HK1218128B - Anti-tfpi antibody variants with differential binding across ph range for improved pharmacokinetics - Google Patents

Description

Anti-TFPI antibody variants with improved pharmacokinetics and differential binding across the pH range

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Application No. 61/798,261, filed March 15, 2013, which is incorporated herein by reference.

Background Art

Current preventive management for hemophilia A and B patients is to replace FVIII or FIX (recombinant or plasma-derived products). These treatments are administered twice or three times a week, placing a heavy burden on patients to comply with their preventive regimens. Despite strict management and strict adherence, patients typically experience occasional breakthrough bleeding and require on-demand treatment. Without proper management, frequent and severe bleeding leads to significant morbidity, particularly hemophilic arthropathy. Although existing drugs for the treatment of hemophilia A and B patients have definite efficacy, most youth, adolescents, and the elderly decide to reduce the burden of prevention by reducing the number of injections taken on a routine basis. This approach further compromises the protection needed to adequately manage bleeding.

Therefore, agents that provide significant protection and require relatively infrequent administration are most desirable. Optimal treatment should provide protection with weekly or less frequent dosing. Given the current competitive landscape, once-weekly treatments administered intravenously (i.v.) or subcutaneously (s.c.) may be feasible within the next 3-4 years. Therefore, agents administered i.v. or s.c. should provide an excellent administration profile with considerable protection. In the event that subcutaneous administration can be achieved, once-weekly dosing may also provide significant value to future therapeutic prospects due to the reduced invasiveness of this approach.

Another major problem in the treatment of hemophilia at present is the development of inhibitory antibodies to factor VIII or factor IX. About 25% of patients treated with FVIII develop inhibitors or neutralizing antibodies to FVIII. Although less frequent, inhibitors have also been found in patients treated with FIX. The development of inhibitors significantly reduces the effectiveness of replacement therapy and has challenged the management of bleeding in hemophilia patients. The current treatment for bleeding in patients with FVIII or FIX inhibitors is a bypass therapy using recombinant factor VIIa or plasma-derived FEIBA. The half-life of rFVIIa is quite short (-2 hours) and therefore preventive treatment in these patients is uncommon [Blanchette, Haemophilia 16 (supplement 3): 46-51, 2010].

To address these unmet medical needs, antibodies against tissue factor pathway inhibitors (TFPI) have been developed as long-acting agents. See WO 2010/017196; WO 2011/109452; WO 2012/135671. TFPI is the main inhibitor of tissue factor that initiates the coagulation pathway, which is intact in hemophilia patients (PWH), and therefore inhibiting TFPI can restore hemostasis in PWH that display inhibitory antibodies to FVIII or FIX. In addition to allowing this goal to be achieved, therapeutic agents of monoclonal antibody (mAb) therapy have been shown to have significantly longer circulation half-lives (up to 3 weeks) than recombinant replacement factors. Antibodies that inhibit TFPI also have significant bioavailability with subcutaneous injection. Therefore, anti-TFPI monoclonal antibody therapy will meet the important unmet medical needs of subcutaneously injected, long-lasting hemostatic protection for PWH and PWH with inhibitors.

However, although inhibition of TFPI has been shown to promote coagulation in hemophilic plasma and hemophilic animals, antibodies against TFPI have relatively short, nonlinear half-lives due to a phenomenon called target mediated drug disposition (TMDD), a process in which the antibody is removed from the circulation due to its interaction with a rapidly cleared target, or sequestered from the plasma due to its colocalization with the target (antibody:antigen complex). Therefore, antibodies that avoid TFPI-mediated TMDD and have extended half-lives would result in less frequent dosing and reduce the amount of material required per dose. In addition, the requirement for fewer doses also makes subcutaneous administration, where the amount of administration is the limiting step, feasible. For example, in non-human primates, the optimized anti-TFPI antibody 2A8-g200 (WO 2011/109452) has a half-life of 28 hours when administered at 5 mg/kg and 67 hours when administered at 20 mg/kg.

