TW201806618A - Antibody formulations - Google Patents

Abstract

High concentration antibody formulations capable of stable long-term storage are disclosed.

Description

Antibody formulation [Cross-reference to related applications]

This case claims the benefit of US Provisional Patent Application No. 62 / 358,404, filed on July 5, 2016. This case also claims the rights of European Patent Application No. 16306090.8 filed on August 30, 2016. Each of these applications is incorporated herein by reference in its entirety.

The present invention relates to antibody formulations having extended storage stability in glass containers.

CXCR5, also known as Burkitt lymphoma receptor (BLR1), CD185, MDR15, and MGC117347, is a G protein-coupled receptor that is a member of the CXC chemokine receptor family. CXCR5 affects B cell migration and tissue localization, as demonstrated by CXCR5 knockout mice lacking peripheral lymph nodes, has fewer Peyer's patches, and has reduced B cell levels. CXCL13, also known as BLC, is a ligand for CXCR5. CXCL13 is a B-cell chemical attractant. Anti-CXCR5 binding agents are therapeutically relevant, and they have been formulated into pharmaceutical products that can be administered to a subject, particularly a human subject, for the treatment of an inflammatory disease.

Pharmaceutical formulations suitable for intravenous or subcutaneous administration containing anti-CXCR5 binding agents must be Highly concentrated (about 20 mg / mL to about 100-150 mg / mL, even up to 250 mg / mL or higher). However, many problems with such drug formulations can occur at high concentrations, including increased viscosity, pH drift, and solution color changes. Furthermore, at high binder concentrations, the likelihood of forming visible and sub-visible particles, binder aggregates, and / or binder semi-molecules is increased. In addition, at high binder concentrations, opportunities for interaction between the drug formulation and its storage container are increased. Therefore, there is still a need for improved pharmaceutical formulations to avoid these limitations.

The invention provides an anti-CXCR5 antibody drug formulation with increased storage stability in a glass storage container.

In a first aspect, the invention provides an antibody formulation suitable for subcutaneous administration to a patient. The formulation includes about 50 to about 250 mg / mL of an anti-CXCR5 antibody; a citrate buffer; greater than about 0.01% (w / v) surfactant; greater than about 50 mM amino acid; and greater than about 1% sucrose. The pH of the formulation is about pH 6.

In a specific embodiment of the first aspect, the anti-CXCR5 antibody or a fragment thereof comprises: (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain comprising SEQ ID NO: 12 Heavy chain variable domain of amino acid sequence; (b) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLAS (SEQ ID NO: 59), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and amino acid sequences of IVY (SEQ ID NO: 63); (c) amino acids comprising SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 The light chain variable domain of the sequence, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; (d) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLAS (SEQ ID NO: 64), MQHLEYPYT ( SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (e) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSNLAS (SEQ ID NO: 65), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amines Amino acid sequence; (f) a variable light chain (VL) comprising an amino acid of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, and an amino acid comprising SEQ ID NO: 23 Variable heavy chain (VH); (g) a variable light chain comprising the amino acid of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, and comprising SEQ ID NO: 33 or SEQ Variable heavy chain of amino acid of ID NO: 34; (h) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), amino acid sequences of VIWGDGTTY (SEQ ID N: 62), and IVY (SEQ ID NO: 63); (i) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLA (SEQ ID NO: 67), MQHLEYPYT ( SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (j) RSSKSLLHSSGKTYLY (SEQ ID NO: 58 ), RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62) And an amino acid sequence of IVY (SEQ ID NO: 63); (k) a variable light chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 37 Heavy chain; (1) a variable light chain comprising the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43, and a variable light chain comprising SEQ ID NO: 45 or SEQ ID NO: 47 A variable heavy chain of an amino acid sequence; (m) a variable light chain comprising an amino acid sequence of SEQ ID NO: 55, and a variable light chain comprising an amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57 Variable heavy chain; or (n) RSSKSLLHSSGKTYLYW (SEQ ID NO: 69), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and the amino acid sequence of IVY (SEQ ID NO: 63).

In another specific embodiment of the first aspect, the amino acid is arginine or methylsulfide Amino acid.

In a specific embodiment of the first aspect, the surfactant is polysorbate.

In a second aspect, the invention provides an antibody formulation suitable for subcutaneous administration to a patient. The formulation includes about 100 to about 175 mg / mL of antibody; about 10 mM citrate buffer; about 0.1% (w / v) surfactant; about 200 mM arginine; and about 4.5-9% sucrose. The pH of the formulation is about pH 6.

In a specific embodiment of the second aspect, the antibody is a fully human anti-CXCR5 antibody.

In another specific embodiment of the second aspect, the antibody includes a heavy chain and a light chain, said heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and said light chain comprising the amine of SEQ ID NO: 32 Amino acid sequence.

In a further specific embodiment of the second aspect, the antibody comprises a single chain Fv.

In another specific embodiment of the second aspect, the antibody is an isolated antibody or a fragment thereof that specifically binds to the extracellular domain of human CXCR5.

In a specific embodiment of the second aspect, the isolated antibody or fragment thereof comprises RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN ( SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences.

In a specific embodiment of the second aspect, the surfactant is a polysorbate.

In a specific embodiment of the second aspect, the polysorbate is polysorbate 20 or polysorbate 80.

In a third aspect, the invention provides an antibody formulation comprising about 175 mg / mL of a humanized IgG4 anti-CXCR5 antibody; about 10 mM citrate buffer; about 1.0 mg / mL polysorbate 80; about 200 mM Arginine HCl; and about 45 mg / mL sucrose. PH of the formulation It is about pH 6.

In a specific embodiment of the third aspect, the humanized IgG4 anti-CXCR5 antibody includes a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and the light chain comprises Amino acid sequence of SEQ ID NO: 32.

In a fourth aspect, the present invention provides a container containing the antibody formulation of any of the foregoing aspects.

In a specific embodiment of the fourth aspect, the container is a pre-filled syringe, vial or auto-injector.

In another specific embodiment of the fourth aspect, the container contains the antibody formulation of any of the foregoing aspects in a lyophilized form.

In a fifth aspect, the present invention provides a kit comprising the container of the fourth aspect and any specific embodiments thereof, and a label or instructions for administering and using the antibody formulation.

In a specific embodiment of the fifth aspect, the administering is performed by injection.

In a sixth aspect, the present invention provides the antibody formulation of any one of the foregoing aspects or embodiments for use in a method of treating or diagnosing a human or animal body.

In a seventh aspect, the present invention provides a method for treating rheumatoid arthritis, which comprises administering the antibody formulation of any one of the foregoing aspects or embodiments to a subject in need thereof.

In an eighth aspect, the invention provides a lyophilized form of the antibody formulation of any of the foregoing aspects or embodiments.

Figure 1 is a photomicrograph of a cellulose filter after placebo filtration of a first anti-CXCR5 antibody formulation. Magnification is 50 times.

Figure 2 is a photomicrograph of a cellulose filter after placebo filtration of a second anti-CXCR5 antibody formulation. Magnification is 50 times.

Figure 3 is a photomicrograph of a cellulose filter after placebo filtration of a third anti-CXCR5 antibody formulation. Magnification is 50 times.

Figure 4 is a photomicrograph of a cellulose filter after filtration of a first anti-CXCR5 antibody drug product (DP; SAR113244) stored in a Nalgene bottle. Magnification is 50 times.

FIG. 5 is a photomicrograph of the cellulose filter of FIG. 4 at 200X magnification.

FIG. 6 is a photomicrograph of a cellulose filter after DP filtration of a second anti-CXCR5 antibody. Magnification is 50 times.

Figure 7 is a photomicrograph of a cellulose filter after DP filtration of a first control anti-CXCR5 antibody. Magnification is 50 times. The first control anti-CXCR5 antibody DP is a "Stardust free" control formulation (SAR252067), which shows similar particle formation as the first anti-CXCR5 antibody DP, but without stardust particles. This situation proves the difficulty of stardust particle analysis.

FIG. 8 is a photomicrograph of the cellulose filter of FIG. 7 at 200X magnification.

Figure 9 is a photomicrograph of the cellulose filter after SAR341403 DP filtration. Magnification is 50 times.

FIG. 10 is a photomicrograph of the cellulose filter of FIG. 9 at 200X magnification.

FIG. 11 is a photomicrograph of a cellulose filter filtered by a second control anti-CXCR5 antibody DP (SAR341403). Magnification is 50 times. The second control anti-CXCR5 antibody DP is another "stardust-free" control formulation that shows similar particle formation to the first anti-CXCR5 antibody DP and further demonstrates the difficulty of stardust particle analysis.

FIG. 12 is a photomicrograph of the cellulose filter of FIG. 11 at 200X magnification.

FIG. 13 is a DLS measurement size distribution diagram of a DP SAR113244 solution (star dust-free solution) stored in a plastic bottle.

Figure 14 is the DLS measurement size distribution of DP solution (including stardust solution) stored in a glass vial Illustration.

Figure 15 shows particles from the sample (top trace) of the FT-IR spectrum; bottom spectrum locus indicating a comparison of the protein; acyl located two broad band at 3300cm ^-1 and 1650cm ^-1, 1520cm ^-1 at Amine bands are typical for proteins.

16 is a Raman spectrum of a particle identified as a protein; a first trace indicates a spectrum of particles from a sample; a second trace indicates a spectrum of particles from a sample having a subtracted background; a third trace indicates Comparison of best matches from database (protein).

FIG. 17 is an FT-IR spectrum of a particle identified as a protein. The first trace indicates the FT-IR spectrum of the particles from the sample; the second trace indicates the best match with the database (polyethylene); the third trace indicates the spectrum of the protein for comparison; the wide bands at 3300 cm ^-1 respectively The two amidamine bands at 1650 cm ^-1 and 1520 cm ^-1 are typical for proteins.

FIG. 18 is a Raman spectrum of a particle identified as a protein. The first trace indicates the spectrum of particles from the sample; the second trace indicates the spectrum of particles from the sample with a subtracted background; the third trace indicates a comparison of the best matches from a database identified as polyethylene.

Figure 19 shows the results of particle formation during the mechanical stress study; dark gray = visible particles, light gray = 1-2 particles, scattered white = mostly clear solution, white = no particle solution; columns from left to right Indication: Formulations A, H, B, C and D (see Table 1).

Figure 20 shows the results of particle formation during the mechanical stress study; dark gray = visible particles, checkered pattern = stardust or stardust-like particles, light gray = 1-2 particles, scattered white = mostly clear solution, White = particle-free solution; columns from left to right indicate: formulations A, H, E, F, and G (see Table 1).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

It should be noted that as used in this specification and the scope of the attached patent application, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise Regulations.

The term "about" or "approximately" means within 10% of a given value or range, such as within 5% (or 1% or less).

The term "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (such as a formulation of the invention) that is present outside the patient's body, such as through the mucosa, intradermally, intravenously Intra, subcutaneous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, administration of the substance usually occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance usually occurs before the onset of the disease or its symptoms.

