (embodiment where the third Fab molecule is a crossover Fab molecule and is preferably identical to the first Fab molecule). The second and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, and in particular, the Fc domain is the IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be further fused to each other.

[0246] In another embodiment, the second and third Fab molecules are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In a specific such embodiment, the antibody consists essentially of the first, second and third Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, the second Fab molecule is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such structures are schematically shown in FIGS. 1C and 1F (embodiment where the third Fab molecule is a conventional Fab molecule and is identical to the second Fab molecule) and FIGS. 1J and 1N (embodiment where the third Fab molecule is a crossover Fab molecule and is identical to the first Fab molecule). The first and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, and in particular, the Fc domain is the IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be further fused to each other.

[0247] In the structure of an antibody where the Fab molecules are fused to the N-terminus of each subunit of the Fc domain via an immunoglobulin hinge region at the C-terminus of the Fab heavy chain, two Fab molecules, the hinge region, and the Fc domain essentially form an immunoglobulin molecule. In certain embodiments, the immunoglobulin molecule is an immunoglobulin of the IgG class. In more certain embodiments, the immunoglobulin is an immunoglobulin of the IgG1 subclass. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In yet more certain embodiments, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

[0248] In some antibodies, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are optionally fused to each other via a peptide linker. Depending on the composition of the first and second Fab molecules, the Fab light chain of the first Fab molecule can be fused to the N-terminus of the Fab light chain of the second Fab molecule at its C-terminus, or the Fab light chain of the second Fab molecule can be fused to the N-terminus of the Fab light chain of the first Fab molecule at its C-terminus. The fusion of the Fab light chains of the first and second Fab molecules further reduces the mispairing of non-matching Fab heavy and light chains and also decreases the number of plasmids required for the expression of some antibodies.

[0249] In certain embodiments, the antibody has a polypeptide where the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and then the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -CH2-CH3(-CH4)), and the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2)-CH2-CH3(-CH4)) polypeptide. In some embodiments, the antibody has a Fab heavy chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH (1) -CL (1) ) and the Fab light chain polypeptide of a second Fab molecule (VL (2) -CL (2) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0250] In certain embodiments, the antibody has a Fab heavy chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule includes a crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region), and the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -CH2-CH3(-CH4)) polypeptide, and the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -CH2-CH3(-CH4)) polypeptide. In some embodiments, the antibody has a Fab light chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL (1) -CH1 (1) ) and the Fab light chain polypeptide of a second Fab molecule (VL (2) -CL (2) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0251] In some embodiments, the antibody has a Fab light chain variable region of a first Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule includes a crossover Fab heavy chain with the heavy chain variable region replaced by the light chain variable region), then the Fab heavy chain constant region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, and then the Fab heavy chain of the second Fab molecule sharing a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -VH (2) -CH1 (2) -CH2-CH3(-CH4)) polypeptide. In other embodiments, the antibody has a Fab heavy chain of a second Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain variable region of a first Fab molecule, then the Fab light chain variable region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule includes a crossover Fab heavy chain with the heavy chain variable region replaced by the light chain variable region), and then the Fab heavy chain constant region of the first Fab molecule sharing a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VL (1) -CH1 (1) -CH2-CH3(-CH4)) polypeptide.

[0252] In some of these embodiments, the antibody has a crossover Fab light chain polypeptide of the first Fab molecule (VH (1) -CL (1) ) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule, and a Fab light chain polypeptide (VL (2) -CL (2)further comprises these. In addition to these embodiments, if appropriate, the antibody has a Fab heavy chain variable region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule, and then the Fab light chain constant region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second Fab molecule (VH (1) -CL (1) -VL (2) -CL (2) ) polypeptide, or the Fab light chain polypeptide of the second Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, and then the Fab heavy chain variable region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL (2) -CL (2) -VH (1) -CL (1) ) further comprises a polypeptide.

[0253] Antibodies according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0254] In some embodiments, the antibody has a Fab heavy chain variable region of the second Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region), then the Fab light chain constant region of the first Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, and then the Fab heavy chain of the second Fab molecule sharing a carboxy-terminal peptide bond with the Fc domain subunit (VH(1) -CL (1) -VH (2) -CH1 (2) -CH2-CH3(-CH4)) polypeptide. In other embodiments, the antibody is such that the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of the first Fab molecule, and then the variable region of the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region), and then the constant region of the Fab light chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CH1 (2) -VH (1) -CL (1) -CH2-CH3(-CH4)) polypeptide.

[0255] In some of these embodiments, the antibody is a crossover Fab light chain polypeptide (VL (1) -CH1 (1) ) of the first Fab molecule, in which the variable region of the Fab light chain shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule, and the Fab light chain polypeptide (VL (2) -CL (2) ) of the second Fab molecule. In other of these embodiments, if appropriate, the antibody is such that the variable region of the Fab light chain of the first Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule, and then the constant region of the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second Fab molecule (VL (1) -CH1 (1) -VL (2) -CL (2) ) polypeptide, or the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of the first Fab molecule, and then the variable region of the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (VL(2) -CL (2) -VH (1) -CL (1) ) further comprises a polypeptide.

[0256] Antibodies according to these embodiments are (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0257] In certain embodiments, the antibody is a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, and then the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region) (VH (1) -CH1 (1) -VL (2) -CH1 (2) ). In some embodiments, the antibody is a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ).

[0258] In certain embodiments, the Fab light chain variable region of the antibody and the second Fab molecule share a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and then the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. The polypeptide (VL (2) -CH1 (2) -VH (1) -CH1 (1) ) is included. In some embodiments, the antibody further includes a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0259] In certain embodiments, the antibody has a Fab heavy chain variable region of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region), and then the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. The polypeptide (VH (2) -CL (2) -VH (1) -CH1 (1) ) is included. In some embodiments, the antibody further includes a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0260] In certain embodiments, the antibody is a polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) ) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, then the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, and then the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region). In some embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In some embodiments, the antibody further comprises the Fab light chain polypeptide of a third Fab molecule (VL (3) -CL (3) ).

[0261] In certain embodiments, the antibody is a polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2)It includes (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further includes the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0262] In certain embodiments, the antibody has a polypeptide (VL (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), then the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and then the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. In some embodiments, the antibody further includes a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further includes the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0263] In certain embodiments, the antibody is a polypeptide (VH (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region), and then the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and then the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. In some embodiments, the antibody further includes a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further includes the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0264] In certain embodiments, the antibody is such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, and then the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and then the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the third Fab molecule, and then the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region) (VH (1) -CH1 (1) -VL (2) -CH1 (2) -VL (3) -CH1 (3) ) polypeptide. In some embodiments, the antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In some embodiments, the antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ).

[0265] In certain embodiments, the antibody has a Fab heavy chain of a first Fab molecule sharing a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of a second Fab molecule, and then the variable region of the Fab heavy chain of the second Fab molecule sharing a carboxy-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain with the heavy chain constant region replaced by the light chain constant region), and then the constant region of the Fab light chain of the second Fab molecule sharing a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of a third Fab molecule, and then the variable region of the Fab heavy chain of the third Fab molecule sharing a carboxy-terminal peptide bond with the constant region of the Fab light chain of the third Fab molecule (i.e., the third Fab molecule includes a crossover Fab heavy chain with the heavy chain constant region replaced by the light chain constant region) (VH (1) -CH1 (1) -VH (2) -CL (2) -VH (3) -CL (3) ) polypeptide. In some embodiments, the antibody further includes a polypeptide in which the variable region of the Fab light chain of the second Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In some embodiments, the antibody further includes a polypeptide in which the variable region of the Fab light chain of the third Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the third Fab molecule (VL (3) -CH1 (3) ).