This relatively short half-life and the need for larger doses to overcome TMDD increase the injection burden on patients, limit formulations for subcutaneous administration, and increase product costs. Pharmacokinetic analysis of these antibodies in non-human primates demonstrated that the circulating half-life was not linear with dose and, particularly at lower doses, was shorter than is characteristic of antibody drugs. A similar pharmacokinetic profile is described in US2011/0318356 A1 for another anti-TFPI antibody. This difference in having a significantly shortened T1/2 at lower doses is characteristic of TMDD, with slower clearance at higher doses due to target saturation.

Therefore, there is an unmet medical need for better prophylactic treatments for moderate to severe hemophilia A and B, particularly for those patients with inhibitors to FVIII or FIX. This need would be met by anti-TFPI antibodies with improved characteristics that can be administered intravenously or subcutaneously and less frequently, preferably once a week or less.

SUMMARY OF THE INVENTION

To increase the half-life of anti-TFPI antibodies and reduce the burden of injection, longer-acting anti-TFPI antibodies without potency loss were produced and tested to confirm improved performance compared to other anti-TFPI antibodies that have been shown to have TFPI binding characteristics and are effective in treating coagulation defects. (See WO 2011/109452). In particular, TMDD was reduced by generating variant anti-TFPI antibodies with reduced affinity at pH 6.0 relative to pH 7.4. Anti-TFPI antibodies can be complexed with their target (TFPI) and taken up by cells through receptors involved in TFPI clearance. One receptor identified by Narita et al. (JBC 270 (42): 24800-4, 1995) is LRP (LDL receptor-related protein), which targets TFPI for degradation in endosomes. However, if this antigen:antibody complex can be destroyed at low pH values (which are characteristic of endosomes), the antibody can be recycled through FcRn binding, thereby increasing its exposure in the circulation. This principle has been shown for antibodies against PSCK9 by Chapparo-Riggers et al. JBC 287(14): 110-7 (2012).

One approach to disrupting antigen:antibody complexes at lower pH is to replace histidine residues near the antigen:antibody interaction surface. The amino acid histidine (His) is protonated at low pH values near pH 6.0, and thus a residue that is neutral at pH 7.4 acquires a positive charge at pH 6.0. This can result in charge repulsion with other amino acids on the antigen:antibody interaction surface and a desired degree of disruption or destabilization.

To identify pH-sensitive residues, CDR amino acids and other amino acids involved in antigen:antibody binding of anti-TFPI antibodies (e.g., 2A8-g200) to TFPI antigen were individually changed to His. Individual His variants demonstrated differential binding at pH 7.4 versus pH 6.0, and combinations of variants with differential binding have been tested for optimization.

After endosomal release, these pH-sensitive anti-TFPI mAb variants bind to nascent FcRN receptors and recirculate into the plasma. Therefore, the combination of a pH-sensitive TFPI-binding site and an Fc domain with increased affinity for FcRN at low pH has a synergistic effect, increasing half-life, reducing the injection burden on patients, and lowering product costs.

Description of the drawings

Figure 1 shows an alignment of the amino acid sequences of 2A8-g200 and anti-TFPI mAbs mutated for histidine substitution (SEQ ID NOs for these sequences can be found in Table 1). A. Variable heavy chain, B. Variable light chain. CDR regions 1-3 are indicated.

Figure 2 shows the synthesis and subcloning of the 2A8-g200 Fab histidine scanning library. The CDR 1-3 regions are indicated by dotted lines. The underlined amino acid residues indicate the positions of the residues that contact TFPI. Asterisks (*) indicate proposed His mutation sites. A) 2A8-g200 heavy chain; B) 2A8-g200 light chain; C) 4B7-gB9.7 heavy chain; and D) 4B7-gB9.7 light chain.

Figure 3 shows the dissociation constants of improved antibodies with exemplary histidine mutations: A. L-L27H and B. L-Y31H at two pH values. Surface plasmon resonance (Biacore T200) was used to measure the dissociation rates of the antibodies.

Figure 4 shows the pK profiles observed over time in HemA mouse plasma for several monoclonal antibodies at a concentration of 2 mg/kg: 2A8-g200 (--■--), histidine-substituted monoclonal antibodies TPP2256 (L-Y31H/Y49H) (-·-■-·-) and TPP2259 (L-Y31H) (-▲-). The pharmacokinetic parameters of the antibodies were determined after intravenous (i.v.) bolus administration of 2 mg/kg to HemA mice.