In the context of a polypeptide, the term "analog" refers to a polypeptide that has similar or identical functions to a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or an anti-CXCR5 antibody, but it does not necessarily include a CXCR5 polypeptide, CXCR5 A fragment of a polypeptide, a CXCR5 epitope, or an anti-CXCR5 antibody having a similar or identical amino acid sequence; or having a structure similar or identical to a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or an anti-CXCR5 antibody.

A polypeptide having a similar amino acid sequence refers to a polypeptide that meets at least one of the following: (a) a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or an amino acid sequence of an anti-CXCR5 antibody that is at least 30% as described herein %, At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, A polypeptide having an amino acid sequence that is at least 95%, or at least 99% identical; (b) a polypeptide encoded by a nucleotide sequence that, under stringent conditions, encodes the CXCR5 polypeptide, CXCR5 polypeptide described herein Fragment, the CXCR5 epitope, or the nucleotide sequence of an anti-CXCR5 antibody (or its VH or VL region) hybridizes at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues Group, at least 20 amino acid residues, At least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino groups Acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues (see, e.g., Sambrook et al. (2001) Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY); and (c) by nucleotide sequence An encoded polypeptide, said nucleotide sequence being at least 30% greater than the nucleotide sequence encoding a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or an anti-CXCR5 antibody (or its VH or VL region) as described herein, 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , Or at least 99% the same. A polypeptide having a structure similar to a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or an anti-CXCR5 antibody refers to a polypeptide that has secondary and tertiary levels similar to a CXCR5 polypeptide, a fragment of a CXCR5 polypeptide, a CXCR5 epitope, or a CXCR5 antibody Or four-level structure. The structure of the polypeptide can be determined by methods known to those skilled in the art, including but not limited to X-ray crystallography, nuclear magnetic resonance, and crystal electron microscopy.

An "antagonist" or "inhibitor" refers to a molecule capable of inhibiting one or more biological activities of a target molecule. Antagonists can be by disabling or killing ligand-activated cells, and / or by interfering with receptor or ligand activation (such as tyrosine kinase activation) or signal transduction after binding of the ligand to the receptor To interfere with receptor binding to the ligand, and vice versa. Antagonists can completely block the receptor-ligand interaction or can significantly reduce this interaction. For the purposes of the present invention, all these intervention points of the antagonist should be considered equivalent.

For example, an "antagonist" or "inhibitor" of CXCR5 refers to a molecule capable of inhibiting one or more biological activities (such as signaling) of CXCR5. Accordingly, included within the scope of the present invention are antagonists (such as neutralizing antibodies) that bind to CXCR5, other ligands of CXCL13 or CXCR5, Complexes of CXCR5 and its ligands (such as CXCL13); amino acid sequence variants or derivatives of CXCR5 or CXCL13 that antagonize the interaction between CXCR5 and ligands such as CXCL13; soluble CXCR5, which is optionally fused to Heterologous molecules such as immunoglobulin regions (eg, immunoadhesins); complexes containing CXCR5 associated with another receptor or biomolecule; synthetic or natural sequence peptides that bind to CXCR5; and so on.

The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein. The term antibody includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv ( scFv) (including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F (ab ' ) fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies Fragment binding to an epitope of any of the above. Specifically, antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen-binding domains or molecules containing an antigen-binding site that specifically binds to CXCR5 antigen (e.g., one or more of an anti-CXCR5 antibody Complementarity determining region (CDR)). Anti-CXCR5 antibodies can be of any kind (e.g. IgG, IgE, IgM, IgD, IgA and IgY), any type (e.g. IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g. IgG2a and IgG2b) Immunoglobulin molecule. In some embodiments, the anti-CXCR5 antibody is humanized, such as a humanized monoclonal anti-CXCR5 antibody. In some embodiments, the anti-CXCR5 antibody is an IgG antibody, specifically a humanized IgG4 antibody.

As used herein, the term "anti-CXCR5 antibody" refers to an antibody that specifically binds human CXCR5 or a derivative thereof (derivative) as defined herein, including but not limited to inhibiting or significantly reducing the binding of CXCR5 to its ligand and A molecule that inhibits CXCR5 activity. For example, encompassed anti-CXCR5 antibodies include isolated antibodies or fragments thereof that specifically bind to the human CXCR5 extracellular domain, such as those disclosed in US Patent No. 8,647,622, which are incorporated herein by reference for all purposes. Specifically, the covered anti-CXCR5 antibody of the present invention has a U.S. patent A variable light chain comprising the amino acid sequence of SEQ ID NO: 32 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 33, No. 8,647,622. In another specific embodiment, an isolated antibody or fragment thereof that specifically binds to the human CXCR5 extracellular domain includes U.S. Patent No. 8,647,622 RSSKLSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ ID NO: 68), MQHLEYPYT ( SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences.

The term "B-cell activity" means higher than normal B-cell levels, which can be local, or evidence of the biological performance or function of B-cells, such as antibody expression, the presence or activity of Bruton's tyrosine kinase, and expression of CD19 Or presence, B cell activation factor expression or presence, etc.

The term "binding agent" refers to any molecule that binds or specifically binds CXCR5, such as an antibody, siRNA, nucleic acid, aptamer, protein or small molecule organic compound, or a variant or fragment thereof.

The term "by-products" includes undesired products that reduce or decrease the proportion of therapeutic / prophylactic binding agents (such as antibodies) in a given formulation. For example, typical by-products include aggregates of antibodies, fragments of antibodies, such as fragments produced by degradation of antibodies by deamination or hydrolysis, or mixtures thereof. Generally, aggregates are complexes having a molecular weight greater than that of the monomeric antibody. Antibody degradation products may include, for example, fragments of antibodies, such as produced by amidation or hydrolysis. Generally, the degradation product is a complex having a molecular weight smaller than that of the monomeric antibody. In the case of IgG antibodies, this degradation product is less than about 150 kD.

The terms "composition" and "formulation" are intended to cover products containing optionally specified amounts of specified ingredients (e.g., anti-CXCR5 antibodies), as well as products produced directly or indirectly from combinations of specified ingredients (optionally in specified amounts). Any product.

The terms "constant region" and "constant domain" refer to the carboxy-terminal portions of the light and heavy chains, which do not directly participate in the binding of antibodies to antigens, but exhibit various effector functions, such as those with Fc receptors interaction. The term refers to the part of an immunoglobulin molecule that has a more conserved amino acid sequence than the rest of the immunoglobulin (a variable domain containing an antigen binding site). The constant domain contains the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

The term "CXCR5" relates to known molecules that occur naturally in lymphocytes, especially B cells, especially naïve B cells; to molecules isolated from these cells; to the use of known materials and methods, and the use of encoding CXCR5 Recombinantly prepared molecules of nucleic acids; and certain parts of CXCR5, such as extracellular (EC) domains, which retain the characteristics and properties relevant to the practice of the invention, such as binding to CXCL13. The soluble CXCR5 molecule may consist essentially of the EC domain of CXCR5, and generally includes approximately the first 60 amino acids of the molecule, ie, the amino terminal portion of CXCR5.

CXCR5 is a non-promiscuous receptor. CXCL13 is a ligand of CXCR5 and is constitutively expressed in basal cells such as follicular dendritic cells and lymphoid tissues. CXCL13 specifically attracts a small subset of B cells and one T cell (which is referred to as B helper follicular T cells (TFH)). Given the many interactions between the T cell and B cell populations in the immune system, this is not unexpected. Moreover, activated T cells induce or up-regulate CXCR5 expression. Infiltration of lymphocytes into the tertiary ectopic germinal center (GC) has been found to be fully associated with exacerbations and tolerance breakdown in certain conditions that previously have such atypical lymph node-like structures. Using in vivo mouse models, such as CXCR5-/-and CXCL13-/-mice, the deletion of receptors or ligands causes the GC fine structure to change due to altered localization of T and B cells and possible interactions. These mice are also protected from severe collagen-induced arthritis (CIA). Since CXCR5 is selectively expressed on mature B cells associated with the onset of RA, blocking this receptor can modulate the arthritic response in affected individuals. The use of biologicals (i.e., anti-TNFα and anti-CD20 antibodies, rituximab) for the treatment of rheumatoid arthritis has been shown to be clinically effective, and in particular, the clinical signs and symptoms of patients receiving B cell-directed therapy have shown long-term improvement . Selectively targeting CXCR5 expressed only on mature B cells and B helper T cells will not affect B cell development or impair patient immunity. versus Rituximab is different in that the antibodies of the invention are neutralizing antibodies that do not mediate cytotoxicity.

"CXCR5 disease" is a disease, disorder, condition, abnormality, etc., which is characterized or induced by the overexpression or increased level of CXCL13 or other CXCR5 ligands, increased B cell levels, increased B cell activity levels, and increased CXCR5 levels High or abnormal CXCR5 metabolism or activity.

The term "epitope" refers to a local region on the surface of an antigen (e.g., a CXCR5 polypeptide or a CXCR5 polypeptide fragment) that is capable of binding to one or more antigen-binding regions of a binding agent (e.g., an antibody), and that Mammals, such as humans, have antigenic or immunogenic activity. An epitope with immunogenic activity is part of a polypeptide that elicits an antibody response in an animal. The epitope with antigen activity is part of the polypeptide to which the antibody specifically binds, and is determined by any method known in the art, such as immunoassay. An epitope is not necessarily immunogenic. An epitope usually consists of a chemically active surface molecule of a molecule, such as an amino acid or a sugar side chain, and has specific three-dimensional structural characteristics and specific charge characteristics. The region of the polypeptide involved in the formation of the epitope may be a continuous amino acid of the polypeptide, or the epitope may be aggregated from two or more non-contiguous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of an antigen. Anti-CXCR5 antibodies can specifically bind to epitopes in the monomeric (denatured) form of CXCR5, epitopes in the trimer (natural) form of CXCR5, or the monomeric (denatured) and trimer (natural) forms of CXCR5 both.

The term "excipient" refers to inert substances commonly used as diluents, carriers, preservatives, adhesives, stabilizers, etc. for drugs, including but not limited to proteins (such as serum albumin, etc.), amino acids ( For example, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (such as alkyl sulfonates, caprylates, etc.), surfactants ( For example, SDS, polysorbate, nonionic surfactants, etc.), sugars (for example, sucrose, maltose, trehalose, etc.) and polyhydric alcohols (for example, mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., Which is incorporated herein by reference in its entirety for all purposes.

