WO2011106272A1 - Novel binding assays useful in identifying antibodies with altered half-lives - Google Patents

Abstract

The present invention relates to methods of identifying target antibodies with desired characteristics. Preferably, the antibody identified according to the methods of the invention exhibit a longer in vivo half life relative to other antibodies with similar or identical Fc region amino acid sequence.

Description

NOVEL BINDING ASSAYS USEFUL IN IDENTIFYING ANTIBODIES WITH

ALTERED HALF-LIVES

This Application claims the benefit of U.S. Provisional Patent Application No. 61/307,182; filed February 23, 2010; which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the identification of target antibodies exhibiting desired in vivo characteristics useful in the prevention and treatment of various diseases. Preferably, the present invention relates to the identification of biomolecules exhibiting improved in vivo half- lives, A feature of the invention relies on the discovery that while antibodies with identical or substantially identical Fc region amino acid sequences were considered to exhibit substantially similar in vivo half lives, such is not always the case. The inventors have discovered a novel way of identifying target antibodies exhibiting longer half-lives using assays disclosed herein notwithstanding the fact that antibodies from within the pool have identical or substantially identical Fc region amino acid sequences. This is contrary to the teachings of the art.

Identifying therapeutic and diagnostic IgGs and other bioactive molecules with increasing half- life of using methods of the invention has many benefits including reducing the amount and/or frequency of dosing of these molecules, for example, in vaccines, passive immunotherapy and other therapeutic and prophylactic methods.

BACKGROUND OF THE INVENTION

Recent years have seen increasing promises of using antibodies as diagnostic and therapeutic agents for various disorders and diseases. Therapeutic monoclonal antibodies (mAbs) have proven to be a promising approach to treat many human diseases. There are currently at least 25 mAbs approved for therapeutic use, all belonging to the IgG family (1, 2). The importance of antibodies in general for diagnostic, research and therapeutic purposes is reflected in the significant amount of effort that has been expended to study, and to modify antibody sequences and structures, from those found in natural antibodies, to achieve desired characteristics. Such attempts are well established in the art. See, for example, U.S. Pat. Nos. 6,165,745; 5,854,027; WO 95/14779; WO 99/25378; Chamow et at, J. Immunol. (1994), 153:4268-4280; Merchant et al, Nature Biotech. (1998), 16:677-681 ; Adlersberg, Ric. Clin, Lab, (1976), 6(3): 191-205. Modifications of antibody sequences, for example those of the framework, are common.

There are five types of immunoglobulins in humans. These groups are known as IgG,

IgM, igD, IgA, and IgE, and are distinguished based on the isotypes of the heavy chain gene (γ, μ, δ, a, and ε respectively). The most common isotype is IgG, and is composed of two identical heavy chain polypeptides and two identical light chain polypeptides. The two heavy chains are covalently linked to each other by disulfide bonds and each light chain is linked to a heavy chain by a disulfide bond. Each heavy chain contains approximately 445 amino acid residues, and each light chain contains approximately 215 amino acid residues.

Each heavy chain contains four distinct domains that are generally referred to as variable domain (VH), constant heavy domain 1 (CHI), constant heavy domain 2 (CH2), and constant heavy domain 3 (CH3). The CHI and CH2 domains are joined by a hinge region (inter- domain sections) that provides the Ig with flexibility. Each light chain contains two distinct domains that are generally referred to as the variable light (VL) and the constant light (CL).

The variable regions of the heavy and light chains directly bind antigen and are responsible for the diversity and specificity of Igs. Each VL and VH has three complementarily- determining regions (CDRs, also known as hyper variable regions). When the VL and VH come together through interactions of the heavy and light chain, the CDRs form a binding surface that contacts the antigen.

Each IgG molecule has an Fc domain and two antigen-binding Fab domains. While the variable regions are involved in antigen binding, the heavy chain constant domains, primarily CH2 and CH3, are involved in non-antigen binding functions. This region, generally known as the Fc region, has many important functions. For example, ADCC and phagocytosis are mediated through interaction of cell-bound monoclonal antibodies with Fc gamma receptors (FcyR), CDC by interaction of cell-bound mAbs with the series of soluble blood proteins that constitute the complement system (e.g., Clq), and for half-life by binding to the neonatal Fc receptor (FcRn). See Presta, Current Pharmaceutical Biotechnology (2002), 237-256. Proper glycosylation of the Fc region of a monoclonal antibody (such as IgG) is thought to be important in conferring wild type effector functions. See, for e.g., Jefferis & Lund, Immunol. Lett. (2002), 82(l-2):57-65; Lisowska, Cell. Mol Life. Sci. (2002), 59(3);445-455; Radaev & Sun, Mol. Immunol. (2002), 38(14): 1073-1083; Mimura et al, Adv. Exp. Med. Biol. (2001), 495:49-53; Rudd et al., Science (2001), 291 (5512):2370-2376; Jefferis et al., Immunol. Rev. (1998), 163:59- 76; Wright & Morrison, Trends Biotechnol. (1997), 15(l):26-32; Jefferis & Lund, Chem.

Immunol. (1997), 65:1 1 1-128. Human IgGs have an average half life of 21 days in healthy human subjects, which is longer than any other known serum protein (3).