Detailed Description of the Invention

As used herein, the term "tissue factor pathway inhibitor" or "TFPI" refers to any variant, isoform, and species homolog of human TFPI that is naturally expressed by cells. In a preferred embodiment of the invention, binding of the antibody of the invention to TFPI reduces blood clotting time.

As used herein, "antibody" refers to a complete antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. The term includes full-length immunoglobulin molecules (e.g., IgG antibodies), which are naturally occurring or formed by recombinant processing of normal immunoglobulin gene fragments, or immunologically active portions of immunoglobulin molecules, such as antibody fragments, which retain specific binding activity. Regardless of the structure, antibody fragments bind to the same antigen recognized by the full-length antibody. For example, an anti-TFPI monoclonal antibody fragment binds to an epitope of TFPI. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the _VL , _VH , _CL , and _CH1 domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the _VH and _CH1 domains; (iv) a Fv fragment consisting of the _VL and _VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a _VH domain; (vi) isolated complementarity-determining domains (CDRs); (vii) minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., III et al., Protein Eng 1997; 10:949-57); (viii) camel IgG; and (ix) IgNAR. In addition, although the two domains of the Fv fragment _{, VL} and _VH, are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, enabling them to be prepared as a single protein chain in which the _VL and _VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are analyzed in the same manner as for intact antibodies.

In addition, it is contemplated that antigen-binding fragments may be included in antibody mimetics. As used herein, the term "antibody mimetics" or "mimetic" refers to a protein that exhibits similar binding to an antibody, but is smaller than an alternative antibody or non-antibody protein. Such antibody mimics may be included on a scaffold. The term "scaffold" refers to an engineered polypeptide platform for a new product with modified functions and features.

As used herein, the terms "inhibit binding" and "block binding" (e.g., referring to inhibition/blocking of binding of a TFPI ligand to TFPI) are used interchangeably and include partial or complete inhibition or blocking. Inhibition and blocking are also intended to include a measurable decrease in the binding affinity of any TFPI to a physiological substrate when contacted with an anti-TFPI antibody, compared to TFPI not contacted with the anti-TFPI antibody, for example, blocking the interaction of TFPI with Factor Xa or blocking the interaction of the TFPI-Factor Xa complex with tissue factor, Factor Vila, or the tissue factor/Factor Vila complex by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

Therapeutic antibodies have been prepared by the hybridoma technology described by Koehler and Milstein in "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256, 495-497 (1975). Fully human antibodies can also be produced recombinantly in prokaryotes and eukaryotes. Recombinant production of antibodies in host cells, rather than hybridoma production, is preferred for therapeutic antibodies. Recombinant production has the advantages of greater product consistency, potentially higher production levels, and controlled manufacturing that minimizes or eliminates the presence of animal-derived proteins. For these reasons, it is desirable to have recombinantly produced monoclonal anti-TFPI antibodies. As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to the preparation of a single molecular composition of antibody molecules. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Generally, therapeutic antibodies for human diseases are produced using genetic engineering to generate murine, chimeric, humanized, or fully human antibodies. Due to their short serum half-life, inability to trigger human effector functions, and production of human anti-mouse antibodies, murine monoclonal antibodies have been shown to have limited use as therapeutic agents (Brekke and Sandlie, "Therapeutic Antibodies for Human Diseases at the Dawn of the Twenty-first Century" Nature 2, 53, 52-62, Jan. 2003). Chimeric antibodies have been shown to elicit human anti-chimeric antibody responses. Humanized antibodies further minimize the mouse component of the antibody. However, fully human antibodies completely avoid the immunogenicity associated with the mouse element. Therefore, it is necessary to develop human or humanized antibodies to a degree that can avoid the immunogenicity associated with other forms of genetically engineered monoclonal antibodies. In particular, due to the need for frequent dosing and long duration of treatment, chronic prophylactic treatments such as those requiring the use of anti-TFPI monoclonal antibodies for hemophilia will have a high risk of developing an immune response to the treatment if antibodies with mouse components or mouse origin are used. For example, antibody treatment for hemophilia A may require weekly dosing for the patient's entire life. This will be a continuous challenge to the immune system. Therefore, there is a need for fully human antibodies for the antibody treatment of hemophilia and related heredity and acquired defects or coagulation defects. Therefore, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity, which has at least a portion of variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific in vitro mutagenesis or by somatic mutations in vivo). These human antibodies include chimeric antibodies, such as mouse/human and humanized antibodies, which retain non-human sequences.