In the context of a peptide or polypeptide, the term "fragment" refers to a sequence that contains less than the full-length amino acid sequence Peptide or polypeptide. Such fragments may, for example, result from truncation of the amino terminus, truncation of the carboxy terminus, and / or deletion of internal residues of the amino acid sequence. Fragments can be generated by alternative RNA splicing or from protease activity in vivo. In some specific embodiments, the hCXCR5 fragment includes a polypeptide comprising at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, and at least 15 consecutive amino acid residues of the amino acid sequence of the CXCR5 polypeptide. Amino acid residues, polypeptides with an amino acid sequence of at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino groups Acid residues, at least 60 consecutive amino residues, at least 70 consecutive amino residues, at least 80 consecutive amino residues, at least 90 consecutive amino residues, at least 100 consecutive amino groups Acid residues of at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues or at least 250 consecutive amino groups Acid residues, or antibodies that specifically bind to CXCR5 polypeptide. In a specific embodiment, a fragment of the CXCR5 polypeptide or an antibody that specifically binds the CXCR5 antigen retains at least one, at least two, or at least three functions of the polypeptide or antibody.

The terms "fully human antibodies" or "human antibodies" are used interchangeably herein and refer to antibodies that include human variable regions and may include human constant regions. In specific embodiments, the term refers to an antibody comprising a constant region and a variable region of human origin. In a specific embodiment, the antibody is a fully human antibody. The term "fully human antibody" includes antibodies having constant and variable regions corresponding to human germline immunoglobulin sequences, such as Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, United States Health And Department of Public Services, NIH Publication No. 91-3242). Methods of producing fully human antibodies are known in the art.

The phrase "recombinant human antibody" includes a human antibody prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vehicle transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, Antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cattle) for human immunoglobulin genes (see e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295), or antibodies produced, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat et al., 1991). However, in some specific embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or when in vivo somatic mutagenesis is performed using transgenic animals directed against human Ig sequences), therefore, the amines of the VH and VL regions of the recombinant antibodies The amino acid sequence is a sequence that may not exist naturally in the human antibody germline library, although it is derived from and related to human germline VH and VL sequences.

"IgG4 binding agent" or "binding agent comprising at least a portion of an IgG4 Fc region" refers to a binding agent described herein that comprises at least a fragment of an IgG4 Fc. In some embodiments, the fragment comprises 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 IgG4 Fc regions. Amino acids. In other specific embodiments, the fragment comprises 10-50, 50-100, 100-150, or 150-200 amino acids of the IgG4 Fc region. In other specific embodiments, a portion of the IgG4 Fc region may have certain homology with the IgG4 Fc region. For example, an IgG4 binding agent can include a portion of a protein that has greater than 50, 60, 70, 80, 90, 93, 95, 96, 97, 98, 99, or 100% homology to the IgG4 Fc region. Exemplary Fc regions of IgG4 are described throughout the specification.

When used in reference to antibodies, the term "heavy chain" refers to five different species, called alpha (α), delta (Δ), epsilon (ε), gamma (γ), and mu (μ), which are based on heavy chains The amino acid sequence of the constant domain. These different types of heavy chains are well known in the art and produce five types of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including the four subtypes of IgG, namely IgG1, IgG1, IgG3, and IgG4. In some embodiments, the heavy chain is a human heavy chain.

A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or a fragment thereof (e.g., an antibody's Fv, Fab, Fab ', F (ab') ₂ or other target binding Subsequence), which contains sequences derived from non-human immunoglobulins compared to human antibodies. Generally speaking, the humanized antibody basically includes all variable regions of one, usually two, wherein all or substantially all CDR regions correspond to non-human immunoglobulin CDR regions, and all or substantially all FRs Region is the FR region of a human immunoglobulin template sequence. The humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a selected human immunoglobulin template. In general, the goal is to have antibody molecules that are least immunogenic in humans. Therefore, it is possible that one or more amino acids in one or more CDRs can also be changed to amino acids that are less immunogenic in the human host, but do not substantially reduce the one or more CDRs to CXCR5. Or CXCL13 specific binding function. Alternatively, the FR may be non-human, but the most immunogenic amino acid is replaced with a less immunogenic amino acid. However, the CDR transplantation discussed above is not the only way to obtain humanized antibodies. For example, it may not be sufficient to modify only the CDR regions, because it is not uncommon for framework region residues to participate in determining the three-dimensional structure of the CDR loop and the total affinity of the antibody and its ligand. Therefore, any method can be implemented so that a non-human parent antibody molecule is modified to an antibody molecule that is less immunogenic to humans, and the global sequence identity with a human antibody is not always necessary. Therefore, humanization can also be achieved, for example, by replacing only a few residues, especially by replacing residues that are exposed outside the antibody molecule and are not embedded within the molecule, making it difficult to access the host immune system. This method is taught herein in terms of replacing "active" or "flexible" residues on an antibody molecule, with the goal of reducing or reducing the immunogenicity of the resulting molecule without compromising the specificity of the antibody for its epitope or determinant Sex. See, eg, Studnicka et al., Prot Eng 7 (6) 805-814, 1994; Lazar et al., Mol Immunol. 2007 Mar; 44 (8): 1986-98 (2007); Sims et al., J Immunol 151: 2296 (1993); Chothia et al., J Mol Biol 196: 901 (1987); Carter et al., Proc Natl Acad Sci USA 89: 4285 (1992); Presta et al., J Immunol 151: 2623 (1993), WO2006 / 042333 and US Patent No. 5,869,619.

An "isolated" or "purified" binding agent (e.g., an antibody) is substantially free of cellular material or other contaminating proteins from cell or tissue sources or protein-derived media, or is substantially free of chemical precursors or Other compounds. For example, the language "substantially free of cellular material" A preparation comprising an antibody in which the antibody is separated from the cellular components of the cell, said antibody being isolated from the cell or produced recombinantly. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, or 5% (dry weight) of a heterologous protein (also known as a contaminating protein). When the antibody is produced recombinantly, it is desirable to be substantially free of culture medium, that is, the culture medium occupies less than about 20%, 10%, or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, in some embodiments, it is substantially free of chemical precursors or other chemical substances, that is, it is separated from chemical precursors or other chemical substances involved in protein synthesis. Therefore, these antibody preparations have less than about 30%, 20%, 10%, or 5% (dry weight) of a compound or chemical precursor other than the antibody of interest. In some embodiments, the anti-CXCR5 antibody is isolated or purified.

The terms "human CXCR5", "hCXCR5" or "hCXCR5 polypeptide" and similar terms refer to polypeptides ("polypeptides", "peptides" and "proteins" disclosed in U.S. Pat. "" Are used interchangeably herein), and related polypeptides, including their SNP variants. Related polypeptides include allelic variants (e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecific homologues, which in some embodiments are retained CXCR5 activity and / or sufficient to produce an anti-CXCR5 immune response. A soluble form of CXCR5 is also encompassed, which is sufficient to generate an anti-CXCR5 immune response. As will be understood by those skilled in the art, an anti-CXCR5 binding agent (such as an antibody) can bind a CXCR5 polypeptide, a polypeptide fragment, an antigen, and / or an epitope because the epitope is part of a larger antigen, and the larger antigen is larger A portion of a polypeptide fragment, which in turn is part of a larger polypeptide.

The term "Kabat numbering" and similar terms are recognized in the art and refer to more variable (i.e. hypervariable) residues than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen binding portion Numbering system for amino acid residues. (See Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat et al. (1991)).

When referring to antibodies, the term "light chain" refers to two different The same type is called kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.

The terms "manage", "managing" and "management" refer to the beneficial effects obtained by a subject from a treatment (eg, a prophylactic or therapeutic agent) that does not lead to the cure of the infection. In some embodiments, the subject is administered one or more treatments (e.g., prophylactic or therapeutic agents, e.g., formulations of the invention) to "manage" a CXCR5-mediated disease (e.g., rheumatoid arthritis) or One or more symptoms to prevent the progress or worsening of the disease.

The term "monoclonal antibody" refers to an antibody obtained from a population of homogeneous or substantially homogeneous antibodies, and each monoclonal antibody typically recognizes a single epitope on an antigen. In some embodiments, a "monoclonal antibody" is an antibody produced by a single fusion tumor or other cell. The term "single strain" is not limited to any specific method for making antibodies. For example, monoclonal antibodies can be prepared by a fusion tumor method as described in Kohler et al .; Nature, 256: 495 (1975) or can be isolated from a phage library. Other methods of making colony cell lines and monoclonal antibodies expressed therefrom are known in the art (see, for example, Short Protocols in Molecular Biology, (2002) fifth edition; edited by Ausubel et al., John Wiley and Sons, New Chapter 11 of York).

The term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the United States Pharmacopeia, European Pharmacopoeia, or other recognized pharmacopoeia for use in animals, especially humans.

"Pharmaceutically acceptable excipient" refers to any inert substance used in combination with an active molecule, such as a monoclonal antibody, to prepare an acceptable or convenient dosage form. "Pharmaceutically acceptable excipients" are excipients that are used in a dose and concentration that are non-toxic to the recipient and compatible with the other ingredients of the formulation containing the monoclonal antibody.

The term "prevention" refers to a CXCR5-mediated disease and / or symptoms associated with it caused by administration or a combination of treatments or treatments provided herein (e.g., a combination of prophylactic or therapeutic agents (such as a formulation of the invention). Total or partial inhibition of progression, relapse, onset or spread.

The term "prophylactic agent" refers to any agent that can completely or partially inhibit the progression, recurrence, onset, or spread of a CXCR5-mediated disease and / or symptoms associated with it in a subject. In some embodiments, the term "prophylactic agent" refers to a formulation of the invention. In some other embodiments, the term "prophylactic agent" refers to an agent other than a formulation of the invention. In some embodiments, the prophylactic agent is known to be useful or has been or is being used to prevent a CXCR5-mediated disease and / or symptoms associated therewith, or to prevent the onset of a CXCR5-mediated disease and / or symptoms associated therewith , Development, progress, and / or severity. In specific embodiments, the prophylactic agent is a fully human antibody, such as a fully human monoclonal antibody or a humanized anti-CXCR5 antibody, such as a humanized anti-CXCR5 monoclonal antibody.

The term "CXCR5 antigen" refers to the portion of a binding agent (such as an antibody) that specifically binds a CXCR5 polypeptide. CXCR5 antigen also refers to an analog or derivative of a CXCR5 polypeptide or a fragment thereof that is specifically bound by a binding agent, such as an antibody. The region of the CXCR5 polypeptide that contributes to the epitope may be a continuous amino acid of the polypeptide, or the epitope may be aggregated from two or more discontinuous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of an antigen. A local region on the surface of the CXCR5 antigen capable of eliciting an immune response is the CXCR5 epitope. An epitope may or may not be a three-dimensional surface feature of an antigen.

The terms "CXCR5-mediated disease" and "CXCR5-mediated disorder" are used interchangeably and refer to any disease that is caused in whole or in part by or as a result of CXCR5. In some embodiments, CXCR5 is abnormally (eg, highly) expressed on the cell surface. In some embodiments, CXCR5 can be abnormally upregulated on specific cell types. In other specific embodiments, normal, abnormal, or excessive cellular signaling is caused by the binding of CXCR5 to a CXCR5 ligand. In some embodiments, the ligand of CXCR5 is CXCL13. In some embodiments, the CXCR5-mediated disease is rheumatoid arthritis (RA).