[0266] In certain embodiments, the antibody has a polypeptide in which the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule includes a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and then the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, and then the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and then the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) ). In some embodiments, the antibody further includes a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In some embodiments, the antibody further includes a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ).

[0267] In certain embodiments, the antibody has a Fab heavy chain variable region of a third Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule includes a crossover Fab heavy chain with the heavy chain constant region replaced by the light chain constant region), then the Fab light chain constant region of the third Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, and then the Fab heavy chain variable region of the second Fab molecule sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain with the heavy chain constant region replaced by the light chain constant region), and then the Fab light chain constant region of the second Fab molecule sharing a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH (3) -CL (3) -VH (2) -CL (2) -VH (1) -CH1 (1) ) polypeptide. In some embodiments, the antibody further includes a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further includes a polypeptide (VL (3) -CH1 (3) ) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule.

[0268] According to any of the above embodiments, the components of the antibody (e.g., Fab molecules, Fc domains) may be directly fused or may be fused via various linkers, particularly peptide linkers containing one or more amino acids (typically about 2-20 amino acids) described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S)n (SEQ ID NO: 21), (SG4) n (SEQ ID NO: 22), or G4 (SG4) n (SEQ ID NO: 23) contains a peptide linker, where n is generally an integer from 1 to 10, typically from 2 to 4.

[0269] 2. Fc domain The anti-CD20 / anti-CD3 bispecific antibody may comprise an Fc domain consisting of a pair of polypeptide chains comprising the heavy chain domains of the antibody molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit thereof comprises CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable binding to each other.

[0270] In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is human.

[0271] (i) Fc domain modifications that promote heterodimerization An anti-CD20 / anti-CD3 bispecific antibody may comprise different components (e.g., antigen-binding domains) fused to one or the other of the two subunits of the Fc domain, and thus the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, in order to increase the yield and purity of such antibodies in recombinant production, it would be advantageous to include in the Fc domain of the antibody modifications that promote the association of the desired polypeptides.

[0272] Accordingly, in certain embodiments, the Fc domain comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The region where the protein-protein interaction between the two subunits of the human IgG Fc domain extends most extensively is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is within the CH3 domain of the Fc domain.

[0273] Several methods for modifying the CH3 domain of the Fc domain to enhance heterodimerization are well described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) is directed not to homodimerize with itself but to heterodimerize with the other CH3 domain engineered in a complementary fashion (as a result, the first CH3 domain and the second CH3 domain heterodimerize and no homodimers between two first CH3 domains or two second CH3 domains are formed). These different methods for improving heavy chain heterodimerization are considered as different alternatives, in combination with heavy chain-light chain modifications (e.g., exchange / replacement of variable or constant regions in one Fab arm, or introduction of substitutions of charged amino acids with opposite charges at the CH1 / CL interface), to reduce light chain mispairing and Bence Jones-type by-products.

[0274] In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification to one of the two subunits of the Fc domain and a "hole" modification to the other one of the two subunits of the Fc domain.

[0275] The "knob-into-hole" technology is described, for example, in U.S. Patent Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng. 9, 617-621 (1996), and Carter, J Immunol Meth. 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") on the contact surface of a first polypeptide and a complementary cavity ("hole") on the contact surface of a second polypeptide such that the protrusion can be positioned within the cavity, promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the contact surface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Complementary cavities of the same or similar size to the protrusion are created on the contact surface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0276] Thus, in one particular embodiment, in the CH3 domain of the first subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion within the CH3 domain of the first subunit that can be positioned within the cavity inside the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit that can accommodate the protrusion inside the CH3 domain of the first subunit.

[0277] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0278] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0279] Protrusions and cavities can be created by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or peptide synthesis.

[0280] In certain embodiments, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, further, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (EU numbering).

[0281] In further embodiments, in the first subunit of the Fc domain, further, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain, further, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (EU numbering). The introduction of these two cysteine residues forms a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0282] In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (EU numbering).

[0283] In certain embodiments, the CD3 antigen-binding portion described herein is fused to the first subunit of the Fc domain (including the "knob" modification). Without wishing to be bound by theory, the fusion of the CD3 antigen-binding portion to the knob-containing subunit of the Fc domain (further) minimizes the generation of bispecific antibodies comprising two CD3 antigen-binding portions (steric clashes of two knob-containing polypeptides).

[0284] Other techniques for enhancing heterodimers are contemplated as alternatives and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0285] In one embodiment, the heterodimerization method described in EP1870459 A1 is used instead. This method is based on introducing charged amino acids having opposite charges at specific amino acid positions in the CH3 / CH3 domain contact surface between the two subunits of the Fc domain. One preferred embodiment is the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutations D399K and E357K in the other CH3 domain of the Fc domain (EU numbering).

[0286] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further comprises the amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (EU numbering).

[0287] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises the amino acid mutations S354C and T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises the amino acid mutations Y349C and T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further comprises the amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (all EU numbering).

[0288] In one embodiment, the heterodimerization method described in WO 2013 / 157953 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (EU numbering). In a further embodiment, the first CH3 domain further comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D, and L368E (preferably L368E) (EU numbering).

[0289] In one embodiment, the heterodimerization method described in International Publication No. WO 2012 / 058768 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain comprises additional amino acid mutations at positions T411, D399, S400, F405, N390, or K392, for example, (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W; (b) D399R, D399W, D399Y or D399K; (c) S400E, S400D, S400R, or S400K; (d) F405I, F405M, F405T, F405S, F405V or F405W; (e) N390R, N390K or N390D; (f) K392V, K392M, K392R, K392L, K392F or K392E (EU numbering). In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y and Y407A, and the second CH3 domain comprises the amino acid mutations T366V and K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A, and the second CH3 domain comprises the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R, and S400R (EU numbering).

[0290] In one embodiment, for example, the heterodimerization method described in International Publication No. WO 2011 / 143545 with amino acid modifications at positions selected from the group consisting of 368 and 409 is alternatively used (EU numbering).

[0291] In one embodiment, the heterodimerization method described in International Publication No. WO 2011 / 090762, which also uses the knob-into-hole technology described above, is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (EU numbering).

[0292] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody, or the Fc domain of the anti-CD20 / anti-CD3 bispecific antibody, is of the IgG2 subclass and the heterodimerization method described in International Publication No. WO 2010 / 129304 is used.

[0293] In alternative embodiments, the modification that promotes the association of the first and second subunits of the Fc domain includes, for example, modifications that mediate an electrostatic steering effect as described in PCT International Publication No. WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the contact surface of the two Fc domain subunits by charged amino acid residues such that homodimer formation is electrostatically less favorable and heterodimerization is electrostatically more favorable. In one such embodiment, the first CH3 domain includes an amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain includes an amino acid substitution of D399, E356, D356 or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain further or alternatively includes an amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E), or aspartic acid (D)) (EU numbering).

[0294] In yet further embodiments, the heterodimerization method described in International Publication No. WO 2007 / 147901 may alternatively be used. In one embodiment, the first CH3 domain includes the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain includes the amino acid mutations D239K, E240K, and K292D (EU numbering).

[0295] In yet another embodiment, the heterodimerization method described in International Publication No. WO 2007 / 110205 may be used.

[0296] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (EU numbering).

[0297] (ii) Fc domain modifications that reduce the binding and / or effector functions of Fc receptors The Fc domain confers desirable pharmacokinetic properties, including a long serum half-life and a desirable tissue-blood distribution ratio, that contribute to good accumulation in target tissues for antibodies such as anti-CD20 / anti-CD3 bispecific antibodies. However, at the same time, the Fc domain can cause unwanted targeting of the antibody to cells that express Fc receptors rather than to the preferred antigen-bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway can lead to cytokine release, which, in combination with other immune-activating properties that the antibody may have and the long half-life of the antibody, can result in over-activation of cytokine receptors and cause severe side effects when administered systemically.