As used herein, an "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to TFPI is substantially free of antibodies that bind antigens other than TFPI). However, an isolated antibody that binds to an epitope, isoform, or variant of human TFPI may have cross-reactivity with other related antigens, e.g., from other species (e.g., TFPI species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, "specific binding" refers to an antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 10 ⁵ and binds to the predetermined antigen with an affinity higher than that of the antibody binding to an unrelated antigen (e.g., BSA, casein) other than the predetermined antigen or a very related antigen, for example, at least twice as high. The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

As used herein, "high affinity" for IgG antibodies refers to a binding affinity of at least about 10 ⁷ , in some embodiments at least about 10 ⁸ , in some embodiments at least about 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or higher, for example, up to 10 ¹³ or higher. However, for other antibody isotypes, "high affinity" binding can vary. For example, "high affinity" binding for IgM isotypes refers to a binding affinity of at least about 1.0×10 ^7. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

"Complementarity determining region" or "CDR" refers to one of the three hypervariable regions within the heavy or light chain variable region of an antibody molecule that form an N-terminal antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Starting from the N-terminus of the heavy or light chain, these complementary determining regions are designated "CDR1," "CDR2," and "CDR3," respectively. CDRs are involved in antigen-antibody binding, and CDR3 includes a unique region specificity for antigen-antibody binding. Thus, an antigen binding site may include six CDRs, which include CDR regions from each heavy or light chain V region.

As used herein, except for the individual or multiple histidine substitutions described below, "conservative substitutions" refer to modifications of a polypeptide that involve one or more amino acid substitutions of amino acids with similar biochemical properties that do not result in a loss of the biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the prior art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is contemplated that the antibodies of the invention may have conservative amino acid substitutions and still retain activity.

The term "substantially homologous" indicates that two polypeptides or designated sequences thereof (with appropriate amino acid insertions or deletions) are identical in at least about 80% of their amino acids, typically at least about 85%, preferably about 90%, 91%, 92%, 93%, 94%, or 95%, and more preferably at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of their amino acids when optimally aligned and compared. The present invention includes polypeptide sequences having substantial homology to the particular amino acid sequences recited herein.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions × 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparisons and determination of percent identity between two sequences can be obtained using a mathematical algorithm such as the AlignX™ module of Vector NT™ (Invitrogen Corporation, Carlsbad, CA). For AlignX™, the default parameters for multiple alignment are: Gap Opening Penalty: 10; Gap Extension Penalty: 0.05; Gap Separation Penalty Range: 8; % Alignment Delay Identity: 40. (For more details, see

http://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Communities/Vector-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/Al ignX-Module-for-Vector-NTI-Advance.reg.us.html)

Another method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also known as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson et al., Nucleic Acids Research, 1994, 2(22):4673-4680), which is based on the algorithm of Higgins et al. (Computer Applications in the Biosciences (CABIOS), 1992, 8(2):189-191). In the sequence alignment, both the query and subject sequences are DNA sequences. The results of the global sequence alignment are expressed as percent identity. The preferred parameters for the CLUSTALW alignment of DNA sequences for calculating percent identity by pairwise alignment are: Matrix = IUB, k-tuple = 1, number of top diagonals = 5, gap penalty = 3, gap open penalty = 10, and gap extension penalty = 0.1. For multiple alignments, the following CLUSTALW parameters are preferred: Gap opening penalty = 10, Gap extension parameter = 0.05; Gap separation penalty range: 8; Alignment delay % identity = 40.