One of the key components controlling mAb pharmacokinetics is the neonatal Fc receptor (FcRn). A mechanism for IgG catabolism was proposed by Brambell's group (Brambell et al, Nature, 203: 1352 1355, 1964; Brambell, Lancet, ii: 1087 1093, 1966). They proposed that a proportion of IgG molecules in the circulation are bound by certain cellular receptors (i. e., FcRn), which are saturable, whereby the IgGs are protected from degradation and eventually recycled into the circulation; on the other hand, IgGs which are not bound by the receptors are degraded. The proposed mechanism was consistent with the IgG catabolism observed in hypergammaglobulinemic or hypogammaglobulinemia patients. Furthermore, based on his studies as well as others (see, e.g., Spiegelberg et al., J. Exp. Med,, 121 :323 338, 1965; Edelman et al., Proc. Natl. Acad. Sci. USA, 63:78 85, 1969), Brambell also suggested that the mechanisms involved in matemofetal transfer of IgG and catabolism of IgG may be either the same or, at least, very closely related (Brambell, Lancet, ii: 1087 1093, 1966). Indeed, it was later reported that a mutation in the Fc-hinge fragment caused concomitant changes in catabolism, matemofetal transfer, neonatal transcytosis, and, particularly, binding to FcRn (Ghetie et al, Immunology Today, 18(12):592 598, 1997).

These observations suggested that portions of the IgG constant domain control IgG metabolism, including the rate of IgG degradation in the serum through interactions with FcRn. Indeed, increased binding affinity for FcRn increased the serum half-life of the molecule (Kim et al, Eur. J. Immunol, 24:2429 2434, 1994; Popov et al, Mol. Immunol., 33:493 502, 1996;

Ghetie et al, Eur. J, Immunol., 26:690 696, 1996; Junghans et at, Proc. Natl. Acad. Sci. USA, 93:5512 5516, 1996; Israel et al, Immunol., 89:573 578, 1996).

FcRn acts as a salvage receptor to protect IgGs from lysosomal degradation. The impact of this pathway on mAb pharmacokinetics is well established. FcRn-binding properties are thus an important part of mAb characterization. FcRn protein is a heterodimer consisting of two polypeptides: 50kD class I major histocompatibility complex (MHCl)-like protein ( -FcRn) and 15kD β2 microglobulin ((β₂ιη) (4). Crystal structure has shown that FcRn binds to the Fc domain of IgG at the Cy2-Cy3 3 interface (6-7). It has been hypothesized that IgGs are subject to fluid-phase pinocytosis by many types of cells in the body (4-5). The pinocytosed IgGs are subsequently transported to the acidic endosomes where they encounter FcRn. The interaction between FcRn and IgG is strictly pH-dependent (4, 6). FcRn binds to IgG with nanomolar affinities at slightly acidic endosomal pH (pH 5-6) and salvages IgG from lysosomal

degradation. FcRn has no detectable binding to IgG at neutral pH, resulting in release of IgG upon encountering the extracellular milieu. The pH-dependent IgG binding is the basis for all functions of FcRn (4-6). Fc mutants with either significantly reduced affinity at acidic pH, or significantly increased FcRn binding at neutral pH, have very short half life in vivo (8-9).

Importantly, Fc mutants with improved FcRn binding properties also show longer half life in vivo (10-12).

The use of immunoglobulins as therapeutic agents has increased dramatically in recent years and has expanded to different areas of medical treatments. Such uses include treatment of agammaglobulinemia and hypogammaglobulinemia, as immunosuppressive agents for treating autoimmune diseases and graft- vs.-host (GVH) diseases, the treatment of lymphoid malignancies, and passive immunotherapies for the treatment of various systemic and infectious diseases. Also, immunoglobulins are useful as in vivo diagnostic tools, for example, in diagnostic imaging procedures.

In general, the art recognizes that certain residues in the constant region of IgG perform critical roles in conferring biochemical and functional characteristics associated with antibodies, and therefore modifications of these residues must be made with care, if at all. One critical issue in these therapies is the persistence of immunoglobulins in the circulation. The rate of immunoglobulin clearance directly affects the amount and frequency of dosage of the immunoglobulin. Increased dosage and frequency of dosage may cause adverse effects in the patient and also increase medical costs.

Modulation of IgG molecules by amino acid substitution, addition, or deletion to increase or reduce affinity for FcR-n is also disclosed in WO 98/23289; however, the publication does not list any specific mutants that exhibit either longer or shorter in vivo half-lives.

Since FcRn plays an important role in extending IgG half life, one would expect that in vitro FcRn binding properties may be indicative of its in vivo pharmacokinetics. Such in vitro tools would be highly valuable for mAb lead design and optimization during drug discovery process. There are conflicting reports, however, on whether in vitro FcRn binding can be used to predict IgG pharmacokinetics in vivo. Initial studies clearly showed that Fc mutants with either significantly reduced affinity at acidic H, or significantly increased FcRn binding at neutral pH, have very short half life in vivo (8-9), Importantly, Fc mutants with improved FcRn binding properties also show longer half life in vivo (10-12). However, there have also been reports of Fc mutants with improved FcRn binding properties that failed to show any half life improvement in vivo (13-14)

In view of the importance of antibodies with increased half-lives or in certain aspects with reduced half-life's and their role in modulating human disease, there is a need in the art for identification of target antibodies that exhibit the desired characteristics, preferably those that have a longer half-life and are therapeutically effective. However, these previous works focused on mAbs with different Fc sequences, mAbs sharing a similar or identical Fc region amino acid sequences are widely believed to bind FcRn similarly and exhibit substantially similar in vivo half lives. Towards this end, the present invention provides methods of identifying antibodies that exhibit improved or longer half-life in vivo in spite of the fact that such antibodies shared a similar or identical Fc region amino acid sequences with other antibodies that surprisingly did not exhibit the desired longer half-life as would be predicted by the prior art.

Despite widespread efforts, there remains a significant and serious need for improved methods based on using antibodies that are capable of exerting the desirable biological effects, yet exhibit reduced undesirable effector function-associated side effects. The invention described herein addresses this need and provides other benefits.