The term "sugar" refers to a class of molecules that are derivatives of polyols. Sugars are often referred to as carbohydrates and can contain varying amounts of sugar (sugar) units, such as monosaccharides, disaccharides, and polysaccharides.

The term "specifically binds" or "specifically binds" means specifically binding to an antigen or a fragment thereof without specifically binding to other antigens. For example, antibodies that specifically bind an antigen can bind to other peptides or polypeptides with lower affinity, such as by, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIACORE®, or other assays known in the art Law determined. An antibody or a variant or fragment thereof that specifically binds an antigen may cross-react with the relevant antigen. In some embodiments, antibodies or variants or fragments thereof that specifically bind to an antigen do not cross-react with other antigens. Antibodies or variants or fragments thereof that specifically bind to the CXCR5 antigen can be identified, for example, by immunoassay, BIACORE® or other techniques known to those skilled in the art. Typically, the specific or selective response is at least twice the background signal or noise, and more usually more than 10 times the background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336.

A "stable" or "stabilized" formulation is one in which a binding agent (such as an antibody) substantially retains its physical stability, identity, integrity and / or chemical stability, identity, integrity when stored. And / or biologically active formulations. Various analytical techniques for measuring protein stability are available in the art and reviewed in, for example, Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. 1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured for a selected period of time at a selected temperature and other storage conditions. Stability can be determined by at least one method selected from the group consisting of: visual inspection, SDS-PAGE, IEF, HPSEC, RFFIT, and κ / λ ELISA methods. For example, if color and / or transparency is visually checked, or measured by UV light scattering, SDS-PAGE, or (high pressure) size exclusion chromatography (HPSEC), it does not show aggregation, precipitation, and / or denaturation. Signs, the antibody "retains its physical stability." In some embodiments, when using the formulation of the present invention, 5% or less, usually 4% or less, usually 3% or less, more usually 2% or less, especially 1 % Or less antibodies form aggregates, such as by HPSEC Or any other suitable method for measuring aggregate formation. For example, an antibody is considered to be a specific formulation if it has a purity of about 90%, usually about 95%, and especially about 98%, after a certain predetermined time under certain storage conditions in the specific formulation. stable. Chemical stability can be assessed by detecting and quantifying chemically altered proteins. Chemical alterations may involve size modification (e.g. shearing), which can be performed using, for example, (HP) SEC, SDS-PAGE and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS) Evaluation. Other types of chemical changes include changes in charge (for example, appearing as a result of amidin), which can be evaluated by, for example, ion exchange chromatography. For example, if the biological activity of an antibody at a given time is at least about 90% (within measurement error) of the biological activity exhibited at the time of preparation of the antibody formulation, as determined in an antigen binding assay or a virus neutralization assay , The antibody "retains its biological activity" in the drug formulation at a given time.

In a specific embodiment, the antibody formulation may be "stable against Stardust particle formation", which means that when stored in glass containers under accelerated conditions for a period of 6 months, An antibody formulation that will form stardust particles.

The terms "subject" and "patient" are used interchangeably. As used herein, the subject is typically a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human), and in some implementations The example is human. In a specific embodiment, the subject is a mammal, such as a human, with a CXCR5-mediated disease. In another specific embodiment, the subject is a mammal, such as a human, at risk for developing a CXCR5-mediated disease.

The term "therapeutically effective amount" refers to an amount of treatment (eg, a formulation of the invention) sufficient to reduce and / or improve the severity and / or duration of a given disease and / or symptoms associated therewith. The term also includes reducing or improving the development or progression of a given disease, reducing or improving the recurrence, development or onset of a given disease, and / or improving or enhancing the preventive or therapeutic effect of another therapy (e.g., in addition to the formulations of the invention External treatment). In some embodiments, treatment of antibodies of the invention A therapeutically effective amount is about 0.1 mg / kg (mg antibody per kg body weight of the subject) to about 100 mg / kg. In some specific embodiments, the therapeutically effective amount of the antibody provided therein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg About 20mg / kg, about 25mg / kg, about 30mg / kg, about 35mg / kg, about 40mg / kg, about 45mg / kg, about 50mg / kg, about 60mg / kg, about 70mg / kg, about 80mg / kg About 90 mg / kg or about 100 mg / kg (or a range thereof). In some embodiments, a "therapeutically effective amount" as used herein also refers to the amount of an antibody of the invention that achieves a particular result (e.g., inhibits the biological activity of CXCR5 of a cell, such as binding to CXCL13).

The term "therapeutic agent" refers to any agent that can be used to treat, manage, or ameliorate a CXCR5-mediated disease and / or symptoms associated therewith. In some embodiments, the term "therapeutic agent" refers to a formulation of the invention. In certain other specific embodiments, the term "therapeutic agent" refers to an agent other than a formulation of the invention. In some embodiments, a therapeutic agent is an agent known to be useful or has been or is being used to treat, manage, or ameliorate a CXCR5-mediated disease or one or more symptoms associated therewith.

The term "therapy" refers to any regimen, method, and / or agent that can be used to prevent, manage, treat, and / or ameliorate a disease (such as IBD or GVHD) or a CXCR5-mediated disease (such as rheumatoid arthritis). In some embodiments, the terms "therapies" and "therapy" refer to CXCR5 mediation known to those skilled in the art, such as medical personnel, for use in the prevention, management, treatment, and / or improvement of Biological therapies, supportive therapies and / or other therapies for your disease.

The terms "treat", "treatment" and "treating" refer to the administration of one or more treatments (including but not limited to the administration of one or more prophylactic or therapeutic agents, such as the formulation of the present invention Reduction or improvement in the progression, severity, and / or duration of a CXCR5-mediated disease (e.g., rheumatoid arthritis) produced by a). In specific embodiments of CXCR5, such terms refer to a reduction or inhibition of the binding of CXCR5 to CXCL13, and / or the inhibition or inhibition of one or more symptoms associated with a CXCR5-mediated disease such as rheumatoid arthritis cut back.

The term "variable region" or "variable domain" refers to a portion of the light and heavy chains, typically about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino groups in the light chain. Acids, whose sequences vary widely between antibodies, and are used for the binding and specificity of each particular antibody for its particular antigen. Variability in sequences is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. Amino acid positions are numbered according to the EU, such as Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C.) 5th edition. ("Kabat, etc."). In some embodiments, the variable region is a human variable region.

As used herein, the terms "stardust" and "stardust particles" are used interchangeably to refer to particles that are present in a glass container in the presence of an antibody formulation within the container.

B. Preparations

The formulations of the present disclosure may be in the form of a liquid and a lyophilized powder comprising an anti-CXCR5 binder. In addition, such formulations may include buffers, surfactants, tonicity agents, amino acids, and other excipients.

Binding agent

In some embodiments of the invention, the anti-CXCR5 antibody is a humanized or fully human antibody. Examples of humanized and fully human antibody isotypes include IgA, IgD, IgE, IgG, and IgM. In some embodiments, the anti-CXCR5 antibody is an IgG antibody. There are four forms of IgG. In some embodiments, the anti-CXCR5 antibody is an IgG4 antibody. In some embodiments of the invention, the anti-CXCR5 antibody is a humanized IgG4 antibody.

Some specific embodiments of the formulations of the invention also include variants of anti-CXCR5 binding agents, such as antibodies. Variants of anti-CXCR5 antibodies can have similar physicochemical properties due to their high similarity, and are therefore included within the scope of the present invention. A variant is defined as an antibody having an amino acid sequence that is at least 95%, at least 97%, such as at least 98% or 99% homologous to an anti-CXCR5 antibody, And can compete for binding to CXCR5 polypeptide, CXCR5 polypeptide fragment or CXCR5 epitope. In some embodiments, these variants improve, neutralize, or otherwise inhibit CXCR5 biological activity (eg, the binding of CXCL13 to CXCR5). Competition to determine target binding can be performed by conventional methods known to those skilled in the art. In some embodiments, the variant is a human antibody, and in some embodiments an IgG4 molecule. In some specific embodiments, the variant is at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to a covered antibody disclosed herein. The term "variant" refers to an antibody that contains an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of an anti-CXCR5 antibody. The variant may have conservative sequence modifications, including amino acid substitutions, modifications, additions, and / or deletions.

Examples of modifications include, but are not limited to, glycosylation, ethionization, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and cell ligand or Connection of other proteins. Amino acid modifications can be introduced into nucleic acids encoding antibodies by standard techniques known in the art, such as site-directed mutagenesis, molecular selection, oligonucleotide-directed mutagenesis, and random PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which amino acid residues are replaced with amino acid residues having similar structural or chemical properties. A family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), uncharged Amino acids with polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains ( For example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chain amino acids (for example, threonine, val Amino acids, isoleucines) and amino acids of aromatic side chains (eg tyrosine, phenylalanine, tryptophan). It is clear to a person skilled in the art that classifications of amino acid residue families other than those used above may also be used. In addition, variants may have non-conservative amino acid substitutions, such as replacing a certain amino acid with an amino acid residue having a different structure or chemical property. Similar minor changes can also include amino acid deletions or insertions, or both. Can use well-known in the art Computer programs to find guidance for determining which amino acid residues can be substituted, modified, inserted or deleted without eliminating immune activity. Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally compare the amino acid sequences to be compared and define similar or identical amino acid residues. The variant may have the same or different (higher or lower) binding affinity than the anti-CXCR5 antibody, but still be able to specifically bind CXCR5, and may have the same, higher, or lower biological activity as the anti-CXCR5 antibody.

Specific embodiments of the invention also include antigen-binding fragments of anti-CXCR5 binding agents (such as antibodies). The terms "antigen-binding domain", "antigen-binding region", "antigen-binding fragment" and similar terms refer to the portion of an antibody comprising an amino acid residue (e.g., an amino acid residue that interacts with the antigen and confers specificity and affinity for the antigen to the binding agent) Complementarity determining region (CDR)). The antigen-binding region may be derived from any animal species, such as rodents (such as rabbits, rats, or hamsters) and humans. In some embodiments, the antigen-binding region is of human origin. Non-limiting examples of antigen-binding fragments include: Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, dAb fragments, and amino acid residues from the hypervariable region of a mock antibody The smallest unit of identification of the basis.

In some embodiments of the invention, the anti-CXCR5 binding agent (or a variant or antigen-binding fragment thereof) will improve, neutralize, or otherwise inhibit the biological activity of CXCR5 in the body (eg, the binding of CXCL13 to CXCR5).

In some embodiments of the invention, the anti-CXCR5 binding agent (or a variant or antigen-binding fragment thereof) is an antagonist that improves, neutralizes, or otherwise inhibits the biological activity of CXCR5 in the body (such as the binding of CXCL13 to CXCR5) Binding agent.