Also provided are pharmaceutical compositions comprising a therapeutically effective amount of an anti-TFPI monoclonal antibody and a pharmaceutically acceptable carrier. As used herein, a "therapeutically effective amount" means an amount of an anti-TFPI monoclonal antibody variant or a combination of such an antibody and Factor VIII or Factor IX that is effective to increase clotting time in vivo, or otherwise provide a measurable benefit in vivo to a patient in need thereof. The precise amount will depend on a variety of factors, including but not limited to the physical properties of the components and therapeutic composition, the intended patient population, individual patient considerations, etc., and can be readily determined by one skilled in the art. A "pharmaceutically acceptable carrier" is a substance that can be added to the active ingredient to aid in formulation or stabilization of the preparation and that does not cause significant adverse toxic effects to the patient, and has no significant adverse toxic effects to the patient. Examples of such carriers are known to those skilled in the art and include water, sugars such as maltose or sucrose, albumin, salts such as sodium chloride, etc. Other carriers are described as examples in Remington's Pharmaceutical Sciences by E.W. Martin. Such compositions will comprise a therapeutically effective amount of at least one anti-TFPI monoclonal antibody.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. The purposes of such media and reagents for pharmaceutically active substances are well known in the art. The composition is preferably formulated for parenteral injection. The composition can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some cases, it will include isotonic agents, for example, sugars, polyols in the composition, such as mannitol, sorbitol or sodium chloride.

As required, sterile injectable solutions can be prepared by mixing a suitable solvent with the active compound of a desired amount and the composition enumerated above or in combination, followed by aseptic microfiltration. Typically, dispersions are prepared by mixing the active compound into a sterile medium containing an alkaline dispersion medium and other components of those required as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of any other desired component of the active component plus a previously sterile filtered solution.

Human monoclonal antibodies can be used therapeutically to treat inherited and acquired deficiencies or defects in blood coagulation. For example, human monoclonal antibodies can be used to block the interaction of TFPI with FXa, or to prevent TFPI-dependent inhibition of TF/FVIIa activity. Furthermore, human monoclonal antibodies can be used to restore TF/FVIIa-driven FXa production, thereby circumventing deficiencies in FVIII- or FIX-dependent FXa expansion.

Human monoclonal antibodies have therapeutic uses in the treatment of hemostatic disorders such as thrombocytopenia, platelet disorders, and bleeding disorders (e.g., hemophilia A, hemophilia B, and hemophilia C). Such disorders can be treated by administering a therapeutically effective amount of an anti-TFPI monoclonal antibody variant to a patient in need thereof. Human monoclonal antibodies also have therapeutic uses in the treatment of uncontrolled bleeding in indications such as trauma and hemorrhagic stroke. Thus, a method for shortening bleeding time is also provided, comprising administering a therapeutically effective amount of an anti-TFPI human monoclonal antibody variant of the present invention to a patient in need thereof.

The antibody can be used as a monotherapy or in combination with other treatments to address hemostatic disorders. For example, co-administration of one or more variant antibodies of the present invention with coagulation factors such as factor VIIa, factor VIII or factor IX is believed to be useful for treating hemophilia. In separate embodiments, factor VIII or factor IX are administered substantially without factor VII. "Factor VII" includes factor VII and factor VIIa.

The method for treating the genetic and acquired deficiency or defect of blood coagulation comprises administering (a) a first amount variant monoclonal antibody in conjunction with human tissue factor pathway inhibitor and (b) a second amount of factor VIII or factor IX, wherein the first and second amounts are together effective for treating the deficiency or defect. Similarly, the method for treating the genetic and acquired deficiency or defect of blood coagulation comprises administering (a) a first amount monoclonal antibody variant in conjunction with human tissue factor pathway inhibitor and (b) a second amount of factor VIII or factor IX, wherein the first and second amounts are together effective for treating the deficiency or defect, and further wherein factor VII is not co-administered. The present invention also includes a pharmaceutical composition comprising a combination of the monoclonal antibody variant of the present invention and factor VIII or factor IX in a therapeutically effective amount, wherein the composition does not comprise factor VII.

These combination therapies may reduce the frequency of infusions of coagulation factors required.By co-administration or combination therapy is meant the administration of two therapeutic agents, each formulated separately or formulated together in one composition, and when formulated separately, at about the same time or at different times, but within the same treatment period.

In some embodiments, one or more antibody variants described herein can be used in combination to address hemostatic disorders. For example, co-administration of two or more antibody variants described herein is believed to be useful in treating hemophilia or other hemostatic disorders.