SUMMARY OF THE INVENTION

Embodiments of this invention are made available by the inventors' identification of a surprising discovery relative to antibodies having a similar or identical Fc region amino acid sequence. The art has long assumed that antibodies with identical Fc sequences bind FcRn similarly. A surprising feature of the invention relies on the discovery that mAbs with wild-type human Fc sequences can interact with FcRn with considerable differences in both binding at acidic pH and dissociation at neutral pH, indicating that the Fab domain may also impact the FcRn interaction. Consistent with these observations, the inventors characterized a group of therapeutic mAbs with identical wild type human Fc sequences and different Fab domains for their ability to bind human or human-like FcRn at pH 6,0 and to dissociate at pH 7.3, and observed an apparent correlation between dissociation at neutral pH and in vivo

pharmacokinetics (PK): mAbs with more slow dissociation fractions tended to show shorter terminal half life (tl/2). To confirm whether the observed in vitro FcRn binding differences had any in vivo implication, the inventors further examined the relationship between in vitro FcRn binding and in vivo pharmacokinetics in both human FcRn mice, non-human primates and humans. Detailed herein is data supporting the hypothesis that IgGs with identical or substantially similar Fc sequences exhibit differences in FcRn-binding. Examples below further show the potential correlations between FcRn binding parameters and in vivo PIC.

Thus, the present invention relates to methods of identifying biomolecules (preferably a protein, more preferable an antibody or a fragment thereof) that has an increased in vivo half- life. In certain embodiments, the methods of the invention propose identifying target antibodies that exhibit longer half-lives relative to antibodies having a similar or identical Fc region amino acid sequence. Such preferred target antibodies identified by the methods of the invention exhibit a distinct neutral pH dissociation kinetics for FcRn.

The present invention relates to methods for evaluating the in vivo half life of antibodies and antigen-binding fragments thereof as well as identifying antibodies with a desired in vivo half life. The method includes performing an antibody/FcRn binding assay and deteniiining the %bound value of the dissociation or the k2/B value of the biexponentiai function that characterizes this event and, on the basis of one or more of these values, evaluating the in vivo half-life of the antibody or antigen-binding fragment thereof. For example, antibodies that are designated as having a "%bound value" that is higher than the high %bound of a control antibody and those that fall in-between the high and low %bound control antibodies are discarded, while those that exhibit a %bound value that is comparable to or lower than that of the low %bound control antibody are considered/identified as target antibodies having the desired characteristics of potential increased or longer half life in vivo.

The present invention provides in part a method for evaluating the in vivo half life of a target antibody comprising (i) separately determining a slow dissociation phase

biexponentiai decline function of target antibody or second antibody binding to FcRn under conditions favoring dissociation of said antibody and FcRn following an initial fast dissociation phase of said antibody and FcRn, using surface plasmon resonance, which function is described by: RU_t = A e ^kl '+ B e^{"fc t} + C, wherein t is time, RUt is surface plasmon resonance response units at time t, A and B are initial values at time zero for the two, faster and slower, dissociation phases respectively; kl and k2, are the apparent first order rate constants for the faster and slower dissociation phases, respectively; and C is the surface plasmon resonance response units at end of dissociation.; and (ii) comparing the value k2/B for the target antibody and the second antibody; wherein the target antibody is determined to have an increased or longer in vivo half life than the second antibody if the k2/B for the target antibody is larger than the k2/B for the second antibody; wherein the target antibody is determined to have a decreased or shorter in vivo half life than the second antibody if the k2/B for the target antibody is smaller than the k2 B for the second antibody; and/or wherein the target antibody is determined to have a similar in vivo half life to the second antibody if the k2/B for the target antibody similar to the k2/B for the second antibody. Though this method makes use of SPR to determine binding between FcRn and antibody or antigen-binding fragment thereof, this method can be adapted to any of several other binding formats. For example, SPR response units at time=t can be replaced with a value that reflects the extent or quantity of antibody/FcRn binding at t; or C can be used to express the extent or quantity of antibody/FcRn binding at the end of dissociation. For example, SPR response units can be replaced with enzyme linked immunosorbent assay (ELISA) units such as color change of the reaction solution at OD₄50nm.

The present invention includes an embodiment of the invention which is a method for evaluating the in vivo half life of a target antibody or antigen-binding fragment thereof (antibody) comprising (i) separately determining a slow dissociation phase biexponential decline function of target antibody or second antibody or antigen-binding fragment thereof (antibody) binding to FcRn under conditions favoring dissociation of said antibody and FcRn following an initial fast dissociation phase of said antibody and FcRn, which function is described by: Q_t = A e ^{k! 1} + B Q ^{k 1} + C, wherein t is time, Qt is quantity of antibody bound to FcRn at time t, A and B are initial values at time zero for the two, faster and slower, dissociation phases respectively; kl and k2, are the apparent first order rate constants for the faster and slower dissociation phases, respectively; and C is the surface plasmon resonance response units at end of dissociation; and (ii) comparing the value k2/B for the target antibody and the second antibody; wherein the target antibody is determined to have an increased or longer in vivo half life than the second antibody if the k2/B for the target antibody is larger than the k2/B for the second antibody; wherein the target antibody is determined to have a decreased or shorter in vivo half life than the second antibody if the k2/B for the target antibody is smaller than the k2/B for the second antibody; and/or wherein the target antibody is determined to have a similar in vivo half life to the second antibody if the k2/B for the target antibody similar to the k2/B for the second antibody.

"Dissociation constant" is a type of equilibrium constant that specifically involves the measure of the propensity of dissociation of a complex molecule into its subcomponents. An example of its application is to describe how tightly a ligand binds to a particular protein, e.g., an antibody binds an antigen.