In some specific embodiments, the anti-CXCR5 binding agent (or a variant or antigen-binding fragment thereof) is at about 50 to about 500 mg / mL, about 25 to about 400 mg / mL, about 50 to about 250 mg / mL, about 5 mg / mL. mL to about 280 mg / mL, about 5 mg / mL to about 200 mg / mL, about 5 mg / mL to about 125 mg / mL, about 5 mg / mL to about 75 mg / mL, about 5 mg / mL to about 50 mg / mL, and about 5 mg An amount of from about 25 mg / mL is present in the formulation. For example, anti-CXCR5 junction The mixture can be about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, and about 50 mg / mL. mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL, about 205 mg / mL, about 210 mg / mL, about 215 mg / mL, about 220 mg / mL, about 225 mg / mL, about 230 mg / mL, about 235 mg / mL, about 240 mg / mL, about 245 mg / mL, about 250 mg / An amount of mL, about 255 mg / mL, about 260 mg / mL, about 265 mg / mL, about 270 mg / mL, about 275 mg / mL, or about 280 mg / mL is present in the formulation.

In alternative embodiments, the anti-CXCR5 binding agent may be at about 5 to about 25 mg / mL, about 26 to about 50 mg / mL, about 51 to about 75 mg / mL, about 76 to about 100 mg / mL, and about 101 to About 125 mg / mL, about 126 to about 150 mg / mL, about 151 to about 175 mg / mL, about 176 to about 200 mg / mL, about 201 mg / mL to about 225 mg / mL, about 226 mg / mL to about 250 mg / mL, about 251 to about 280 mg / mL, about 5 to about 25 mg / mL, about 40 to about 60 mg / mL, about 75 to about 85 mg / mL, or about 90 to about 110 mg / mL are present in the formulation.

In a first specific embodiment, the antibodies covered are isolated antibodies or fragments thereof that specifically bind to the extracellular domain of human CXCR5. The antibody or fragment thereof may include: (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 12; (b) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLAS (SEQ ID NO: 59), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (c) comprising SEQ ID NO: 13, SEQ ID NO: 14, or The light chain variable domain of the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; (d) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLAS ( (SEQ ID NO: 64), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; ( e) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSNLAS (SEQ ID NO: 65), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 62) SEQ ID NO: 63); (f) a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, and comprising Variable heavy chain (VH) of the amino acid sequence of SEQ ID NO: 23; (g) Variable light chain comprising the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32 Chain, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34; (h) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID N: 62), and IVY (SEQ ID NO : 63) of the amino acid sequence; (i) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLA (SEQ ID NO: 67), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY ( (SEQ ID NO: 62), and amino acid sequence of IVY (SEQ ID NO: 63); (j) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60) ), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (k) the amino acid sequence comprising SEQ ID NO: 35 A variable light chain, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 37; (1) an amino acid sequence comprising SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43 Variable A light chain, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47; (m) a variable light chain comprising the amino acid sequence of SEQ ID NO: 55, and comprising SEQ ID NO: 56 or a variable heavy chain of the amino acid sequence of SEQ ID NO: 57; or (n) RSSKSLLHSSGKTYLYW (SEQ ID NO: 69), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60 ), The amino acid sequences of GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63).

In a second specific embodiment, the antibodies covered are isolated antibodies or fragments thereof that specifically bind to the extracellular domain of human CXCR5. The antibody or fragment thereof comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 32, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 33 .

In a third specific embodiment, the antibodies covered are isolated antibodies or fragments thereof that specifically bind to the extracellular domain of human CXCR5. The antibody or fragment thereof comprises RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), And the amino acid sequence of IVY (SEQ ID NO: 63).

In any one of the first, second, or third specific embodiments described above, the antibody or fragment thereof may further include one or more constant regions. The constant domains may be one or more of C _H1, C _H2, C _H3 and / or C _L composition. The one or more constant regions may be from an IgG antibody, which may be, for example, an IgG4 antibody.

In any of the first, second or third specific embodiments described above, the antibody or fragment thereof may be a single chain Fv.

In a specific embodiment, a pharmaceutical composition is encompassed, comprising a therapeutically effective amount of an antibody or fragment thereof of any one of the first, second or third specific embodiments described above and a pharmaceutically acceptable carrier.

Buffer

Formulations of the invention may include a citrate buffer as a buffer. Other buffers can also be used. The buffer maintains a physiologically suitable pH. In addition, the buffer enhances the isotonicity and chemical stability of the formulation. In some embodiments, the citrate buffer is present in the formulation at a concentration of about 0.5 mM to about 50 mM, such as about 5 mM to about 15 mM. For example, the citrate buffer can be at about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM , About 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, approximately 36mM, approximately 37mM, approximately 38mM, approximately 39mM, approximately 40mM, approximately 41mM, approximately 42mM, approximately 43mM, approximately 44mM, approximately 45mM, approximately 46mM, approximately 47mM, approximately 48mM, approximately 49mM, or approximately 50mM In the formulation. In some embodiments, the citrate buffer is present in the formulation at a concentration of about 7 mM to about 13 mM, such as about 9 mM to about 11 mM. In some embodiments, the citrate buffer is present at a concentration of about 10 mM.

In some embodiments, the formulation of the present invention has a pH value below pH 6. In some embodiments, the pH of the formulation ranges from about 5.0 to about 6.0. For example, the pH of the formulation may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0. In some embodiments, the pH range of the formulation may be from about 5.5 to about 6.0. In some embodiments, the pH is about 5.5 or about 6.0. The pH of the formulation can be measured by any means known to those skilled in the art. The means for measuring pH is to use a pH meter with a microelectrode. The pH of the formulation can be adjusted using any method known in the art. Exemplary chemicals for changing the pH of a formulation are hydrochloric acid (HCl) and sodium hydroxide (NaOH).

In some embodiments, the formulations of the invention have a pH that is equal to or lower than the isoelectric point (pI) of a binding agent, such as an antibody. The isoelectric point is the pH at which a specific molecule or surface does not carry a net charge. anti- The pI of the CXCR5 binding agent can be determined by any method known to those skilled in the art. In some embodiments, the pI of the anti-CXCR5 antibody is determined by denaturing isoelectric focusing. The pI range of a fully human IgG4 antibody encompassed herein may be from about 6.9 to about 9.1.

For example, covered formulations include anti-CXCR5 binding agents and citrate buffers, where the pH of the formulation is equal to or lower than about pH 6 and the isoelectric point (pI) of the binding agent. The formulations of the present invention provide a significant improvement over conventional IgG4 binding agent formulations, such as phosphate buffered saline (PBS) formulations, which can form unwanted by-products when increasing the binding agent concentration in the formulation. In particular, the formulations of the invention have reduced amounts of aggregates, semi-molecules, degradation products, low molecular weight proteins (LMWP), high molecular weight proteins (HMWP), and the acidity, basicity, and neutrality of the binding agents in the formulation Rearrangement of isotypes.

Surfactant

The formulations of the invention may optionally further comprise a surfactant, which is also referred to as a stabilizer. Surfactants / stabilizers are chemical compounds that interact in a formulation and stabilize biomolecules and / or general pharmaceutical excipients. In some embodiments, surfactants can be used in conjunction with lower temperature storage. Surfactants generally protect the binding agent from stresses induced by the air / solution interface and stresses induced by the solution / surface, which may otherwise lead to protein aggregation. Surfactants may include, but are not limited to, polysorbate, glycerol, dicarboxylic acid, oxalic acid, succinic acid, fumaric acid, phthalic acid, and combinations thereof. Those skilled in the art know that other surfactants, such as non-ionic or ionic detergents, can be used as long as they are pharmaceutically acceptable and suitable for administration to a subject. In some embodiments, the surfactant is a polysorbate. Examples of the polysorbate include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

In an exemplary embodiment, the surfactant is present in the formulation in an amount of about 0.001% to about 0.1% (w / v). For example, the surfactant can be at about 0.001% (w / v), about 0.002% (w / v), about 0.003% (w / v), about 0.004% (w / v), and about 0.005% (w / v ), About 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.02% (w / v) , About 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% ( w / v), about 0.09% (w / v), and about 0.1% (w / v) are present in the formulation. In specific embodiments, the surfactant is present at about 0.003% to about 0.05% (w / v), about 0.004% to about 0.025% (w / v), or about 0.005% to about 0.02% (w / v). , For example, in an amount of about 0.005% (w / v) in the formulation. For example, polysorbate 20 may be at about 0.001% to about 0.1% (w / v), about 0.002% to about 0.01% (w / v), about 0.003% to about 0.008% (w / v), and about 0.004% to about 0.006% (w / v), such as in an amount of about 0.005% (w / v). In alternative specific embodiments, polysorbate 20 is present at about 0.001% to about 0.1% (w / v), about 0.005% to about 0.05% (w / v), and about 0.0075% to about 0.025% ( w / v), for example, in an amount of about 0.01% (w / v). In other alternative embodiments, polysorbate 20 is present at about 0.001% to about 0.1% (w / v), about 0.005% to about 0.05% (w / v), and about 0.01% to about 0.03%. (w / v), for example, in an amount of about 0.02% (w / v).

Tonicity agent

The formulations of the invention may optionally further comprise a tonicity agent. In general, tonicity agents are used to adjust or maintain the osmolality of the formulation to bring it closer to the osmotic pressure of a body fluid, such as blood or plasma. Tonicity agents can also maintain binding agent levels in the formulation. In part, tonicity agents help maintain the level, ratio, or proportion of the therapeutically active adhesive present in the formulation. As used herein, the term "tension" refers to the behavior of a biological component in a fluid environment or solution. Isotonic solutions have the same osmotic pressure as plasma and can be infused intravenously into a subject without altering the osmotic pressure of the subject's plasma. In fact, in some embodiments of the invention, the tonicity agent is present in an amount sufficient to make the formulation suitable for intravenous infusion. Generally, tonicity agents are also used as fillers or stabilizers. Thus, tonicity agents may allow the binding agent to overcome various stresses, such as freezing and shearing. Tonicity agents can include, but are not limited to, sugars, sugars, glycerol, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride, and other inorganic salts. Those skilled in the art know that other tonicity agents can be used as long as it They are pharmaceutically acceptable, that is, suitable for administration to a subject.

In some embodiments, the tonicity agent is present in the formulation in an amount of about 0.1% to 10% (w / v). For example, the tonicity agent can be at about 0.1% (w / v), about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5% (w / v) , About 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1% (w / v), about 2% ( w / v), about 3% (w / v), about 4% (w / v), about 4.5% (w / v), about 5% (w / v), about 5.5% (w / v), About 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), and about 10% (w / v) are present in the formulation In. Alternatively, the tonicity agent may be present in the formulation in an amount of about 2% to about 8% (w / v), about 3% to about 7% (w / v), and about 4% to about 6% (w / v) In. In other alternative embodiments, the tonicity agent may be present in the formulation in an amount of about 0.1% to about 1%, about 0.1% to about 0.5%, about 0.1 to about 0.3%, and about 0.2%.