The pharmaceutical composition may be administered parenterally to a subject with hemophilia A or B, at dosages and frequencies that vary with the severity of the bleeding episode or, in the case of prophylactic treatment, with the severity of the patient's coagulation defect.

The compositions can be used as push or by continuous infusion to the patient of needs.For example, the push administration of the antibody variant presented with Fab fragment can be the amount of 0.0025 to 100mg/kg body weight, 0.025 to 0.25mg/kg, 0.010 to 0.10mg/kg or 0.10-0.50mg/kg.For continuous infusion, the antibody variant presented with Fab fragment can be used for 1-24 hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours or 1-2 hours with 0.001 to 100mg/kg body weight/minute, 0.0125 to 1.25mg/kg/minute, 0.010 to 0.75mg/kg/minute, 0.010 to 1.0mg/kg/minute or 0.10-0.50mg/kg/minute. For the administration of antibody variants presented as full-length antibodies (with complete constant regions), the dosage can be about 1-10 mg/kg body weight, 2-8 mg/kg, or 5-6 mg/kg. Such full-length antibodies are generally administered by infusion over a period of 30 minutes to 3 hours. The frequency of administration depends on the severity of the condition. The frequency range can be three times a week to once every two weeks to six months.

Additionally, the composition can be administered to the patient by subcutaneous injection.For example, a dose of 10 to 100 mg of the anti-TFPI antibody can be administered to the patient by subcutaneous injection weekly, biweekly, or monthly.

Variant monoclonal antibodies to human tissue factor pathway inhibitor (TFPI) are provided. Isolated nucleic acid molecules encoding the same are further provided. Pharmaceutical compositions comprising the variant anti-TFPI monoclonal antibodies and methods for treating inherited and acquired deficiencies or defects in blood coagulation, such as hemophilia A and B, are also provided. Methods for shortening bleeding time by administering the anti-TFPI monoclonal variants to patients in need thereof are also provided. Methods for producing variant monoclonal antibodies that bind to human TFPI according to the present disclosure are also provided.

The therapeutic composition includes an antibody having a binding region that differs from the sequence of a parent TFPI-binding antibody by intentional selection or engineering of one or more amino acid histidine (H, HIS) substitutions for at least one naturally occurring amino acid as defined in the parent sequence. The amino acid changes confer a longer circulating half-life, T1/2, relative to the parent molecule.

Disclosed are antibodies specific for TFPI that bind TFPI at pH 6.0 with at least 20% less efficiency than at pH 7.0 and exhibit an approximately 400% improvement in circulating T1/2. The beneficial effect of reducing TMDD has been demonstrated for antibodies or antibody binding regions other than target antibodies, such as 2A8-g200 or 4B7-gB9.7, by substituting at least one naturally occurring amino acid in the amino acid histidine (H, HIS) relative to the parent sequence. In particular, variants of 2A8-g200 may have any of the following substitutions: VL-Y31H, VH-Y102H, VH-Y100H, VH-Y32H, VL-F48H, VL-S50H, VL-Y49H, VL-L27H, VL-V45H, VL-W90H, and combinations thereof.

The anti-TFPI antibody 2A8-g200 and 4B7-gB9.7 variants can bind to TFPI with high affinity and specificity in vivo (see WO 2011/109452). Figure 1 shows the amino acid sequence information of 2A8-g200 and 4B7-gB9.7, as well as other 2A8 and 4B7 variants described in WO 2011/109452. Table 1 shows the corresponding SEQ ID NOs for the variable heavy and variable light chains of the 2A8 and 4B7 variants shown in Figure 1.