[0298] Thus, in certain embodiments, the Fc domain of the anti-CD20 / anti-CD3 bispecific antibody exhibits a reduced binding affinity for Fc receptors and / or a reduced effector function when compared to the negative IgG1 Fc domain. In such an embodiment, the Fc domain (or a molecule comprising said Fc domain, such as an antibody) exhibits a binding affinity for Fc receptors that is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG1 Fc domain (or the corresponding molecule comprising the native IgG1 Fc domain), and / or exhibits an effector function that is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG1 Fc domain (or the corresponding molecule comprising the native IgG1 Fc domain). In one embodiment, the Fc domain (or a molecule comprising said Fc domain, such as an antibody) does not substantially bind to Fc receptors and / or does not induce effector function. In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits a binding affinity for the neonatal Fc receptor (FcRn) that is substantially similar to that of the native IgG1 Fc domain. A binding affinity for FcRn that is substantially similar is achieved when the Fc domain (or a molecule comprising said Fc domain, such as an antibody) exhibits a binding affinity for FcRn that is greater than about 70%, particularly about 80%, and even more particularly about 90% of the native IgG1 Fc domain (or the corresponding molecule comprising the native IgG1 Fc domain).

[0299] In certain embodiments, the Fc domain is engineered such that its binding affinity for Fc receptors and / or effector functions are reduced as compared to a non-engineered Fc domain. In certain embodiments, the Fc domain comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors and / or effector functions. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors. In certain embodiments, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors by at least one-half, at least one-fifth, or at least one-tenth. In embodiments where there are more than one amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain for Fc receptors by at least one-tenth, at least one-twentieth, or even at least one-fiftieth. In one embodiment, a molecule comprising the engineered Fc domain, such as an antibody, exhibits a binding affinity for Fc receptors that is less than 20%, particularly less than 10%, and even less than 5% compared to its corresponding molecule comprising a non-engineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components (specifically, the binding affinity for C1q) is also reduced. In one embodiment, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain for said receptor, is achieved when the Fc domain (or a molecule comprising said Fc domain, such as an antibody) exhibits an affinity that exceeds about 70% of the binding affinity of the non-engineered form of the Fc domain (or the corresponding molecule comprising said non-engineered form of the Fc domain) for FcRn. The Fc domain, or a molecule comprising said Fc domain (such as an antibody), can exhibit an affinity that exceeds about 80%, and in some cases about 90%, of such affinity. In certain embodiments, the Fc domain is engineered to have reduced effector function. Reduction of effector function can include, but is not limited to, reduction of complement-dependent cytotoxicity (CDC), reduction of antibody-dependent cell-mediated cytotoxicity (ADCC), reduction of antibody-dependent cell phagocytosis (ADCP), reduction of cytokine secretion, reduction of immune complex-mediated antigen uptake by antigen-presenting cells, reduction of binding to NK cells, reduction of binding to macrophages, reduction of binding to monocytes, reduction of binding to polymorphonuclear cells, reduction of direct signaling that induces apoptosis, reduction of cross-linking of target-binding antibodies, reduction of dendritic cell maturation, or reduction of T cell priming, and can include one or more of these. In one embodiment, the reduction of effector function is one or more selected from the group consisting of reduction of CDC, reduction of ADCC, reduction of ADCP, and reduction of cytokine secretion. In a particular embodiment, the reduction of effector function is reduction of ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by the non-engineered Fc domain (or the corresponding molecule comprising the non-engineered Fc domain).

[0300] In one embodiment, the amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for the Fc receptor are amino acid substitutions. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (EU numbering). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (EU numbering). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (EU numbering). In such embodiments, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (EU numbering). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (EU numbering). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In certain embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (EU numbering). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G (“P329G LALA”). In such embodiments, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The combination of amino acid substitutions “P329G LALA” almost completely abrogates Fcγ receptor (and complement) binding of the human IgG1 Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

[0301] IgG4 antibodies exhibit a reduced binding affinity for Fc receptors and a reduced effector function as compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (EU numbering). In one embodiment, to further reduce its binding affinity and / or its effector function for Fc receptors, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (EU numbering). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (EU numbering). In certain embodiments, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, specifically the amino acid substitutions S228P, L235E, and P329G (EU numbering). Such IgG4 Fc domain variants and their Fcγ receptor binding characteristics are described in PCT International Publication No. WO 2012 / 130831, which is hereby incorporated by reference in its entirety.

[0302] In certain embodiments, the Fc domain that exhibits a reduced binding affinity for Fc receptors and / or a reduced effector function as compared to the native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (EU numbering).

[0303] In certain embodiments, the N-glycosylation of the Fc domain is removed. In such an embodiment, the Fc domain comprises an amino acid substitution at position N297, particularly an amino acid substitution that replaces asparagine with alanine (N297A) or aspartic acid (N297D) or glycine (N297G) (EU numbering).

[0304] In addition to the Fc domains described in this specification and International Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector functions also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (EU numbering). Such Fc variants include Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant having substitutions of alanine at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0305] Variant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be verified, for example, by sequencing.

[0306] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard devices such as a BIACORE® instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain, or a molecule containing an Fc domain, for an Fc receptor may be evaluated using cell lines known to express a particular Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0307] The effector functions of an Fc domain, or a molecule (e.g., an antibody) containing an Fc domain, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA. 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA. 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be utilized (e.g., ACTI for flow cytometry TM See non-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in an animal model (such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998)), for example, in i.

[0308] In some embodiments, binding of the Fc domain to complement components, specifically binding to C1q, is reduced. Thus, in some embodiments where the Fc domain is engineered to have reduced effector function, the reduction in said effector function includes a reduction in CDC. A C1q binding assay can be performed to determine whether an Fc domain, or a molecule (e.g., an antibody) containing the Fc domain, can bind to C1q and thereby have CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg and Glennie, Blood 103:2738-2743 (2004)).

[0309] 3. Substitutions, Insertions, and Deletions In certain examples, the anti-CD20 / anti-CD3 bispecific antibody variants of the pharmaceutical compositions provided herein have one or more amino acid substitutions. Target sites for mutagenesis by substitution include HVRs and FRs. Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions". More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF0007701982000003.tif168170

[0310] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0311] Non-conservative substitutions involve exchanging a member of one of these classes for another.

[0312] Certain substitution variants involve substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies and can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, the variant antibody is displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0313] Modifications (e.g., substitutions) may be made, for example, in the HVRs to improve antibody affinity. Such modifications can be made in “hot spots” within the HVRs, i.e., residues encoded by codons that mutate frequently during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by construction of a secondary library and reselection therefrom is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some examples of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants having the desired affinity. Another method for introducing diversity includes techniques directed to HVRs in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0314] In certain examples, substitutions, insertions, or deletions can occur within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind to its antigen. For example, conservative modifications (e.g., conservative substitutions as described herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications can be, for example, outside of the antigen contact residues within the HVRs. In certain examples of the variant VH and VL sequences described above, each HVR is either unmodified or has one, two, or three or fewer amino acid substitutions.

[0315] A useful method for identifying residues or regions of an antibody that can be targets for mutagenesis is what is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (such as charged residues like Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (such as alanine or polyalanine) to determine whether the interaction between the antigen and the antibody is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition to this, the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they have the desired properties.

[0316] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing over 100 residues, as well as in-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N-terminal or C-terminal of the antibody to an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0317] 4. Glycosylation In certain examples, the anti-CD20 / anti-CD3 bispecific antibody contained in the pharmaceutical composition of the present invention can be modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the anti-CD20 / anti-CD3 bispecific antibody can be readily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0318] When an antibody contains an Fc region, the carbohydrates attached thereto can be modified. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some instances, modification of the oligosaccharides in an antibody can be performed to generate antibody variants with improved specific properties.