In some exemplary embodiments, the tonicity agent is a sugar. Examples of sugar include glucose, sucrose (which is also called sucrose), maltose, trehalose, dextrose, xylitol, fructose, and mannitol. For example, mannitol may be present at about 1% to about 10% (w / v), about 2% to about 8% (w / v), or about 3% to about 5% (w / v), such as about 4% ( w / v). Alternatively, sucrose (which is also referred to as sucrose) can be at about 1% to about 10% (w / v), about 3% to about 8% (w / v), or about 4% to about 6% (w / v) , For example, in an amount of about 4.5, 5, 5.5, or 6% (w / v).

In some other exemplary embodiments, the tonicity agent is sodium chloride. For example, sodium chloride may be at about 0.1% (w / v), about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5 to about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), and about 1% (w / v). Alternatively, sodium chloride may be present in the formulation in an amount of about 0.1% to about 1%, about 0.1% to about 0.5%, about 0.1 to about 0.3%, and about 0.2%.

In additional exemplary embodiments, the formulation may include one or more tonicity agents. For example, the formulation may contain one or more of the aforementioned tonicity agents at the aforementioned concentrations. In some specific embodiments, the formulation may include sucrose and sodium chloride, wherein the sucrose and sodium chloride concentrations are each It is about 0.1% to about 10% (w / v). In some embodiments, the sucrose concentration is about 6% and the sodium chloride concentration is about 0.2%. Alternatively, the sucrose concentration is about 4.5% and the sodium chloride concentration is about 0.2%.

In certain embodiments of the present invention, the osmolality of the formulation ranges from about 200 mOsm / kg to about 350 mOsm / kg, about 270 mOsm / kg to about 330 mOsm / kg, and about 280 mOsm / kg to about 320 mOsm / kg. , Or about 290 mOsm / kg to about 310 mOsm / kg, such as about 300 mOsm / kg. In other words, in some embodiments, the formulation of the present invention is substantially isotonic, that is, it has substantially the same osmotic pressure as human blood. The osmolality can be measured by any method known to those skilled in the art, such as using a vapor pressure or a freeze-type osmometer. The osmolality of the formulations of the invention can be adjusted, for example, by one or more tonicity agents described herein.

Amino acid

The formulations of the invention may optionally further comprise amino acids. Examples of amino acids include, but are not limited to, glycine, alanine, aspartic acid, lysine, serine, tyrosine, cysteine, glutamine, methionine, spermine Acid and proline. In an exemplary embodiment, the amino acid is present in the formulation in an amount of about 0.1% to 5% (w / v). For example, the amino acid can be at about 0.1% (w / v), about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5% (w / v ), About 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v) , About 1.7% (w / v), about 1.8% (w / v), about 1.9% (w / v), about 2.0% (w / v), about 3% (w / v), about 4% ( w / v), and about 5% (w / v) are present in the formulation. Alternatively, the amino acid is present in the formulation in an amount of about 1.3% to about 1.8% (w / v) or about 1.4% to about 1.6% (w / v), such as about 1.5% (w / v). In other alternative embodiments, the amino acid is at about 0.5% to about 1.5% (w / v), or about 0.8% to about 1.2% (w / v), such as about 1.0% (w / v ) Is present in the formulation. Exemplary amino acids are proline or arginine. For example, proline can be from about 1% to about 2% (w / v), from about 1.3% to about 1.8% (w / v), from about 1.4% to about An amount of 1.6% (w / v), such as about 1.5% (w / v), is present in the formulation. Alternatively, arginine can be present in the formulation in an amount of about 0.5% to about 1.5% (w / v) or about 0.8% to about 1.2% (w / v), such as about 1.0% (w / v).

Other excipients

In addition, the formulations of the present invention may include other excipients, including but not limited to water for injection, diluents, solubilizers, soothing agents, additional buffers, inorganic or organic salts, antioxidants, and the like. However, in some specific embodiments, in addition to those described above, the formulations of the present invention do not include other excipients. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980), can be included in the formulation, provided they do not Adversely affects the desired characteristics of the formulation. In a specific embodiment, the formulation is substantially free of preservatives, but in alternative embodiments, a preservative can be added as needed. For example, a lyoprotectant or lyoprotectant may be included in the lyophilized formulation.

Liquid or lyophilized formulation

The formulations of the invention can be liquid formulations or lyophilized formulations. In some embodiments, the liquid formulation may be used for injection or diluted before injection. Alternatively, the formulation may be a lyophilized powder. In some embodiments, the lyophilized powder is prepared to be combined with one of multiple solvent volumes to reach the desired concentration just prior to administration.

Exemplary formulations

In a specific embodiment of the present invention, the present invention provides a stable liquid antibody formulation suitable for subcutaneous administration, the formulation comprising: a) a fully human being greater than about 100 mg / mL, such as about 100 to about 175 mg / mL An anti-CXCR5 antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 33 and a light chain containing the amino acid sequence of SEQ ID NO: 32; b) about 10 mM citrate buffer; c) about 0.1% (w / v) polysorbate 20 or polysorbate 80; and d) about 200 mM spermine; e) about 4.5-9% sucrose, wherein the pH of the formulation is about pH 6.0.

In a specific embodiment of the present invention, the present invention provides a stable antibody formulation comprising: a) a humanized IgG4 anti-CXCR5 antibody comprising about 150 to about 175 mg / mL, comprising a polypeptide comprising SEQ ID NO: 33; The heavy chain of the amino acid sequence and the light chain containing the amino acid sequence of SEQ ID NO: 32; b) about 10 mM citrate buffer; c) about 0.1% (w / v) polysorbate 80; and d) about 200 mM spermine; e) about 4.5% sucrose, wherein the pH of the formulation is about pH 6.0.

In a specific embodiment of the present invention, the present invention provides a stable antibody formulation comprising: a) 175 mg / mL humanized IgG4 anti-CXCR5 antibody, which comprises an amino acid comprising SEQ ID NO: 33 The heavy chain of the sequence and the light chain containing the amino acid sequence of SEQ ID NO: 32; b) about 10 mM citrate buffer; c) about 0.1% polysorbate 80; and d) about 200 mM arginine; e) about 4.5% sucrose, wherein the pH of the formulation is about pH 6.0.

stability

Covered formulations are stable at 5 ° C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer, usually at least about 12, 18 or 24 months or longer. In an exemplary embodiment, they are stable at 5 ° C for at least about 6 months or more. In other exemplary embodiments, they are stable at 5 ° C for at least about 9 months. In a further exemplary embodiment, they are stable at 5 ° C. for at least about 1 year or more, and are generally greater than about 2 years or greater than about 4 years.

Dosing model

In some specific embodiments of the invention, the formulation is suitable for parenteral, intravenous, intramuscular, intradermal, subcutaneous, or a combination thereof. The formulations of the invention are suitable for delivery by various techniques.

Dosage and dosage form

The effective dosage of the formulation of the present invention varies depending on many different factors, including the means of administration, the target site, the physiological state of the subject, whether the subject is a human or an animal, other drugs administered, and treatment for prevention Sex is still therapeutic. Typically, the subject is human, but non-human mammals including transgenic mammals can also be treated. Therapeutic doses may need to be titrated to optimize safety and efficacy.

The formulations of the invention can be administered multiple times. The interval between single administrations can be daily, weekly, bi-weekly, monthly or yearly. The interval can also be irregular. In some methods, the dose is adjusted to achieve a certain plasma binding agent such as an antibody concentration. The dose and frequency will vary depending on the half-life of the anti-CXCR5 binding agent (eg, an antibody) in the subject. Generally, human antibodies show the longest half-life, followed by humanized, chimeric, and non-human antibodies.

In a further specific embodiment, the invention provides a pharmaceutical unit dosage form comprising a therapeutically effective amount of a formulation of the invention for treating one or more diseases in a subject by administering the dosage form to the subject. In some specific embodiments, the subject is a human. A person can be an adult or can be an infant. The term "pharmaceutical unit dosage form" means a physically discrete unit suitable as a uniform unit dose of a subject to be treated, each unit containing a predetermined amount of The calculation combined with the required citrate buffer and pH produces the desired therapeutic / preventive effect.

The unit dosage form may be a container containing a formulation. Suitable containers include, but are not limited to, sealed ampoules, vials, bottles, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (eg, the container may be a vial with a stopper pierceable by a hypodermic needle). In some embodiments, the container is a vial. Generally, the container should maintain the sterility and stability of the formulation.

In a specific embodiment, the formulation is packaged in a 4 mL vial (2R ISO vial) made of transparent, colorless Type I glass and closed with a flip-of cap with a flange (polypropylene) The plug (fluoropolymer coated bromobutyl) was closed. In some embodiments, the vial is filled with 1.7 mL of formulation such that the vial has an excess volume of about 0.2 mL per vial, and an extractable volume of 1.5 mL. For example, this means that the dose intensity of the antibody (eg 175 mg / mL) would be contained in a 1.5 mL solution.

In a specific embodiment, the formulation is repackaged in a container (eg, a cardboard box) that protects the vial from light.

Set

Some specific embodiments of the invention include kits comprising a formulation of the invention. The kit may also contain one or more containers containing pharmaceutically acceptable excipients, and include other materials (including filters / needles and syringes) required from the market and user point of view. Associated with the kit may be instructions that are normally included in a commercial package of a therapeutic, prophylactic or diagnostic product, which contains, for example, indications, usage, dosage, manufacture, administration, regarding the use of such therapeutic, preventive or diagnostic product , Contraindications, and / or warnings. The kit can also be associated with a label, which can be any kind of data carrier (such as a leaflet, sticker, wafer, print, or barcode) containing information. In some specific embodiments, the above indications and the like may be included in or on a label. The kit may further include a device for administering the formulation, in particular a device containing the formulation, ie a pre-filled device such as, but not limited to, a pre-filled syringe or a pre-filled auto-injector. Sets can also be packaged Contains a container that contains a formulation, ie, a pre-filled container, such as a pre-filled vial, cartouche, sachet, or ampoule.

Combination of different specific embodiments

In the context of the present invention, any of the specific embodiments described herein may be combined with one or more of the embodiments described herein unless explicitly stated otherwise. Specifically, any of the binding agents and antibodies described herein and the formulations described herein can be used in combination with any kit, pre-filled device, or pre-filled container, or can be used in a therapeutic method or medical application, such as Related to each antibody described herein (eg, a stable formulation comprising an anti-CXCR5 antibody can be combined with any of the kits, containers, or devices described herein). Any binding agent that specifically binds an antigen as described herein (eg, a binding agent that specifically binds CXCR5) can also be used in a therapeutic method related to each antibody (ie, anti-CXCR5) as described herein, and vice versa.

Examples

The following examples illustrate specific embodiments of the invention. They are for illustrative purposes only and should not be considered as limiting the invention.

Example 1: Analysis of Stardust Particle Formation

A) Introduction

The drug product (DP) named SAR113244 by Sanofi is a humanized IgG monoclonal antibody (mAb) that selectively binds human CXCR5 and is disclosed in U.S. 8,647,622. It was developed for parenteral and subcutaneous administration as a solution for injection at a concentration of 100 mg / mL. The DP solution for injection was packaged in a 2 mL transparent colorless vial (type I glass) with a stopper (bromobutyl rubber coated with a fluoropolymer) and a flip-off cap with flange (aluminum) (poly Acrylic) closed.