Table 1: Corresponding SEQUENCE ID NOs of the variable heavy and variable light chains of the human anti-TFPI antibodies shown in Figure 1.

chain MAb SEQ ID NO: VH 2A8 SEQ ID NO: 1 2A8-127 SEQ ID NO: 5 2A8-143 SEQ ID NO: 6 2A8-200 SEQ ID NO: 7 2A8-216 SEQ ID NO: 8 2A8-227 SEQ ID NO: 9 2A8-g200 SEQ ID NO: 10 2A8-g216 SEQ ID NO: 11 4B7 SEQ ID NO: 3 4B7-B18.5 SEQ ID NO: 19 4B7-B2.0 SEQ ID NO: 20 4B7-B27.1 SEQ ID NO: 21 4B7-B32.5 SEQ ID NO: 22 4B7-B41.2 SEQ ID NO: 23

4B7-B9.7 SEQ ID NO: 24 4B7-gB9.7 SEQ ID NO: 25 4B7-gB9.7-IgG SEQ ID NO: 26 VL 2A8 SEQ ID NO: 2 2A8-127 SEQ ID NO: 12 2A8-143 SEQ ID NO: 13 2A8-200 SEQ ID NO: 14 2A8-216 SEQ ID NO: 15 2A8-227 SEQ ID NO: 16 2A8-g200 SEQ ID NO: 17 2A8-g216 SEQ ID NO: 18 4B7 SEQ ID NO: 4 4B7-B18.5 SEQ ID NO: 27 4B7-B2.0 SEQ ID NO: 28 4B7-B27.1 SEQ ID NO: 29 4B7-B32.5 SEQ ID NO: 30 4B7-B41.2 SEQ ID NO: 31 4B7-B9.7 SEQ ID NO: 32 4B7-gB9.7 SEQ ID NO: 33 4B7-gB9.7-IgG SEQ ID NO: 34

pH-sensitive variants of 2A8-g200 and 4B7-gB9.7 were generated by analyzing the binding characteristics of selected sites after mutagenesis by making both the CDR domains and the residues contact TFPI. Figure 2 shows the positions of possible His mutations: A. 2A8-g200 (designated A200 in Figure 2A) variable heavy chain variable region; B. 2A8-g200 (designated A200 in Figure 2B) variable light chain; C. 4B7-gB9.7 variable heavy chain; and D. 4B7-gB9.7 variable light chain. A histidine residue is substituted for each amino acid in 1) the residue underlined in Figure 2 indicating the amino acid contacting TFPI or 2) the residue indicated by an asterisk in Figure 2 for CDRs 1-3 of the anti-TFPI antibodies 2A8-g200 and 4B7-gB9.7. As shown in Figures 2A and 2B, 40 residues from the heavy chain and 29 residues from the light chain were identified as mutagenesis positions in 2A8-g200. As shown in Figures 2C and 2D, 40 heavy chain and 32 light chain variants were identified in 4B7-gB9.7.

A 2A8-g200 Fab histidine scanning library was synthesized. The library contained 69 members. The 2A8-g200 Fab histidine library was cloned into a bacterial expression vector and the amino acid sequence was verified.

69 clones from the His scan library were transformed into Escherichia coli (E.coli) ATCC strain 9637 and grown on a selective medium containing carbenicillin (100 μg/mL). Single colonies were inoculated into LB-carbenicillin-100 medium. The culture was grown to OD600=0.5 at 37°C, induced with 0.25mM IPTG, and cultivated overnight at 30°C. The bacterial expression culture was harvested by centrifugation at 5,000 × g for 15 min at 4°C. The expression medium was decanted from the pellet. The pellet and the cleaned expression medium were frozen at -20°C. The His mutant protein was purified from the expression medium using Protein A. The purified mutant protein was analyzed by SDS-PAGE and the concentration was obtained by A280.

MaxisorbTM microtiter plates were coated with 1 μg/ml of human TFPI. 100 μl of expression culture medium from each member of the His-scan library was added to two wells on the plate in pairs. The plate was incubated on a shaker for 1 hour at room temperature. The plate was washed three times with PBST. PBS (pH 7.0) was added to one of a pair of wells, and 100 mM pH 6.0 citrate buffer was added to the other of the same pair of wells. The plate was incubated on a shaker for 1 hour at 37°C. The plate was washed three times with PBST and developed with amplex red. A pH 7.0/pH 6.0 ratio was established to arrange sensitive mutant proteins. The ratio of wild-type 2A8-g200Fab was 1.0. 10 clones with a ratio greater than 1.78 between pH 7.0 and pH 6.0 are shown in Table 2 below.