[0319] In one example, an anti-CD20 / anti-CD3 bispecific antibody variant has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined, for example, as described in WO 2008 / 077546, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total of all sugar chain structures attached to Asn297 (e.g., complex structures, hybrid structures, and high mannose structures) measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, US 2003 / 0157108 A1 (Presta, L.); US 2004 / 0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108 A1; WO 2000 / 61739 A2; WO 2001 / 29246 A2; US 2003 / 0115614 A1; US 2002 / 0164328 A1; US 2004 / 0093621 A1; US 2004 / 0132140 A1; US 2004 / 0110704 A1; US 2004 / 0110282 A1; US 2004 / 0109865 A1; WO 2003 / 085119 A2; WO 2003 / 084570 A2; WO 2005 / 035586 A2; WO 2005 / 035778 A2; WO 2005 / 053742 A2; US 2002 / 031140 A1; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines having the ability to produce defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Publication No. 2003 / 0157108, Presta, L; and International Publication No. 2004 / 056312, Adams et al., particularly Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0320] From the above perspectives, in some embodiments, the pharmaceutical composition of the present invention comprises an anti-CD20 / anti-CD3 bispecific antibody variant comprising an aglycosylation site mutation. In some examples, the aglycosylation site mutation reduces the effector function of the antibody. In some examples, the mutation at the aglycosylation site is a substitution mutation. In some examples, the antibody comprises a substitution mutation in the Fc region that reduces the effector function. In some examples, the substitution mutation is at amino acid residues N297, L234, L235, and / or D265 (EU numbering). In some examples, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some examples, the substitution mutation is at amino acid residue N297. In a preferred example, the substitution mutation is N297A.

[0321] Anti-CD20 / anti-CD3 bispecific antibody variants have a bisected oligosaccharide, for example, a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; US Pat. No. 6,602,684; and US Patent Application Publication No. 2005 / 0123546. Other antibody variants contain at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0322] 5. Antibody derivatives In certain examples, the anti-CD20 / anti-CD3 bispecific antibodies of the pharmaceutical compositions provided herein are further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under the conditions in which it is defined, and the like.

[0323] In another example, conjugates of antibodies and non-proteinaceous moieties can be selectively heated by exposure to radiation. In one example, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that heat the non-protective moiety to a temperature at which cells proximal to the non-protective site of the antibody are killed, but which do not harm normal cells.

[0324] C. Recombinant production methods The anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 TCBs, e.g., glofitamab) of the pharmaceutical compositions of the present invention can be produced using recombinant methods and compositions such as those described in U.S. Patent No. 4,816,567, which is hereby incorporated by reference in its entirety.

[0325] For the recombinant production of anti-CD20 / anti-CD3 bispecific antibodies, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0326] Suitable host cells for the cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the present invention may be isolated from the soluble fraction of the bacterial cell paste and further purified.

[0327] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeast are suitable as cloning or expression hosts for vectors encoding antibodies, including strains and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of antibodies having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0328] Also, host cells suitable for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified and can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0329] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0330] Vertebrate cells are also used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cells transformed with SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 cells or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor cells (MMT060562); TRI cells described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include (DHFR - Chinese hamster ovary (CHO) cells, including CHO cells expressing DHFR (Urlaub et al. Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0331] V. Therapeutic Methods and Uses The pharmaceutical composition comprising the anti-CD20 / anti-CD3 bispecific antibody described herein can be formulated for use as a medicament for treating various diseases and disorders. Accordingly, the present invention features a method comprising intravenous administration of the pharmaceutical composition to a subject in need thereof, such as a subject having a disease or disorder such as cancer. The pharmaceutical composition of the present invention can be used for treating or delaying the progression of a cell proliferative disorder in a subject in need thereof (e.g., a human subject in need thereof), or for enhancing the immune function in a subject having a cell proliferative disorder (e.g., cancer).

[0332] In one aspect, the present invention provides the pharmaceutical composition described herein for use in treating a cell proliferative disorder or delaying its progression. In one aspect, the present invention provides the use of the pharmaceutical composition described herein in the manufacture of a medicament for treating or delaying the progression of a cell proliferative disorder. In one aspect, the present invention provides a method for treating or delaying the progression of a cell proliferative disorder in a subject in need thereof, comprising administering the pharmaceutical composition described herein to the subject.

[0333] In one embodiment, the cell proliferative disorder is a cancer that is non-Hodgkin lymphoma (NHL). In some embodiments, the NHL is non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, Burkitt-like lymphoma with 11q abnormality, B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system DLBCL, primary cutaneous DLBCL (lower leg type), Epstein-Barr virus (EBV)-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, large B-cell lymphoma arising in HHV8-related multicentric Castleman disease, B-cell leukemia, breast cancer, colorectal cancer, non-small cell lung cancer, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, follicular lymphoma (FL), in situ follicular neoplasm, mantle cell lymphoma (MCL), in situ mantle cell neoplasm, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell lymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasmacytoma, solitary bone plasmacytoma, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodular marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, primary pleural effusion lymphoma, selected from the group consisting of.In certain embodiments, the cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenström macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt lymphoma (BL).

[0334] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and glioblastoma.

[0335] The anti-CD20 / anti-CD3 bispecific antibody can be formulated for administration to a subject at a dose of 0.5 mg, 2.5 mg, 10 mg, or 30 mg.

[0336] For all of the methods and pharmaceutical formulations described herein, anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to the medical practitioner. Anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) need not be formulated with one or more of the drugs currently being used to prevent or treat the disease in question, but optionally may be formulated together. The effective amount of such other drugs will depend on the amount of anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) present in the formulation, the type of disorder or treatment, and the other factors discussed above. Anti-CD20 / anti-CD3 bispecific antibodies (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) can be appropriately administered to a patient over a series of treatments.

[0337] V. MANUFACTURED ARTICLE In another aspect of the invention, there is provided a manufactured article containing a substance useful for the treatment, prevention and / or diagnosis of the above-described disorders. The manufactured article includes a container and a label or package insert on or associated with the container. Suitable containers include, by way of example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container holds a pharmaceutical composition for use alone or in combination with another composition effective to treat, prevent, and / or diagnose a condition and may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by an intravenous solution bag or a hypodermic needle). At least one active agent in the composition is an anti-CD20 / anti-CD3 bispecific antibody as described herein (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab). The label or package insert indicates that the composition is to be used for treating a selected condition (e.g., cancer) and further includes information related to at least one of the dosing regimens described herein.

[0338] The pharmaceutical composition can be provided in a container having a volume of from 1 ml to 100 ml (e.g., from 1 ml to 5 ml, 5 ml to 10 ml, 10 ml to 15 ml, 15 ml to 20 ml, 20 ml to 25 ml, 25 ml to 30 ml, 30 ml to 40 ml, 40 ml to 50 ml, 50 ml to 60 ml, 60 ml to 70 ml, 70 ml to 80 ml, 80 ml to 90 ml, or 90 ml to 100 ml, e.g., about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, or about 100 ml).

[0339] In some embodiments, the container is a stainless steel container or nickel steel alloy container such as a tank, mini-tank, canister, can, etc. (e.g., HASTELLOY®). In some examples, the pharmaceutical composition in such a container is the drug substance (DS) and can be further diluted before use to, for example, a pharmaceutical product (DP) (e.g., final vial configuration). Alternatively, the pharmaceutical composition in the container is the DP. In some embodiments, the DP is in a container such as an IV bag or syringe (e.g., for delivery via a syringe pump).

[0340] In some embodiments, the manufactured article includes a vial having a volume of about 1 ml or more, such as about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about 19 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 50 ml, or more. In some embodiments, the container is a vial having a volume of about 10 ml. In some embodiments, the vial is disposable. In some embodiments, the vial contains an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab) of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, or more. In some embodiments, the container closure system includes one or more, or all, of a glass vial, stopper, and cap.