The formulation of the SAR113244 monoclonal antibody developed a formulation for phase I clinical research, which contains 10 mg / mL of spermine, 2 mg / mL of NaCl, 0.01% of polysorbate 20, and 10 mM of citric acid. Salt buffer, and has a pH of 6 (hereinafter referred to as "DP solution"). The The compounds are stable in terms of chemical stability and sub-visible particles. However, after about a year, when the formulation was stored in a glass primary packaging material, such as a vial or a pre-filled syringe, the formation of visible particles occurred. When the main packaging material is plastic (bag or bottle), the formation of visible particles does not occur.

Visible particles were found in the DP solution stored in a 2 R ISO glass vial (Schott, Elmsford, NY) during the compatibility study for the Phase 1 clinical research program. The particles are very small, shiny and suspended, so they are called "stardust particles." The discovery of stardust particles led to the cessation of the study and the DP solution was quarantine in batches. An immediate action plan was implemented, and an online filtering scheme was investigated and applied to clinical research. This is summarized in the revised text, which was approved by the health department without any comments, and the study can be restarted. However, for further clinical research, it is desirable to develop formulations without stardust particles. Here we describe a method for preparing a highly concentrated formulation containing SAR113244 without stardust particles.

Stardust particles are not present in DP solutions stored in bags or in Nalgene® bottles. There is also no stardust particles in the DP solution stored directly in glass vials after the manufacturing process. Stardust particles in glass vials are believed to have occurred about one year after storage. Particles of other products (such as protein therapeutics) filled in the same vial of the same type using the same equipment, using the same filling line, using the same stoppers, and never had any stardust particles checked.

Sub-Visible particle measurements using Microflow® imaging or light transmission in DP solutions are always in the acceptable range, regardless of the presence of stardust particles. Samples and long-term storage times obtained directly after manufacture in stability studies No difference was detected in the sub-visible particles of the 1-year sample. To remove stardust particles, a new, stardust-free formulation was subsequently developed for use in a phase II clinical trial. Finally, the formulation showed stability in a glass container for 6 months under accelerated conditions (40 ° C) and 12 months at 2-8 ° C.

B) Method

Visual inspection of DP solution: After completion of DP batch, DP is performed directly by trained personnel Visual inspection of the solution. Test the vial by inverting the vial once or twice and then observing the vial for 5 to 15 seconds (Lab 1: Liquid Inspection Viewer Apollo II; Lab 2: Black Box) or observing the rotating vial without inversion (Laboratory 3: Sidenader vial inspection machine with magnifying glass). The light source is 2000-3750Lux.

Glass layering in a DP solution container: Four different batches of glass layering were analyzed (3 clinical batches, one technical batch). To this end, each batch of 10 mL of DP solution was tested to determine the amount of glass elements (ie, Si, B, Ca, and Al) that penetrated into the solution. Four sets of containers were used in this study: SCHOTT Type I plus (SiO ₂ coated vials), SCHOTT Standard Fiolax, SCHOTT DC (Layered Control) and BD pre-filled syringes. The different solutions were stored at 60 ° C and analyzed at three different time points (after 1, 4, and 12 weeks). For the presence of "flake-like" and / or "non-flaky" particles, visual inspection was performed with an eye nitrile and a magnifying camera. Ten containers were tested at each time point. In addition, five empty containers of each control sample group were optically inspected using a stereo microscope with extended focal depth to qualitatively determine if there were any signs of glass corrosion or reaction zones present on the inner surface of the container. In addition, 10 empty containers and pull points of each sample group were analyzed by a stereo microscope with an extended focal depth to determine if there were any glass corrosion or reaction zones present on the inner surface of the container. sign. SEM cross-section analysis was performed on the inner surfaces of the two "worst" containers at each extraction point of each group. In total, a 10 mL placebo solution (containing 10 mM citrate buffer, 2 g / L NaCl, 10 g / L arginine-HCl, 45 g / L sucrose ( 4.5%) and 0.01% polysorbate 20) were analyzed to quantitatively determine the amount of glass element that penetrated into the solution.

Mechanical stability study: In order to determine whether mechanical stress contributes to the formation of stardust particles in DP solution, a mechanical stress study was performed. During the manufacturing process, the DP solution was added to a mixing vessel and excipients were added. Eight different formulations with a volume of 10 mL (see Table 1 below, each composition based on citrate buffer) were stirred at 200 rpm for 30 minutes or 2 hours. After mixing the excipients, check for visible particles in the solution. The solution was then filtered and filled into 2R glass vials. Subsequently Visual inspection was performed, and the solution was stored at 40 ° C for 4 weeks and checked weekly.

DP-drug product (SAR113244)

RapID studies (filtration, Raman and FT-IR): During the course of this study, 5 samples of DP solution were analyzed. Visual inspection was first performed on the DP solution in a closed container. The solution of each sample was filtered on a separate gold-coated polycarbonate filter (pore size 0.8 μm) with a 4 mm diameter active area. The empty vial was rinsed with 2 × 2 mL of particle-free water, and the stopper was rinsed with 1 × 2 mL of particle-free water. To ensure that all particles are transferred to the filter, the funnel inside the filtration device is rinsed with 8 mL of particle-free water. A picture of the observed particles was taken with a video microscope. Different spectroscopic methods were used to further analyze some of the largest particles (> 50 μm). Raman spectroscopy studies were performed manually by image analysis using a Single Particle Explorer (SPE) manufactured by rap.ID Particle Systems GmbH. The device operates at a laser wavelength of 532 nm. For the FT-IR and / or ATR spectra, an FT-IR spectrometer LUMOS (serial number 190) manufactured by Bruker was used. One of the analyzed particles was further investigated by a scanning electron microscope SEM-EDX (TM 3000) manufactured by Bruker.

Filtration-microscopy: Particles were separated from 5 bottles per batch using a nitrocellulose filter with a pore size of 0.45 μm. The obtained particles were analyzed by light microscopy. Using IR Microscopy by Infrared Spectroscopy The mirror Hyperion 2000 further characterizes particles at 15x magnification.

Measurement of sub-visible particles:

1. Optical particle counter: An optical particle counter (HIAC, Hach) is used to measure the amount of sub-visible particles in a protein formulation. As required by the Pharmacopoeia (USP, Ph. Eur.), Particles larger than 10 μm and 25 μm were investigated. The detection of the particles is performed by a photodetector which detects the optical interference of the illumination caused by the particles in the apparent volume.

2. Flow imaging microscopy: Flow imaging microscopy (microflow imaging (MFI), protein samples)) is used to measure the amount of sub-visible particles in protein formulations. As required by the Pharmacopoeia (USP, Ph. Eur.), Particles larger than 10 μm and 25 μm were investigated. Detect particles with a digital camera.

3. SEC: Use size exclusion chromatography (SEC) to measure the relative amount of soluble aggregates in the nanometer size range. In addition, the purity or relative amount of the monomer is reported.

4. DLS: Use Dynamic Light Scattering (DLS) (Zetasizer Nano-ZS, Malvern) to measure the presence of aggregates and particles in the nanometer size range. Measure hydrodynamic diameter and polydispersity.

C) Results and discussion:

Filtration experiment:

To understand the phenomenon of stardust particles, we tried to separate particles from the SAR113244 drug product (DP solution). The separation of the particles in the suspension can be achieved by filtration or by trying to remove the particles with a pipette or spatula. However, the characterization of stardust particles is very challenging because they cannot be separated. Filtration on a cellulose filter does not separate stardust particles (see Table 2 and Figures 1-3). Although filtering the three placebo solutions did not cause any particles on the filter, the DP solution from the Nalgene bottle showed a significant amount of glitter particles (Figures 4 and 5). Although the absolute number of particles on the filter appeared to be lower than the samples affected by Stardust, the shape and size were not significantly different from the previously separated particles. Therefore, the effectiveness of vacuum filtration to separate "stardust" particles must be questioned, as the DP solution from the Nalgene bottle did not show any stardust particles. The drug substance of SAR113244 also shows particles on the filter (Figure 6), even if the drug substance is starless

Dust particles: The drug substance is stored in a plastic bag only in citrate buffer (old drug substance from stage 1 process) and does not show particles when visually inspected, but can be seen on the filter after filtration To the particles.

Samples from different protein-based therapeutics (SAR252067 and SAR341403) again showed the presence of particles on the filter (Figures 7-12), although the solution was clear (no stardust particles). These findings show that vacuum filtration of highly concentrated antibody solutions has the risk of solution components settling on the filter and makes it impossible to identify possible sub-visible or visible particles present in the solution.

Measured by DLS:

The measurement results of protein aggregates measured by dynamic light scattering did show that the DP solution was stored in plastic bottles without stardust particles (Figure 13), and had a lower polydispersity index than the stardust-containing DP solution stored in glass vials. (Figure 14). This may be an indication that a conformational change in the SAR113244 molecule may be a potential cause of stardust particle formation. In addition to visual inspection, this result indicates that glass containers have a role in the formation of stardust.

Stratified research:

One possible cause of the formation of stardust particles is delamination that occurs from glass vials. therefore, A stratified study was conducted. A stratified study was performed with a placebo solution containing 10 mM citrate buffer, 2 g / L NaCl, 10 g / L arginine-HCl, 45 g / L (4.5%) sucrose, and 0.01% polysorbate 20. The placebo solution was stored in different kinds of glass vials outlined in Table 3.

No delamination was found after 1, 4, or 12 weeks under accelerated conditions (60 ° C) (see Tables 4A and 4B).

Table 4A.

Table 4B. Overview of glass corrosion (glass erosion) and delamination

^a Confirmed stratification: sharp edge or stratified area (SEM); ^b Early indicator: reaction area (SEM) on the inner surface, the coloration observed by visual inspection is not less than "weak" (SM), Si / B concentration ratio Less than or equal to 5 and Si concentration (ICP) higher than 10mg / mL (1mL up to 2mL fill volume); ^d Other: shallow pits, shallow hills, small holes, sediments (SEM), weak to medium observed by visual inspection Scattering (SM), localized reaction zone (horizontal size below 20 μM) (SEM).

However, an increase in the concentration of glass components (Al, Si, B, Ca) was detected in the solution during long-term storage (12 weeks, 60 ° C) (see Table 5).

Table 5. Concentrations of glass elements dissolved under accelerated conditions (ICP analysis)

Based on these observations, it is likely that the glass component (Al, Si, B, Ca) induced particle formation, as this was observed when comparing the formulation in a Nalgene® bottle to the mAb formulation in a vial. The only difference. The placebo study showed no stratification, but some ingredients It can also be used in a DP solution with a protein. Moreover, this study determined that stardust particles do not occur without protein.