Table 2: pH 6.0 TFPI dissociation ELISA

mutation TFPI ELISA ratio Sorting pH 7.0 pH 6.0 pH7.0/pH6.0 wt gA200Fab 5248 5354 0.98 1 VL-Y31H 913 133 6.84 2 VH-Y102H 3310 1079 3.07 3 VH-Y100H 2545 1431 1.78 4 VH-Y32H 3500 2585 1.38 5 VL-F48H 2068 1551 1.33 6 VH-S50H 2159 1637 1.32 7 VL-Y49H 2661 2044 1.3 8 VL-L27H 3422 2637 1.3

9 VL-V45H 2197 1771 1.24 10 VL-W90H 1833 1509 1.21

The purified 2A8-g200 variants of the Fab form (referring to the wt gA200 Fab in Table 1) were tested using surface plasmon resonance (Biacore). Surface plasmon resonance (Biacore T200) was used to measure the dissociation rate of the antibody. Human TFPI (American Diagnostica) was amine coupled to a CM4 or CM5 chip using the method suggested by Biacore, resulting in 100 to 300 RU of immobilized TFPI. The purified 2A8-g200 variants were injected and then dissociated for 40 minutes at pH 7.4 or pH 6.0 buffer. The antibody was diluted in HBS-P buffer at different concentrations and the flow rate was set at 50 μl/minute. After each round of antibody injection, the chip was regenerated by injecting 90 μl of glycine at pH 1.5. The data set was evaluated using BIAevaluation software.

The dissociation constant (kd) for each 2A8-g200 variant antibody was determined using a model with the following formula:

R＝R ₀ e ^{−kd(t−t0)} +offset

Where R is the response at time t, _R0 is the response at time _t0 (dissociation start), and offset allows for the residual response at the end of dissociation. The ratio of kd at pH 6.0 to kd at pH 7.4 was calculated for each 2A8-g200 variant. Mutations observed with a ratio of 2 were considered pH-sensitive mutations and can be used to construct IgG variants of 2A8-g200.

For example, referring to FIG3 , shown are changes observed in the dissociation constant response at two different pHs (pH 6.0 and pH 7.4) in a biocore assay for two exemplary 2A8-g100 light chain histidine substitution mutations: A.L-L27H and B.L-Y31H.

The pharmacokinetic parameters of the antibodies were determined after administration to HemA mice at 2 mg/kg intravenous (i.v.) bolus. All pharmacokinetic parameters were calculated using a non-segmented model using WinNonLin software version 5.3.1 (Pharsight Corporation, Mountain View, CA). The effect of the histidine mutations on the half-life of the anti-TFPI monoclonal antibodies observed in mouse plasma is shown in Figure 4. 2A8-g200 with histidine mutations TPP2256 (L-Y31H/Y49H) and TPP2259 (L-Y31H) increased the observed pK profiles over a time span of 500 hours compared to the pK profiles of the corresponding 2A8-g200 without any histidine substitutions. Table 3 quantifies the increased half-lives observed in the data of Figure 4.

Table 3: pK parameters

Antibody dose T1/2 (mg/kg) (hr)

2A8-g200 2 71 TTP-2256 2 ~331 TTP-2259 2 ~437

Thus, the above-specified antibodies that reduce TFPI-mediated TMDD and have prolonged T1/2 would result in less frequent dosing and a reduced amount of material required per dose. Furthermore, the low dose requirement also makes subcutaneous administration feasible where the volume of administration is the limiting step (the process of removing the antibody from the circulation by treatment or sequestration from the plasma due to its interaction with rapidly cleared targets, or due to its co-localization with its target targets).

There will be various modifications, improvements, and applications of the disclosed invention that will be apparent to those skilled in the art, and this application includes those embodiments to the extent permitted by law. Although the invention has been described in the context of certain preferred embodiments, the full scope of the invention is not limited thereto, but rather is based on the scope of the following claims. All references, patents, or other publications are expressly incorporated herein by reference.

Claims (2)

1. An isolated human monoclonal IgG antibody, wherein the antibody is a histidine-substituted monoclonal antibody comprising the human heavy chain of SEQ ID NO: 10 and the human light chain of SEQ ID NO: 17, and wherein the histidine substitution is VL-Y31H or VL-Y31H/Y49H in the light chain. 2. A therapeutic composition comprising the isolated human monoclonal IgG antibody of claim 1.