[0341] Furthermore, the manufactured product may include (a) a first container containing therein a composition comprising an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) described herein, and (b) a second container containing therein a composition comprising a further cytotoxic agent or other therapeutic agent. Alternatively or additionally, the manufactured product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The manufactured product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0342] A further aspect of the invention relates to the invention described herein.

[0343] Embodiment Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments.

[0344] I. A liquid pharmaceutical composition, having a pH in the range of about 5.0 to about 6.0, from about 1 to 25 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody; from about 10 to 50 mM of a buffer; from about ≧200 mM of an isotonic agent; from about 0 - 15 mM of methionine; and from about ≧0.2 mg / ml of a surfactant; comprising, wherein the anti-CD20 / anti-CD3 bispecific antibody is: a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 comprising a heavy chain variable region, and (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 A light chain variable region comprising: At least one antigen-binding domain that specifically binds to CD20 and comprises: b) At least one antigen-binding domain that specifically binds to CD3, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 A heavy chain variable region comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 A light chain variable region comprising: At least one antigen-binding domain that specifically binds to CD3 and comprises: A liquid pharmaceutical composition comprising:

[0345] II. The liquid pharmaceutical composition according to Embodiment I, wherein the concentration of the anti-CD20 / anti-CD3 bispecific antibody is in the range of about 1 to 5 mg / ml.

[0346] III. The liquid pharmaceutical composition according to Embodiment I or II, wherein the concentration of the anti-CD20 / anti-CD3 bispecific antibody is in the range of about 0.9 - 1.1 mg / ml.

[0347] IV. The liquid pharmaceutical composition according to any one of Embodiments I to III, wherein the concentration of the anti-CD20 / anti-CD3 bispecific antibody is about 1 mg / ml.

[0348] V. The anti-CD20 / anti-CD3 bispecific antibody is a) At least one antigen-binding domain that specifically binds to CD20 and comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8, b) At least one antigen-binding domain that specifically binds to CD3 and comprises the heavy-chain variable region sequence of SEQ ID NO: 15 and the light-chain variable region sequence of SEQ ID NO: 16; The liquid pharmaceutical composition according to any one of Embodiments I to IV, comprising the same.

[0349] VI. The anti-CD20 / anti-CD3 bispecific antibody is a) A first Fab molecule that specifically binds to CD3, particularly CD3 epsilon, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are exchanged with each other; b) A second Fab molecule and a third Fab molecule that specifically bind to CD20, wherein in the constant domain CL of the second Fab molecule and the third Fab molecule, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted with lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second Fab molecule and the third Fab molecule, the amino acid at position 147 is substituted with glutamic acid (E) (EU numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (EU numbering); c) An Fc domain composed of a first subunit and a second subunit capable of stable association; The liquid pharmaceutical composition according to any one of Embodiments I to V, comprising the same.

[0350] VII. The anti-CD20 / anti-CD3 bispecific antibody is glofitamab, and the liquid pharmaceutical composition according to any one of Embodiments I to VI, comprising the same.

[0351] VIII. The buffer is a histidine buffer, optionally a histidine HCl buffer, and the liquid pharmaceutical composition according to any one of Embodiments I to VII, comprising the same.

[0352] IX. A liquid pharmaceutical composition according to any one of Embodiments I to VIII, wherein the concentration of the buffer is from about 15 to 25 mM.

[0353] X. A liquid pharmaceutical composition according to any one of Embodiments I to IX, wherein the concentration of the buffer is about 20 mM.

[0354] XI. A liquid pharmaceutical composition according to any one of Embodiments I to X, wherein the buffer provides a pH of from about 5.2 to about 5.8.

[0355] XII. A liquid pharmaceutical composition according to any one of Embodiments I to XI, wherein the isotonic agent is selected from the group consisting of salts, saccharides, and amino acids.

[0356] XIII. A liquid pharmaceutical composition according to Embodiment XII, wherein the isotonic agent is either sucrose or sodium chloride.

[0357] XIV. A liquid pharmaceutical composition according to Embodiment XIII, wherein the isotonic agent is sucrose at a concentration of about 200 mM or more.

[0358] XV. A liquid pharmaceutical composition according to Embodiment XIII or XIV, wherein the isotonic agent is sucrose at a concentration of about 200 mM to 280 mM.

[0359] XVI. A liquid pharmaceutical composition according to any one of Embodiments XIII to XV, wherein the isotonic agent is sucrose at a concentration of about 240 mM.

[0360] XVII. A liquid pharmaceutical composition according to any one of Embodiments I to XVI, wherein the concentration of methionine is from about 5 to 15 mM.

[0361] XVIII. A liquid pharmaceutical composition according to Embodiment XVII, wherein the concentration of methionine is about 10 mM.

[0362] XIX. A liquid pharmaceutical composition according to any one of Embodiments I to XVIII, wherein the concentration of the surfactant is from about 0.2 to 0.8 mg / ml.

[0363] XX. The liquid pharmaceutical composition according to any one of Embodiments I to XIX, wherein the surfactant is polysorbate 20 or poloxamer 188.

[0364] XXI. The liquid pharmaceutical composition according to Embodiment XX, wherein the surfactant is polysorbate 20 at a concentration of 0.2 - 0.8 mg / ml.

[0365] XXII. The liquid pharmaceutical composition according to Embodiment XXI, wherein the surfactant is polysorbate 20 at a concentration of about 0.5 mg / ml.

[0366] XXIII. A liquid pharmaceutical composition, at a pH of about 5 to about 6, about 1 to 5 mg / ml of an anti-CD20 / anti-CD3 bispecific antibody; about 15 - 25 mM of a histidine buffer; about 200 - 280 mM of sucrose; about 0 - 15 mM of methionine; about 0.2 - 0.8 mg / ml of PS20; and comprising the liquid pharmaceutical composition according to any one of Embodiments I to XXII.

[0367] XXIV. A liquid pharmaceutical composition, at a pH of about 5.5, about 1 mg / ml of glofitamab; about 20 mM of a histidine buffer; about 240 mM of sucrose; about 10 mM of methionine; about 0.5 mg / ml of PS20; and comprising the liquid pharmaceutical composition according to any one of Embodiments I to XXIII.

[0368] XXV. Use of the liquid pharmaceutical composition according to any one of Embodiments I to XXIV for the preparation of a medicament useful for treating a cell proliferative disorder.

[0369] XXVI. A pharmaceutical composition according to any one of embodiments I to XXIV for use in a subject in need of treatment of a cell proliferative disorder or delay of its progression.

[0370] XXVII. A method of treating or delaying the progression of a cell proliferative disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to any one of embodiments I to XXIV.

[0371] XXVIII. Use, liquid pharmaceutical composition for use, or method according to any one of embodiments XXV to XXVII, wherein the cell proliferative disorder is cancer.

[0372] XXIX. The invention described above.

Examples

[0373] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments can be implemented on the premise of the general description provided above.

[0374] Example 1: In silico analysis of grofitamab RO7082859 / glofitamab is a T-cell bispecific humanized monoclonal antibody (TCB) that binds to human CD20 on tumor cells and to the human CD3 epsilon subunit (CD3ε) of the T-cell receptor complex (TCR) on T cells. It is composed of two different heavy chains and two different light chains. Point mutations in the CH3 domain ("knob-into-hole") facilitate the assembly of the two different heavy chains. The exchange of the VH and VL domains in the CD3-binding Fab ("CrossMab approach") and point mutations in the CH and CL domains in the CD20-binding Fab ("charged variants") facilitate the correct assembly of the two different light chains with the corresponding heavy chains. The "knob-into-hole" mutations consist of amino acid exchanges Y349C, T366S, L368A, and Y407V in heavy chain HC1 and amino acid exchanges S354C and T366W in heavy chain HC2 (Kabat EU index numbering). The "charged variant" mutations consist of amino acid exchanges E123R and Q124K in light chain LC2 (Kabat numbering) and amino acid exchanges K147E and K213E in heavy chains HC1 and HC2 (Kabat EU index numbering).