The binding ability of IgGs and molecules comprising an IgG constant domain of Fc fragment thereof to FcRn can be characterized by various in vitro assays. PCT publication WO 97/34631 by Ward discloses various methods in detail and is incorporated herein in its entirety by reference.

In general, the term " assoc" or "Ka", is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" or "Kd," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

The term "K_D" "K , is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of ¾ to K_a (i.e. ¾/Κ_β) and is expressed as a molar concentration (M). ¾ values for antibodies can be determined using methods well established in the art. One of the methods for determining the _D of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore.™ system.

The term "K._0ff as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

The term "%bound" antibody effectively describes antibodies exhibiting the desired neutral pH FcRn "dissociation pattern" meaning the level of slow dissociation fraction.

As such, the aim of the invention is to identify target antibodies that have less slow dissociation fraction. In other words, those antibodies exhibit little or no "slow dissociation fractions" when they dissociate from FcRn at neutral pH. This "slow dissociation fraction" can be measured in numerous ways by one skilled in the art, two of which are described herein as the "%bound" method and biexponential model fitting method. In preferred embodiments, the characteristics include a low "slow dissociation fraction" and increased or longer half life.

The term "Affinity" refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody "arm¹¹ interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

As used herein, the term "Avidity" refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valency of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.

The term "surface plasmon resonance" (SPR), as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BlAcore.™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). The interaction between mAbs and FcRn can be measured by SPR measurement using, for example, a BIAcore T100 (GE Healthcare). In this method, FcRn molecules are coupled to a BIAcore sensor chip (e.g., CMS chip) and the binding of mAbs to the immobilized FcRn is measured at a certain flow rate to obtain sensorgrams using BIAevaluation software, To mimic the in vivo IgG-FcRn interaction process, the mAbs were allowed to reach equilibrium binding with FcRn at pH 6.0 and then exposed to pH 7.3 for dissociation. A series of report points during the dissociation phase are recorded and used for analysis of the dissociation process (method described in detail in Example).

Relative affinities of modified IgGs or fragments thereof, and the wild type IgG for FcRn can be also measured by a simple competition binding assay. Unlabeled modified IgG or wild type IgG is added in different amounts to the wells of a 96-well plate in which FcRn is immobilized. A constant amount of radio-labeled wild type IgG is then added to each well. Percent radioactivity of the bound fraction is plotted against the amount of unlabeled modified IgG or wild type IgG and the relative affinity of the modified hinge-Fc can be calculated from the slope of the curve.

Furthermore, affinities of modified IgGs or fragments thereof, and the wild type IgG for FcRn can be also measured by a saturation study and the Scatchard analysis,

The half-life of mAbs can be measure by pharmacokinetic studies in human FcRn mice (Petcova et al, Int Immuno. 18:1759, 2006). According to this method, target antibodies or fragments thereof are injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at 1 hour to 15 days after the injection. The PK profile thus obtained should be biphasic, that is, a-phase and β-phase. For the determination of the in vivo half-life of the mAbs or fragments thereof, the mAb elimination phase terminal half lives are determined with non-compartmental model using data points in β- phase (e.g. between day 3 and day 15 post dose). See example section.

Another broad aspect of the invention provides an antibody identified in accordance with the methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Dissociation of mAbs from immobilized human FcRn at pH 7.3 with

Biacore.

Figure 2. Dissociation curve - using a bi-phasic model to capture the pH 7.3 IgG- FcRn dissociation curve

Figure 3. In vitro-in vivo correlation using % bound. (A) Human FcRn mice. (B) Monkeys.

Figure 4. In vitro/In vivo correlation using Kd/C

Figure 5. Successful application of the FcRn assay in identifying potential target antibodies. Figure 6. (A) Dissociation curves for mAbs with either high or low %bound values with R >0. ,9999 ddeeffiinniinngg 1 biexponential function; (B) analysis of k₂/Q as it relates to in vivo terminal t_\a in humans. DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

The reference works, patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences that are referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art- understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

This section presents a detailed description of the many different aspects and embodiments that are representative of the inventions disclosed herein. This description is by way of several exemplary illustrations, of varying detail and specificity. Other features and advantages of these embodiments are apparent from the additional descriptions provided herein, including the different examples. The provided examples illustrate different components and methodology useful in practicing various embodiments of the invention. The examples are not intended to limit the claimed invention. Based on the present disclosure the ordinary skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a genetic alteration" includes a plurality of such alterations and reference to "a probe" includes reference to one or more probes and equivalents thereof known to those skilled in the art, and so forth.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al.

Molecular Cloning: A Laboratory Manual, 2d ed._s Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The following terms, unless otherwise indicated, shall be understood to have the following meanings: A gene or protein marker is "informative" for a condition, phenotype, genotype or clinical characteristic if the expression of the gene marker is correlated with the condition, phenotype, genotype or clinical characteristic to a greater degree than would be expected by chance.

The term "antibody" is used herein in the broadest sense and covers fully assembled antibodies, antibody fragments which retain the ability to specifically bind to the antigen (e.g., Fab, F(ab')2, Fv, and other fragments), single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, and the like. The term

"antibody" covers both polyclonal and monoclonal antibodies. As well, the term includes an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

As used herein, the term "about" refers to an approximation of a stated value within an acceptable range. Preferably the range is +/-5% of the stated value.

The term "or" is used herein to mean, and is used interchangeably with, the term

"and/or", unless context clearly indicates otherwise.

"Sequence identity" or "identity" in the context of two nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum

correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions.

The term "substantial identity" or "homologous" in their various grammatical forms in the context of polynucleotides generally means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.

A "native sequence Fc region" or "wild type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region commonly found in nature.