RapID-Study: The purpose of this study is to determine what star dust particles consist of. Previous filtration studies were unsuccessful because it was not possible to separate particles on the filter. RapID provides a solution with a gold filter. Here, the DP solution was filtered on a gold filter, and then analyzed using FT-IR (Fig. 15) and Raman spectroscopy (Fig. 16). The results showed that protein particles were detected in 3 of the 5 vials. In other vials, polyethylene has been detected (Figures 17 and 18). Samples containing polyethylene most likely also contained proteins as typical absorption peaks, as proteins above 3000 cm ^-1 were also found in FT-IR analysis (Figure 17). It must be stated that the sample is very old (4 years-shelf life = 3 years). Therefore, additional studies are needed on samples where stardust particles have just been detected. However, it is believed that protein aggregation is at least partly responsible for the formation of stardust particle formulations. Therefore, new antibody formulations designed to avoid protein aggregation are considered to be able to avoid stardust particle formulations.

In addition, although stardust particles have been found and described in the context of specific anti-CXCR5 antibodies, it is believed that other antibodies may exhibit similar storage issues and therefore be able to potentially extract from the proposed antibody formulations described herein Benefit.

Example 2: Development of a Stardust-Free Particle Formulation

a) Introduction:

To avoid the formation of stardust particles, new formulations must be developed. In this process, chemical stability is as important as the absence of sub-visible particles.

B) Methods and results:

In order to obtain robust formulations that do not easily accumulate over a long period of time, mechanical stress studies are performed on various experimental samples. A magnetic stirrer was used to apply stress to the samples, and the samples were tested for formation of stardust particles (visual inspection) after filtration and storage under accelerated conditions. By mechanical stress studies, it can be shown that increasing the concentration of polysorbate 20 or polysorbate 80 decreases the particle size in antibody formulations. the amount. Based on these results, a stability study was performed in which highly concentrated SAR113244 formulations were tested in glass vials and pre-filled syringes. When the polysorbate 20 or polysorbate 80 concentration was 0.1%, no star dust particles were observed for any SAR113244 concentration. Even under accelerated conditions (6 months, 40 ° C), no visible particles were present. Therefore, highly concentrated formulations containing 100-175 mg / mL are stable without forming stardust particles. When the formulation contains about 100-175mg / mL SAR113244, about 200mM spermine, about 4.5-9% sucrose, about 0.1% polysorbate 20 or about 0.1% polysorbate 80, and about 10mM citrate buffer , Chemical stability is achieved.

Mechanical stress study: first add the drug (SAR113244) to the mixing container and then add excipients (see Table 1 above) to prepare experimental formulations. After the excipients were mixed, the solution was filtered and added to a glass vial. Finally, the final formulation is checked visually.

Mechanical stress studies were performed on vials as described above. The results are shown in FIGS. 19 and 20. Mechanical stress studies have shown that particles are formed even after filtration during storage. However, this visible particle formation process can be reduced or even terminated by increasing the polysorbate concentration. Based on these results, it is speculated that star dust-free particle formulations can be obtained by increasing the polysorbate concentration. Moreover, polysorbate 20 or polysorbate 80 can be used to obtain star dust-free particle formulations.

Stability studies: The inclusion of surfactants in protein-containing drug formulations is well known. Common surfactants are polysorbate 20 and polysorbate 80. Amino acids such as arginine and methionine are also known as stabilizers. However, the inventors have observed that, for the SAR113244 antibody, particle formation occurs only after storage (12 months) when the main packaging material is composed of glass. In order to identify the stable formulation of SAR113244, a drug-related formulation of the antibody was prepared with a polysorbate concentration above 0.01% and an amino acid concentration above 50mM (see Table 6 below). These formulations were stored in glass vials or glass syringes, and stability studies were performed under accelerated conditions (40 ° C), and particle formation was measured (see Table 7).

The results of the stability studies show that even with a polysorbate content of 0.01%, a large amount of spermine Acid (200 mM) also produces a formulation that is stable for 6 months without stardust particles. It is desirable to obtain monoclonal antibodies at even higher concentrations and to obtain stable formulations over a long period of time. Earlier results showed that high concentrations of arginine (Table 5) and large amounts of polysorbate (Figures 19 and 20) in the formulation were beneficial. These observations were taken into account and new stability studies were performed using large amounts of arginine and polysorbate.

The samples of Table 6 were stored in 2R ISO vials at different temperatures (5 ° C, 25 ° C, 40 ° C, and -20 ° C) for 2 weeks (T0.5), 1 month (T1), 3 months (T3), and 6 months (T6) to determine its stability using the method described in Example 1. A control sample was also measured on day 0 (TO). The results are shown in Table 7.

In subsequent tests, new formulations based on formulation 124D with even higher monoclonal antibody concentrations were prepared and tested for stability as described above. In addition, due to the results related to Table 5, increased polysorbate concentrations were also tested. The new formulations are described in Table 8 below, where the formulations Formulations A and B were stored in 2R ISO vials, and formulations C and D were stored in pre-filled syringes (1 mL Hypak BD).

The stability of the formulations of Table 8 was tested, and the results are shown in Table 9.

Based on the results of the stability study, it was shown that a formulation with high antibody concentration, a preparation without stardust particles, was obtained. In fact, antibody concentrations of up to 175 mg / mL are stable for 6 months under accelerated conditions (40 ° C) (see, for example, Formulation B). The amount of particles detected when using a pre-filled syringe as the primary packaging material was slightly higher than the 2R ISO vial, but still far below the regulatory acceptance standards. An experimental formulation with such a high concentration of easily aggregated protein is being tested. The realization of almost no sub-visible particles under accelerated conditions (40 ° C, 6 months) was absolutely unexpected and a huge breakthrough in the establishment of highly stable high-concentration antibody formulations. Indeed, such a high antibody concentration formulation may allow the use of an autoinjector to administer the antibody formulation. In addition, this antibody formulation can achieve an even longer shelf life for antibodies stored at high concentrations in glass containers for extended periods of time.

Having described the invention in detail and by reference to specific embodiments thereof, it is apparent that modifications and variations can be made without departing from the scope of the invention as defined by the scope of the appended patents. More specifically, although some aspects of the invention are considered to be particularly advantageous herein, it is contemplated that the invention is not necessarily limited to these specific aspects of the invention. In alternative specific embodiments, the percentages disclosed herein may vary by an amount of ± 10, 20, or 30% from the values disclosed herein.

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Claims (22)

An antibody formulation suitable for subcutaneous administration to a patient, the formulation comprising: a) about 50 to about 250 mg / mL of an anti-CXCR5 antibody or fragment thereof; b) citrate buffer; c) greater than about 0.01% w / v) a surfactant; d) an amino acid greater than about 50 mM; and e) greater than about 1% sucrose, wherein the pH of the formulation is about pH 6. The antibody formulation of claim 1, wherein the anti-CXCR5 antibody or fragment thereof comprises: (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and an amine comprising SEQ ID NO: 12 Heavy chain variable domain of amino acid sequences; (b) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLAS (SEQ ID NO: 59), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62) and IVY (SEQ ID NO: 63); (c) a light amino acid sequence comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Chain variable domain, and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; (d) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLAS (SEQ ID NO: 64), MQHLEYPYT (SEQ ID NO : 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62) and IVY (SEQ ID NO: 63) amino acid sequences; (e) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSNLAS ( (SEQ ID NO: 65), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (f ) Contains an amino acid of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21 Variable light chain (VL) of sequence, and variable heavy chain comprising amino acid sequence of SEQ ID NO: 23 (VH); (g) a variable light chain comprising the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, and comprising SEQ ID NO: 33 or SEQ ID NO: 34 (H) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID N: 62) and IVY (SEQ ID NO: 63) amino acid sequences; (i) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSNLA (SEQ ID NO: 67), MQHLEYPYT (SEQ ID NO: 60), the amino acid sequences of GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62) and IVY (SEQ ID NO: 63); (j) RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62), and IVY (SEQ ID NO: 63) amino acid sequences; (k) A variable light chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 37; (l) comprising SEQ ID NO: 39, SEQ ID NO: 41 Or a variable light chain of the amino acid sequence of SEQ ID NO: 43, and an amine comprising SEQ ID NO: 45 or SEQ ID NO: 47 A variable heavy chain of an acid sequence; (m) a variable light chain comprising an amino acid sequence of SEQ ID NO: 55, and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 57 Chain; or (n) RSSKSLLHSSGKTYLYW (SEQ ID NO: 69), RMSNLA (SEQ ID NO: 66), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO: 61), VIWGDGTTY (SEQ ID NO: 62) , And IVY (SEQ ID NO: 63) amino acid sequences. The antibody formulation of claim 1, wherein the amino acid is arginine or methionine. The antibody formulation of claim 1, wherein the surfactant is a polysorbate. An antibody formulation suitable for subcutaneous administration to a patient, the formulation comprising: a) about 100 to about 175 mg / mL of antibody; b) about 10 mM citrate buffer; c) about 0.1% (w / v) Surfactants; d) about 200 mM arginine; and e) about 4.5 to 9% sucrose, wherein the pH of the formulation is about pH 6. The antibody formulation of claim 5, wherein the antibody is a fully human anti-CXCR5 antibody. The antibody formulation of claim 6, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 32. The antibody formulation of claim 5, wherein the antibody comprises a single chain Fv. The antibody formulation of claim 5, wherein the antibody is an isolated antibody or a fragment thereof that specifically binds to the extracellular domain of human CXCR5. The antibody formulation of claim 9, wherein the isolated antibody or fragment thereof comprises RSSKSLLHSSGKTYLY (SEQ ID NO: 58), RLSSLA (SEQ ID NO: 68), MQHLEYPYT (SEQ ID NO: 60), GFSLIDYGVN (SEQ ID NO : 61), amino acid sequences of VIWGDGTTY (SEQ ID NO: 62) and IVY (SEQ ID NO: 63). The antibody formulation of claim 5, wherein the surfactant is a polysorbate. The antibody formulation of claim 5, wherein the polysorbate is polysorbate 20 or polysorbate 80. An antibody formulation comprising: a) about 175 mg / mL of a humanized IgG4 anti-CXCR5 antibody; b) about 10 mM citrate buffer; c) about 1.0 mg / mL polysorbate 80; d) about 200 mM Arginine HCl; and e) about 45 mg / mL sucrose, wherein the pH of the formulation is about pH 6. The antibody formulation of claim 13, wherein the humanized IgG4 anti-CXCR5 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 33 and a light chain containing the amino acid sequence of SEQ ID NO: 32. A container comprising an antibody formulation according to any one of claims 1-14. The container of claim 15, wherein said container is a pre-filled syringe, vial or auto-injector. A container comprising an antibody formulation according to any one of claims 1-14 in a lyophilized form. A kit comprising a container as in any of claims 15, 16 or 17 and a label or instructions for administering and using the antibody formulation. The kit of claim 18, wherein said administering is by injection. The antibody formulation of any one of claims 1-14, for use in a method of treating or diagnosing a human or animal body. A method for treating rheumatoid arthritis, comprising administering an antibody formulation according to any one of claims 1-14 to a subject in need thereof. The lyophilized form of the antibody formulation of any of claims 1-14.