[0375] Binding to human CD20 occurs in a bivalent binding mode with high affinity, whereas binding to CD3ε is monovalent and of low affinity. RO7082859 is a human IgG1 that has a modification ("PG LALA" mutation) in the Fc region that inhibits in vitro binding to Fc gamma receptors (FcγR) and prevents FcγR-mediated co-activation of innate immune effector cells including natural killer (NK) cells, monocytes / macrophages, and neutrophils, and the functional binding to FcRn (neonatal Fc receptor) is unchanged. The "PG LALA" mutation consists of amino acid exchanges P329G, L234A, and L235A ("PG LALA", Kabat EU index numbering) in heavy chain HC1 and heavy chain HC2.

[0376] The recombinant antibody is produced in CHO cells and consists of two heavy chains (449 and 674 amino acid residues, respectively) and three light chains (232 and 219 (2 copies) amino acid residues, respectively), which are asymmetrically arranged as shown in Figure 2.

[0377] Overview of the active hot spot Regarding the CD3-binding portion of the molecule, in silico prediction showed that there were two Asn residues that were easily decomposed in the CDR3 of the heavy chain and one exposed Trp residue. In a 14-day stress experiment, no significant change in the target-binding activity was observed after incubation at pH 6.0, but a strong decrease in the target-binding activity was observed after incubation at physiological pH (PBS pH 7.4, data not shown).

[0378] Example 2: Introduction of golphitinib formulation development GLP Tox and human studies Screens were performed according to the scheme shown in Table 4. During the screen, the formulations were exposed to the following conditions: storage for 3 and 6 weeks (5 °C, 25 °C, and 40 °C), shaking and freeze / thaw (F / T) stress (5 cycles) at 5 °C and 25 °C for 1 week. The designated formulations were then followed up to 52 weeks. TIFF0007701982000004.tif73170

[0379] After storage at 5 °C, 25 °C, and 40 °C for 6 weeks, all formulations showed no significant changes in most of the physical properties tested, namely visible particles and particles not visible to the naked eye, color, turbidity, pH, and protein content. Data from CE-SDS (capillary electrophoresis sodium dodecyl sulfate) are not shown as they were not important for the designation.

[0380] In the visible particle analysis by the Seidenader method, no visible particle formation was observed for any of the formulations under all storage conditions. The number of particles invisible to the naked eye was small (not shown). Under mechanical stress conditions, F2 - F5 showed many particles at both 5°C and 25°C. F1 had no particles under either condition. When using EP and Optima, except for F3 and F4 (both with P188), there were virtually no particles (particle 0) in all compositions, and particles were observed but below the limit value (not shown). In the shaking at 5°C, the particles invisible to the naked eye were significantly worse for F3 (P188 + Met) than for F4 (P188). All other formulations showed similar numbers under each condition (not shown).

[0381] Turbidity and color showed no significant changes in all formulations under all conditions after 6 weeks. The surfactant content was stable at 5°C and 25°C and also at 40°C for the formulations containing P188 (F3, F4). In all PS20-containing active formulations (F1, F2, and F5), a decrease in the surfactant content was observed at 40°C regardless of whether the formulation contained methionine or not.

[0382] The beneficial effect of methionine was only observed in the placebo formulation containing PS20, and the decrease in the PS content at 40°C was only for P2 (Figure 3). In the biochemical property evaluation, the differences in the formulations became clear only after storage at 40°C.

[0383] In size exclusion chromatography (SEC), the loss of monomers was more significant in F2 and F5 and was correlated with the increase in the HMW (high molecular weight) area. New HMW species appeared, which were slight in F3 and F4, stronger in F1, and very large in F2 and F5. It was found that the LMW (low molecular weight) species increased at approximately the same rate in all formulations (Figure 4). A similar trend was observed in ion exchange chromatography (IEC), where the basic peak area increased overall, and the increase in the acidic area was more significant in F2 and F5 (Figure 5).

[0384] In summary, the data clearly excluded F2 and F5, indicating that F1, F3, and F4 were equally stable and that none of the three was clearly preferred. F1 (5 mg / ml of golimumab, 20 mM histidine / histidine HCl, pH 5.5, 240 mM sucrose, 10 mM methionine, 0.05% (w / v) of PS20) was designated. An overview of all the analysis results for F1 can be seen in Figure 6.

[0385] Example 3: Introduction to GLP Tox / Human Studies Binding by BIACORE® The aforementioned purity results were also reflected in the loss of CD20 binding at 40 °C for F2 and F5 and the strong loss up to 50% of CD3 binding in their formulations, whereas the loss for the remaining formulations was between 10 and 20% (Figures 7A and 7B).

[0386] Example 4: Development Studies for Phase III and Commercial Formulations In this example, an overview of the pharmaceutical development of the golimumab formulation is provided. As a result of this development, the golimumab medicament is provided as a sterile concentrate for IV infusion. The medicament consists of 1 mg / ml of golimumab, 240 mM sucrose, 10 mM L-methionine, 0.5 mg / ml of polysorbate 20 in a 20 mM L-histidine / L-histidine hydrochloride (HCl) buffer at pH 5.5. Golimumab is the only active ingredient in the drug substance and the medicament. The formulation development studies confirmed that the dosage form and formulation are suitable for the intended use. The formulation is sufficiently robust to ensure that the medicament is stable during manufacture, storage, transport, and administration.

[0387] Formulations with higher protein concentrations (e.g., 5, 25, or 50 mg / ml of golphitinib) were also tested, but were not followed up subsequently due to the formation of subvisible and visible particles due to the degradation of PS20. The release of free fatty acids (lauric acid and myristic acid) at levels increasing with protein concentration confirmed that the root cause of the formation of subvisible and visible particles was due to the hydrolysis of PS20.

[0388] The selection of a liquid dosage form reduced the handling procedures while ensuring the quality of the product during manufacture and until the end of the shelf life of the pharmaceutical product.

[0389] The golphitinib pharmaceutical product will be commercially available in two vial configurations of two strengths, filling 2.5 mg / vial into 6 ml disposable glass vials and 10 mg / vial into 15 ml disposable glass vials, in order to minimize product waste and to conform to the required clinical dosages of 2.5 mg, 10 mg, and 30 mg. In the commercially available pharmaceutical formulation, the concentration of golphitinib was reduced to 1 mg / ml without changing the additive composition.

[0390] The formulation development studies provided the basis for selecting the appropriate dosage form, protein concentration, surfactant concentration, buffer species, solution pH, stabilizer, isotonic agent, and vial configuration of the pharmaceutical product. The formulation of the active ingredient was optimized taking into account facility compatibility, dilution, and storage considerations.

[0391] Selection of Dosage Form The selection of a liquid dosage form to provide a concentrate for infusion reduced the handling procedures while ensuring the quality of the product during manufacture and until the end of the shelf life of the pharmaceutical product.

[0392] Selection of Protein Concentration A protein concentration of 5 mg / ml was selected in Phase I and maintained until Phase III. Subsequently, based on formulation development studies and the latest clinical dosing requirements, a protein concentration of 1 mg / ml was selected as the commercial formulation.