The present invention provides various assays for screening antibodies with the desired characteristics. Screening assays may be used to find or confirm such target

polypeptides exhibiting the desired characteristic or property. For example, a pool of antibodies with similar or identical Fc amino acid sequences may be screened to identify target antibodies with altered Fc n binding, e.g., altered "slow dissociation fraction". A variety of assay types may be employed to evaluate or confirm the desired properties attendant the target antibody or antibodies.

In certain embodiments, a target antibody is one being evaluated using a method of the present invention, e.g., one which exhibits improved or increased in vivo half life relative to other antibodies that share a similar or identical Fc region amino acid sequence. In other embodiments, the desired characteristics of the target antibody include a lower "slow

dissociation fraction" at neutral pH (e.g. low %bound). In yet other embodiments, the desired characteristics of the target antibody include a higher "slow dissociation fraction" at neutral pH (e.g. high %bound).

The binding capability of the target antibodies to FcRn may also be determined using techniques such as ELISA, fluorescence activated cell sorting (FACS) analysis or

radioimmunoprecipitation (RIA). For example, binding of Fc receptors such as Fc.yRI,

Fc.yRIIa, Fc.y.RIIb, Fc.y.RHI, FcRn, etc., can be measured by, for example, titrating a variant polypeptide and measuring bound variant polypeptide using an antibody which binds to the variant polypeptide in an ELISA format (see Examples below). For example, a variant that comprises an antibody may be screened in a standard ELISA assay to determine binding to an FcRn at pH 6.0 and pH 7.0 or pH 7.4. A solid surface coated with streptavidin or neutravidin may be used to capture biotin labeled FcRn from any species, such as mouse or human. After blocking, the capture receptor can be incubated with variant polypeptides (e.g., antibodies) diluted in buffers at pH 6.0 or pH 7.0. In the following step a molecule specific for human antibodies is added (e.g., goat (Fab')i anti-human-Fab conjugated to an enzyme). Thereafter a substrate may be added in order to determine the amount of binding of the variant polypeptide to the immobilized FcRn at pH 6.0 or pH 7.0 or pH 7.4. The results of this assay can be compared to the control (having identical or substantially similar Fc region amino acid sequence) polypeptide's ability to bind the same FcR. In other preferred embodiments, the components for carrying out an ELISA (e.g., with FcRn) to screen variants are packaged in a kit (e.g., with instructions for use).

"Variants" as used herein include target antibodies that exhibit either longer or shorter in vivo half lives relative to control antibodies having identical or substantially similar Fc region amino acid sequences.

The binding affinity o an antibody to FcRn and the off-rate of an antibody-FcRn interaction can also be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled FcRn (e.g., .sup.3H or .sup.1251) with the antibody of interest in the presence of increasing amounts of unlabeled FcRn, and the detection of the antibody bound to the labeled FcRn. The affinity of the antibody of the present invention or a fragment thereof for the antigen and the binding off-rates can be determined from the saturation data by Scatchard analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the FcRn is incubated with an antibody of the present invention or a fragment thereof conjugated to a labeled compound (e.g., ³H or ^I25I) in the presence of increasing amounts of an unlabeled second antibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies to FcRn. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antibody from chips with immobilized FcRn on their surface (see the Example section infra).

Methods of making and using the ADDL-specific antibody designated MK-7305 is described in PCT application PCT/US2005/038125, filed October 21, 2005, which published as WO 2006/055178 on May 26, 2006. See also US 2006-0228349.

EXAMPLES

The methods and compositions discussed in this section constitute a part of the present invention.

Example 1: Development of in vitro FcRn binding assay for determination of antibody in vivo half life.

Materials and Methods

Expression and Purification of FcRn Proteins

Soluble human, rhesus macaque and hybrid human-mouse FcRn were engineered to be expressed using the Bac-to-Bac™ baculovirus expression system from Invitrogen (Carlsbad, CA). Briefly, the cDNAs of the a-subunit (heavy subunit) were truncated to include only the leader peptide and extracellular domains (codons 1 -290) to generate the soluble forms of FcRn (15). To facilitate purification, a His6 tag was added to the C-terminus of each α-subunit. The corresponding full-length β chain (β2ηα) cDN A was co-expressed using a separate baculovirus expression vector. Viruses containing FcRn a- and β-subunits (human a + β, rhesus a + β, or human a + mouse β) were co-transfected and expressed in Sf 9 cells for 72 hours following which the FcRn-containing culture media was collected by centrifugation. The media was neutralized to pH 7.5 using I M Tris pH 8.0 and then filtered using 0.22 μΜ filterware. The heterodimeric FcRn proteins were isolated from media using a QIAGEN Ni-NTA Superfiow (Valencia, CA) with a BioRad Econo-column (Hercules, CA) according to the manufacturer's recommendations. Purified FcRn proteins were filtered and concentrated using a Millipore Amicon-Ultra Centrifugal filter unit (Billerica, MA). SDS-PAGE was performed to confirm presence of the truncated a chain and β2πι. The FcRn proteins were > 99% pure based on SDS- PAGE (data not shown). Purified FcRn proteins were dialyzed into 6 mM sodium phosphate, 100 mM NaCl, pH 7.4 and 0.05% surfactant P-20, dispensed into single-use aliquots and stored at -70°C.

Monoclonal Antibodies

The monoclonal antibodies used to study the relationship between in vitro FcRn binding and in vivo pharmacokinetics were obtained either from Merck's internal programs or commercial sources (adalimumab, basiliximab, bevacizumab, cetuximab, omalizumab, palivizumab and trastuzuma, obtained from yoderm, Norristown, PA).