[0393] To prepare the protein concentration to match the clinical need, the stability of a formulation at pH 5.5 containing 20 mM L-histidine / L-histidine hydrochloride, 10 mM L-methionine, 240 mM D-sucrose, and 0.5 mg / ml polysorbate 20 (PS20) was tested at golimumab concentrations of 1 mg / ml, 5 mg / ml, and 25 mg / ml. These formulations were evaluated at the initial time point (T0), several intermediate time points, and at the end of the study after storage at 2 °C to 8 °C for 104 weeks by assessing the purity of golimumab by SE-HPLC and IE-HPLC, the PS20 content, and the formation of visible particles / invisible particles to the naked eye.

[0394] The purity by SE-HPLC and IE-HPLC was equivalent between the 1 mg / ml formulation and the 5 mg / ml formulation throughout the study (Figures 8A and 8B). The number of invisible particles to the naked eye was also comparable. Furthermore, the 1 mg / ml formulation did not show PS20 degradation exceeding the method variability compared to the 5 mg / ml formulation and the 25 mg / ml formulation (see Figure 12 and "Evaluation of Polysorbate 20 Degradation" below). Based on these results and the current clinical dosage regimens of 2.5 mg, 10 mg, and 30 mg, the 1 mg / ml formulation was selected as the commercial formulation.

[0395] The concentration range of 0.9 - 1.1 mg / ml protein was further evaluated in subsequent multivariate formulation robustness studies (see Example 5, Formulation Robustness Study). In this study, acceptable stability behavior was confirmed within this concentration range.

[0396] Selection of pH, Buffer, Stabilizer, and Isotonic Agent Based on the formulation development studies, a 20 mM solution of L-histidine / L-histidine hydrochloride at pH 5.5 was selected as the buffer, 10 mM L-methionine was used as the stabilizer, and 240 mM D-sucrose was used as the isotonic agent in combination in Phase I and retained for Phase III and the commercial formulation.

[0397] Studies with 5 mg / ml of golimumab were tested by setting the pH range of 20 mM L-histidine / L-histidine hydrochloride buffer from 5.5 to 6.0 and the levels of L-methionine at 0 and 10 mM. Additionally, a comparison of 240 mM D-sucrose and 130 mM sodium chloride was also conducted.

[0398] The effects of pH and stabilizers were evaluated by assessing the purity of golimumab by SE-HPLC and IE-HPLC and the formation of visible particles / invisible particles to the naked eye at the initial time point (T0) and after storage at 40 °C for 6 weeks. The selection of the isotonic agent was evaluated by measuring by SE-HPLC and IE-HPLC and determining the formation of visible particles / invisible particles to the naked eye at the initial time point (T0) and after storage at 25 °C for 26 weeks. The combination of 20 mM L-histidine / L-histidine hydrochloride buffer (pH 5.5) and 10 mM L-methionine showed the least formation of high molecular weight species (HMWS) (Figure 9A) and change in charged variants (Figure 9B) compared to the corresponding formulation without stabilizer or the combination of 20 mM L-histidine / L-histidine hydrochloride buffer / 10 mM L-methionine (pH 6). The concentration of 20 mM L-histidine / L-histidine hydrochloride monohydrate was shown to be sufficient to maintain during the manufacture of the pharmaceutical product and during the storage of the active pharmaceutical ingredient and the pharmaceutical product.

[0399] 240 mM D-sucrose was selected based on the comparison of 240 mM D-sucrose and 130 mM sodium chloride. The number of invisible particles to the naked eye was comparable among the formulations. No formation of invisible particles to the naked eye was observed after storage at 25 °C for 26 weeks in the D-sucrose-containing formulation, while visible particles were observed in the NaCl-containing formulation (Figure 10).

[0400] Selection of Surfactant PS20 at a concentration of 0.5 mg / ml was selected for Phase I based on the results of the stability study and maintained until commercial formulation. In a study of golphitinib at 50 mg / ml in 20 mM L-histidine / L-histidine hydrochloride buffer, pH 5.5, 10 mM L-methionine, and 240 mM D-sucrose, the stabilizing effect of poloxamer 188 (P188) on PS20 was investigated. P188 was tested at levels of 0.5, 0.7, and 1.0 mg / ml, and PS20 was tested at levels of 0.1, 0.3, and 0.5 mg / ml.

[0401] The influence of the added surfactant was evaluated by assessing the purity of golphitinib by SE-HPLC and IE-HPLC and the formation of visible / noncvisible particles at the initial time point (T0) and after shaking at 25 °C for 7 days.

[0402] Visible particle formation was observed at all P188 concentrations. Therefore, P188 was excluded as a suitable surfactant for glofitamab (Figure 11). After shaking at 25°C for 7 days, no visible particles were detected in the formulations containing PS20 (Figure 11). Compared with the 0.5 mg / ml PS20-containing formulation after shaking at 25°C for 7 days, a significant increase in HMWS and charged variants was observed in the 0.1 mg / ml PS20-containing formulation, while a slight increase in the levels of HMWS and charged variants was observed in the 0.3 mg / ml PS20-containing formulation (Figure 11). The number of particles not visible to the naked eye was comparable among different PS20 concentrations. Compared with the 0.5 mg / ml PS20-containing formulation after shaking at 25°C for 7 days, a significant increase in HMWS and charged variants was observed in the 0.1 mg / ml PS20-containing formulation, while a slight increase in the levels of HMWS and charged variants was observed in the 0.3 mg / ml PS20-containing formulation (Figure 11). Therefore, the 0.5 mg / ml PS20-containing formulation was selected. A level of 0.5 mg / ml of polysorbate 20 was shown to be sufficient to protect glofitamab from stresses that can occur during processing (such as agitation, freeze-thaw, or shear stress), handling, storage, and transportation. The concentration range of PS20 from 0.2 to 0.8 mg / ml was further evaluated in subsequent multivariate formulation robustness studies (see Example 5, Formulation robustness study). In this study, acceptable stability behavior was confirmed within this concentration range.

[0403] Example 5: Formulation robustness study The composition of the active pharmaceutical ingredient and the drug product can vary within a range based on manufacturing factors such as the metering tolerance of buffer components. A multivariate formulation robustness study was conducted, and it was demonstrated that the relevant quality attributes (QAs) of glofitamab are acceptable at the boundaries of these composition ranges. A multivariate stability study was conducted at two levels for three factors that were identified as potentially affecting critical quality attributes (CQAs) during the storage of the drug product. The following three formulation parameters were evaluated: 1. Protein concentration 2. pH 3. PS20 concentration. Furthermore, in the univariate stability study, three formulation parameters were evaluated individually: 4. Buffer strength 5. L-Methionine concentration 6. D-Sucrose concentration.

[0404] Multivariate formulation robustness studies demonstrated that the relevant CQAs of glofitamab were acceptable across the full range of the claimed formulation compositions.

[0405] Study Design Risk assessments were conducted to identify the critical drug substance and pharmaceutical formulation parameters important for maintaining product quality during the storage period. Multivariate and univariate analyses were accordingly set up.

[0406] Multivariate analysis (F6 to F12) Three formulation parameters, protein concentration, pH, and PS20 concentration, were used as input factors, and a fractional factorial design (resolution III) stability study at two levels was conducted.

[0407] Univariate analysis (F13 to F20) The concentrations of L-methionine and D-sucrose (low and high concentrations), as well as the buffer strength (low and high levels), were tested.

[0408] One formulation with low protein concentration, low pH, and low PS20 concentration was evaluated by direct comparison with the corresponding formulation with high pH, high protein concentration, and high PS20 concentration.

[0409] One formulation with a PS20 concentration of 0.3 mg / ml was included to support the setting of acceptance criteria.