Surface Plasmon Resonance (SPR) Assay

The interaction between human IgG (mAbs) and FcRn was measured by SPR using a Biacore T-100 instrument (GE Healthcare Biosciences, Piscataway, NJ), Purified FcRn protein was immobilized onto a Biacore CMS biosensor chip via amine coupling to reach a density of -200 response units (RU). The kinetics experiment was conducted at 25 °C using a pH 6.0 running buffer (50 mM NaP0₄, 150 mM NaCl and 0.05% (v/v) Surfactant 20) with a flow rate of 30 μΐ/min. The mAbs were diluted with the pH 6.0 running buffer to 25, 50, and 100 nM respectively, allowed to bind FcRn for 3 min to reach equilibrium and followed by 2 min of dissociation. Two 30-second pulses of pH 7.5 running buffer were used to regenerate the chip and the return of sensorgram to baseline was verified before next run. To determine FcRn binding affinity (dissociation constant, KD) at pH 6.0, the data from all three concentrations was used simultaneously to fit a two-state reaction model found in the Biacore T-l 00 Evaluation software.

The neutral pH dissociation experiment was conducted similarly except using a pH 7.3 running buffer. The mAbs were diluted to 100 nM using pH 6.0 running buffer to allow binding and then quickly exposed to pH 7.3 running buffer for dissociation. Use of Biacore T- 100 instrument (kinetics injection) and impeccable maintenance of instrument are critical for the neutral pH dissociation assay. A series of report points during the dissociation phase were recorded. In particular, one report point (Binding) was inserted at 2 seconds before the pH 7.3 dissociation phase begins and another report point (Stability) was inserted at 5 seconds into the dissociation phase, and "%bound" was calculated as RUs_ta_bmty U Binding (%)· In addition, the whole dissociation process was captured by a series of reporting points from 2 to 110 seconds following dissociation. The resulting dissociation curve was found to be best described by a biexponential decline function: RU_t = A e^{"fa t} + B e^{"fo t} + C, where t is time post dissociation, RU_t is RU at time t, A and B are initial values at time zero for the two (faster and slower) dissociation phases, kj and A¾ are the apparent first order rate constants for the faster and slower dissociation phases, and C is the RU at end of dissociation.

Both Vobound and the biexponential model fitted parameters were used to establish potential correlations with in vivo PK

When applying this method to identify mAbs with longer terminal half life (tl/2) in vivo, the inventors also included 2 control mAbs in every run (an anti-ILl3R antibody characterized by a point mutation that effectively negates its ability to bind the receptor (L- 002082824) as a low "%bound" control, and an anti-ADDL antibody (MK-7305) as a high "%bound" control), and selected mAbs with "% bound" values lower than that of the high control, and preferably similar to or lower than that of the low control. Pharmacokinetics study in human FcRn mice

The heterozygous Tg276 human FcRn mice used in this study were obtained from Jackson Laboratory (Bar Harbor, ME). They are deficient in mouse FcRn-α chain and carry a human FcRn-α chain gene (16). For pharmacokinetics studies, each animal (3-4/group) received a single intravenous injection of mAb at 10 mg/kg via tail vein. A series of 10 μL· of blood was collected at specified time points (every 24h until day 4 and every 2-3d until day 15) via tail vein with a positive displacement pipette and the blood was mixed with 1 of 55 mM EDTA immediately. The mixture was then diluted with 90 μL of PBS and centrifuged. The resulting diluted plasma was subsequently used for immunoassays to quantify mAb levels. All studies were approved by the Institutional Animal Care and Use Committee (IACUC).

An anti-human IgG immunoassay with GyroLab was used to determine all mAb levels. Briefly, a biotinylated mouse anti-human kappa or lambda chain monoclonal antibody (BD Pharmingen) was used for capture and an ALEXA-647 labeled mouse monoclonal antibody specific for human Fc domain (Southern Biotech) was used for detection. The mAb

concentrations were determined from standard curves, The lower limit of quantitation (LLOQ) was 100 ng mL. The entire immunoassay was automated using the Gyros® Bioaffy workstation and Bioaffy 200 compact disks (Gyros, Uppsala, Sweden).

WinNonlin (Enterprise Version 5.01, Pharsight Corp, Mountain View, CA) was used for pharmacokinetics analysis. The mAb elimination phase terminal half lives were determined with non-compartmental model using data points from the terminal phase, usually between day 3 and day 15 post dose. These data points generally fitted well to a mono-exponential decay function.

The pharmacokinetics data in monkeys and humans were obtained from either Freedom of Information (FOI) from FDA/EMEA or internally.. Results

Characterization of the pH 7.3 dissociation between mAb and FcRn

The rapid dissociation of IgG from FcRn at neutral pH is essential for its in vivo half life. The inventor's developed a Surface Plasmon Resonance (SPR) assay using BIAcore to compare the ability of mAbs to dissociate from FcRn at neutral pH. To mimic the in vivo IgG- FcRn interaction process (4), the mAbs were allowed to reach equilibrium binding with FcRn in PBSP, pH 6.0 and then exposed to PBSP, pH 7.3 for dissociation. Under these conditions, all tested mAbs had a very fast initial dissociation phase. However, following the initial rapid dissociation, different mAbs were found to have various amounts of "slow dissociation" fractions (Fig 1).

That the observed "slow dissociation" fraction of a mAb was not due to aggregation was confirmed by the inventors when they examined the aggregation status of mAbs used in the study by size-exclusion chromatography. The inventors observed that all the mAbs had greater than 97% monomer contents and thus no relationship between "%bound" and the trace amounts of aggregation could be found. The amount of "slow dissociation" fraction did not correlate with a mAb's equilibrium binding level at pH 6.0 either.

The difference in "slow dissociation" fraction of a mAb can be quantified with a simple "%bound" parameter: "%bound" = RUStability/RUBinding (%), where "stability" is a report point inserted at 5 seconds after the end of mAb binding (Fig. 1). The relative difference in "%bound" among mAbs were highly reproducible. However, due to the extremely fast initial dissociation, the absolute "%bound" values were not necessarily comparable from run to run. As shown in Fig 2A, the whole pH 7.3 IgG-FcRn dissociation curve could be captured by a biphasic model consisted of an initial fast phase (linear) followed by a slow phase (biexponential). The biexponential function could describe the slow phase well for mAbs with high and low

"%bound" values (Fig. 2B).

The neutral pH dissociation of mAbs from FcRn was measured by BIAcore. Briefly, purified FcRn protein was immobilized onto a BIAcore CM5 biosensor chip. The mAbs were diluted with PBSP (50 mM NaP04, 150 raM NaCl and 0.05% (v/v) Surfactant 20), pH 6.0 to 100 nM, allowed to bind FcRn for 3 min to reach equilibrium and followed by 2 min of dissociation in PBSP, pH 7.3 running buffer. A report point called "Stability" was inserted at 5 seconds after the dissociation phase began and "%bound" was calculated as

RUStability/RUBinding (%).

That the mAbs with higher "%bound" tended to exhibit shorter terminal half life

(tl/2) in vivo is also shown. When using this method to identify mAbs with longer terminal half life (tl/2) in vivo, the assay may include using 2 control mAbs in every run (L-002082824 as a low "%bound" control, and M -7305 as a high "%bound" control), and selecting mAbs with "% bound" values lower than that of the high control, and preferably similar to or lower than that of the low control.

Correlation of in vitro FcRn Undine and in vivo PK

In order to assess the relationship between the observed in vitro FcRn

binding/dissociation differences and in vivo pharmacokinetics of the mAbs, the inventors chose a group of Merck mAbs with IgGl and IgG2 backbones (both IgGl s and IgG2s, all have identical human Fc sequences within subtypes) together with readily-available commercial IgGl mAbs (adalimumab, basiliximab, bevacizumab, cetuximab, omalizumab, palivizumab and trastuzumab, all with wild type human IgGl Fc sequences) for further study. Since the interactions between IgG and FcRn are species-specific and mouse FcRn binds to human IgG with considerable differences (16, 18), the inventors used human FcRn mice as a surrogate system to investigated the impact of pH 7.3 FcRn dissociation on in vivo pharmacokinetics (16). The so-called human FcRn mice actually had a "hybrid" FcRn (human α/mouse β). This hybrid FcRn was expressed from a baculo virus system and showed that the "hybrid" FcRn have comparable human IgG binding characteristics as that of human and monkey FcRn. To minimize the impact of target-mediated clearance, a 10 mg/kg dose was used because it was unknown whether the mAbs used in the study would bind a mouse ligand of very low abundance. As shown in Fig 3 A, these mAbs exhibited a range of terminal half lives in human FcRn mice, despite their wild type human Fc sequences and the fact that no major impact of target-mediated clearance was observed. Importantly, when the %bound hybrid FcRn was plotted against terminal half life in human FcRn mice, an apparent correlation between higher "%bound" and shorter terminal half life was observed (Fig 3 A). The trend was similar for mAbs in either IgGl or IgG2 backbones.

To further determine whether the observed pH 7.3 FcRn dissociation differences had any implication for in vivo PK, the inventors collected all available non-human primate terminal half life data at a saturating dose (so no major impact of target mediated clearance on mAb PK was expected) for the mAbs used in the above human FcRn mice study (Table I). The data was from either rhesus or cynomolgus monkeys. Notwithstanding the fact that there is a single amino acid difference between rhesus and cynomolgus monkey FcRn, the inventors were unable to find any difference between the purified rhesus and cynomolgus FcRn for its ability to bind human IgG. As a consequence, rhesus FcRn was used as the representative monkey FcRn.

When the monkey in vitro FcRn binding properties and in vivo pharmacokinetics were compared, a similar trend was observed between dissociation at neutral pH and in vivo PK: mAbs with higher %bound when dissociated from monkey FcRn tend to have shorter terminal tl/2 in vivo (Fig 3B).

Table I. Summary of in vitro hybrid/monkey FcRn binding parameters and in vivo half lives

MK-2444 IgG2 9.9% 44 8.1% 48

K-4721 IgGl 3.1% NA* 2.1% 271

M -6105 IgGl 3.1% 99 3.2% 140

MK-7305 IgG l 12.9% 44 12.1% 56

adaliraumab IgGl 7.9% 86 9.8% NA

basiliximab IgGl 3.7% 120 4.6% 132

bevacizumab IgGl 11.6% 76 10.9% 210

ceruximab IgGl 3.2% 66 0.3% NA

trastuzumab IgG l 6.9% 81 6.6% 189

*: MK-0646 and MK-4721 exhibited aberrantly high clearance in human FcRn mice and their t_m cannot be determined

As shown in Fig 2, the pH 7.3 dissociation curve of mAbs can be described as a linear fast phase followed by a biexponential slow phase. The dissociation curves of mAbs differ mostly in the slow phase. That biexponential model derived parameters can lead to better in vitro-in vivo correlation is detailed in Table II. For this demonstration, the inventors collected all available in vivo half life data in humans, human FcRn mice and monkeys of 11 mAbs (Table II). Their dissociation from human FcRn at pH 7.3 was measured as described previously and the slow phases were each fitted with a biexponential function. Model derived parameters, either alone or in combinations, were evaluated for potential correlation with in vivo PK. The inventors identified Kd/C as the most promising combination to predict in vivo PK. "Kd/C" may also be referred to herein as "k2/B". The results are shown in Fig 4, a correlation between Kd/C and in vivo half-life from all three species was observed and the correlation coefficients (R2) were 0.75, 0.63 and 0.84 in humans, human FcRn mice and monkeys respectively.