[0410] The ranges of the formulation parameters tested were defined to cover either the acceptance criteria of the pharmaceutical specifications and / or the manufacturing acceptance ranges, as shown in Table 5. Table 6 shows a design plan including 15 experiments with three center points, and the three center points correspond to the target commercial formulation composition. TIFF0007701982000005.tif44170TIFF0007701982000006.tif104170

[0411] The stability of golphitinib in the pharmaceutical compositions described in Table 6 was evaluated as follows: ● Stability study: 〇 Storage conditions: Real time (2°C - 8°C), accelerated (25°C) 〇 Test frequency: Storage for 0, 4, 13, 26 (end of storage at 25°C), 39, 52, 78, and 104 weeks under the above storage conditions ● Stress test: 〇 Five freeze - thaw cycles, 〇 Vibration for 1 week at 2 - 8°C and vibration for 1 week at 25°C ● Stability to support DS: Storage at - 40°C for 0, 26, 52, and 104 weeks Evaluated QAs: 〇 HMWS (high - molecular - weight species) and main peak by SE - HPLC 〇 LMWS (low - molecular - weight species) and main peak by non - reducing CE - SDS 〇 Acidic peaks 2 and 3, acidic region, basic region, and main peak by IE - HPLC 〇 Protein content by ultraviolet - visible spectrophotometry 〇 Polysorbate 20 content by HPLC - ELSD 〇 Concentrations of L - methionine and L - histidine by RP - HPLC 〇 Oxidation and isomerization by peptide mapping (LC - MS) 〇 Potency by bioassay 〇 Visible particles 〇 Particles not visible to the naked eye 〇 Color, transparency / protein turbidity 〇 pH 〇 Osmotic pressure 〇 Density

[0412] Overall Data Analysis Procedure For each formulation, data on all quality attributes were collected over time. The relative changes over time of each QA were evaluated.

[0413] Multivariate analysis: For each quality attribute and each formulation, simple linear regression is applied over time. In this way, the degradation rates of each quality attribute and each formulation are calculated. If not specified, the degradation rate is reported as the degradation rate per week. These degradation rates are evaluated as responses in a design of experiments (DoE) study to examine the effects of three parameters, protein concentration, pH, and PS20 concentration, on these degradations. If the quality attribute did not show a significant change over time compared to the target formulation, regression analysis and effect estimation were not performed. For quality attributes that showed a significant change over time, the main effects of the three factors on the degradation rate were estimated using linear regression. Furthermore, main effect plots were shown to graphically display these effects.

[0414] Univariate analysis: For the parameters tested in the univariate analysis, the results after storage at 2 °C to 8 °C for 39 weeks were evaluated by comparison with T0 to identify potential changes. If a change was identified, the degradation rate was calculated and compared with the degradation rate of the target formulation to estimate the effect of the investigated formulation parameters at the boundary. In some cases, the degradation rate per week was converted to the degradation rate observed at 104 weeks by multiplying it by a factor of 104. Regression analysis was performed using JMP® software (SAS Institute, Cary, NC, Version 10.0 or higher).

[0415] Stability of robust formulations under the recommended storage conditions (2 °C to 8 °C): Table 7 shows an overview of the evaluation of the relative changes compared to the target formulation after storage at 2°C to 8°C for 39 weeks. For all formulations (F8, F9, F20) formulated at pH 6, an increase in the level of acidic variants (acidic region and acidic peak 2 by IE-HPLC) was observed. The observed increase in acidic variants was reflected by a corresponding decrease in the IE-HPLC main peak in the affected formulations. After storage at 2°C to 8°C for 39 weeks, no changes were observed in any other CQAs of all other formulations. As a conclusion, it was confirmed that pH is an important formulation parameter. All other formulation parameters, protein content, PS20, concentrations of L-methionine and D-sucrose, and buffer strength did not affect the tested CQAs within the investigated range.

[0416] Stability of robust formulations under accelerated storage conditions (25°C): Similar to the 2°C to 8°C data, an increase in the level of acidic variants due to deamidation (acidic region and acidic peak 2 by IE-HPLC) was observed in all formulations (F8, F9, F20) formulated at pH 6, which was reflected by a decrease in the IE-HPLC main peak in the affected formulations. Furthermore, in F1 and F2 formulated at pH 5, an increase in the fragmentation level was observed due to an increase in LMWS by CE-SDS. This increase was reflected by a decrease in the CE-SDS main peak. After storage at 25°C for 26 weeks, no changes were observed in any other CQAs of all other formulations.

[0417] As a conclusion, the 25°C data supported that pH is an important formulation parameter. All other formulation parameters did not affect the CQAs. TIFF0007701982000007.tif149170

[0418] Stability of robust formulations under the recommended API storage conditions (-40°C): To support the stability of the active pharmaceutical ingredient (API) across the claimed formulation composition range, a stability study of the API robust formulation stored at -40°C was conducted. The study results confirmed that no significant changes were observed in the tested quality attributes when the formulation was stored at -40°C for 26 weeks under the recommended API storage conditions.

[0419] Stability of robust formulations after shaking and freeze / thaw stress: The formulation was shaken at 2°C - 8°C or 25°C for 1 week. Further, the formulation was evaluated after undergoing 5 freeze / thaw cycles between -40°C and 5°C. All samples had virtually no visible particles due to shaking or freeze / thaw stress.

[0420] In all formulations, particles not visible to the naked eye did not change due to shaking and freeze / thaw stress. Formulations with a low content of PS20 (0.2 mg / ml, F7, F8, F13) showed no impact on product quality after shaking and freeze / thaw stress compared to all other formulations containing PS20 at levels of 0.3 - 0.8 mg / ml.

[0421] This result confirms that a level of polysorbate 20 of ≧0.2 mg / ml is sufficient to protect the protein from shaking and freeze / thaw stress. Similarly, formulations with a low D-sucrose content (200 mM, F19) showed no impact on product quality after shaking and freeze / thaw stress compared to all other formulations containing D-sucrose at levels of 240 - 280 mM. This result confirms that a level of D-sucrose of ≧200 mM is sufficient to protect the protein from freeze / thaw stress. No significant changes due to shaking or freeze / thaw stress were seen in other quality characteristics compared to the control samples.

[0422] Linear regression analysis of identified CQAs based on data at recommended storage conditions (2°C - 8°C): For the affected CQAs (solution pH, protein concentration, and PS20 concentration), a simple linear regression analysis was performed. It was confirmed that pH had the main effect. The calculated degradation rate per week was extrapolated to the expiration of shelf life (EoS) over 104 weeks (= 24 months). The extrapolated results are summarized in Table 8.

[0423] Linear regression analysis demonstrated that the tested pH range had no significant effect on the identified CQAs since all CQAs were within the stability acceptance criteria. However, to control the increase in the acidic region, the pH acceptance criteria at drug release were tightened to 5.2 - 5.8. TIFF0007701982000008.tif78170

[0424] Conclusion: The ex...

Claims

1. At a pH of 5.5, 1 mg / ml of golphitinib; 20 mM of histidine buffer; 240 mM of sucrose; 10 mM of methionine; 0.5 mg / ml of polysorbate 20; A liquid pharmaceutical composition comprising the same.

2. The liquid pharmaceutical composition according to Claim 1, wherein the molar ratio of polysorbate 20 to golphitinib is less than 100.

3. The liquid pharmaceutical composition according to Claim 2, wherein the molar ratio of polysorbate 20 to golphitinib is between 50 and 100.

4. The liquid pharmaceutical composition according to Claim 3, wherein the molar ratio of polysorbate 20 to golphitinib is 79.

5. Use of the liquid pharmaceutical composition according to Claim 1 for the preparation of a medicament useful for treating cell proliferative disorders.

6. The use according to Claim 5, wherein the cell proliferative disorder is cancer.

7. The liquid pharmaceutical composition according to Claim 1 for use in a subject in need of treatment of a cell proliferative disorder or delay in its progression.

8. The liquid pharmaceutical composition according to Claim 7, wherein the cell proliferative disorder is cancer.

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