Table II, Summary of in vitro human FcRn binding parameters and in vivo half lives

MK-7305 0.42 19.1% ND 44 56

1A09 0.18 28.8% ND 24 ND

L-824 0.48 8.8% ND 93 ND

*: palivizumab had no measurable slow phase from this particular run.

To better define the human in vitro-in vivo correlation, we used a model fitting for more accurate quantification of the differences in pH 7.3 FcRn dissociation for mAbs. All human IgGs we examined exhibited extremely fast dissociation from FcRn following exposure to pH 7.3 buffer. This initial phase (0-2 sec) can only be described by a linear function and is similar between different mAbs. A more pronounced difference in the dissociation process between mAbs can be seen following the initial linear phase, and the subsequent dissociation curve (2-110 sec) could be described by a biexponential decay function: RU_t = A€^kl ' + B e^{"t2 t} + C, where A and kj described the earlier "faster dissociation phase", B and ¾ described the later "slower dissociation phase". The biexponential function could describe the dissociation curves for mAbs with either high or low %bound values with R² >0.99 (Fig 6A), i.e. all mAbs have both fast and slow dissociation phases, and they only differ in parameters describing these two phases.

We next investigated whether the biexponential model derived parameters, either alone or in combinations, could lead to a better in vitro-in vivo correlation in humans. Following extensive evaluation, A B was identified as the most promising combination. Since is the apparent first order rate constants for the slower dissociation phase and B is the intercept of the slower dissociation phase at time zero, either lower A¾ or higher B will lead to higher "%bound". Apparently, lower k₂ or higher B will also lead to lower ¾ B. Therefore AyB should be inversely related to "%bound". But unlike %bound, fo/B was derived from the whole dissociation curve, so it can better describe the neutral pH dissociation property. In fact, we did find the correlation trend between in vitro human FcRn binding dissociation and human terminal ti_/2 significantly more reproducible using A B than using %bound. When a group of mAbs with relatively big difference in %bound, such as those in hFcRn mice and NHP studies, were evaluated, the advantage of k₂/B over %bound was not as apparent (data not shown). A correlation between A B and in vivo terminal ti ₂ in human was shown in Fig 6B, The results suggested that the in vitro-in vivo correlation we observed in human FcRn mice and monkeys is also true in humans.

Discussion

The role of FcRn in extending the half life of IgG has been well established (4, 5). However, the present inventors have demonstrated that mAbs with identical Fc sequences can interact with FcRn differently. The steady state location of FcRn is endosomal, where it binds to IgG with high affinity and protects it from lysosomal degradation. The FcRn-bound IgGs are recycled to the plasma membrane and released upon exposure to neutral pH (4). This model of FcRn/IgG recycling provides an explanation as to why mAbs with slower dissociation from FcRn at neutral pH may have shorter in vivo half life. The present finding is also consistent with previous observations that the lack of binding at neutral pH is essential, if not more important than binding at pH 6.0, for IgGs to be salvaged by FcRn (8, 13). Nevertheless, it was an unexpected finding because it had been widely assumed that IgGs with wild-type Fc sequences would interact with FcRn similarly. In fact, human IgGs of the same subclass from different commercial sources had been reported to have different affinity to FcRn, but the difference was attributed to allotypic variations within a single subclass of human IgG (17). In this study, the inventors systematically examined the relationship between in vitro FcRn-binding and in vivo pharmacokinetics with a group of mAbs with identical Fc sequences and the data supports the hypothesis that the Fab domain may also impact FcRn interaction.

Regardless of the mechanism of how the Fab domain might impact FcRn binding, it is highly valuable to have an in vitro system that can be indicative of in vivo pharmacokinetic behavior of therapeutic mAbs. Modulating pharmacokinetics is an important aspect of therapeutic mAb development. Therapeutic mAbs generally are administered parenterally (2). The ability to develop a mAb with extended half life and thus less frequent dosing is often crucial for the success of a product. In addition, some types of therapeutic mAbs, i.e. those used for immunotoxicotherapy, may benefit from a shorter half life. Since engineering of the conserved Fc sequences for half life purpose may raise additional immunogenicity concern, the possibility of modulating mAb half life through Fab region is an attractive alternative. The proposed in vitro FcRn binding assay may also find use as a complementary tool for mAb PK assessment when incorporated into early lead optimization process, where it can be used to identify mAb leads with desired pharmacokinetics properties without compromising its pharmacodynamic activity. As well, the these assays can also be used to identify Fc fusion proteins with desired PK properties.

Example 2: Application of the FcRn assay to identify target antibodies with the desired characteristics.

Anti-ADDL Backup mAb program

A total of > 10 anti-ADDL antibodies have been screened by the in vitro FcRn dissociation assay detailed herein. The results of 2 leading candidates are shown in Figure 5. The pharmacokinetics of candidate 1 and candidate 2 were determined in human FcRn mice. Consistent with predictions of the in vitro FcRn dissociation assay, candidate 1 had a short tl/2 of 29hr, while candidate 2 has a much longer tl 2 of 77hr. PCSK9 specific antibodies were also screened using the in vitro assay(s) detailed herein to identify those with a longer in vivo half life. Results similar to the above referenced anti-ADDL antibody leads were obtained (data not shown).

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Claims

WHAT THIS CLAIMED IS:

1. A method for evaluating the in vivo half life of a target antibody or antigen-binding fragment thereof comprising

(i) separately determining a slow dissociation phase biexponential decline function of target antibody or fragment or second antibody or antigen-binding fragment thereof binding to FcRn under conditions favoring dissociation of said antibody or fragment and FcRn following an initial fast dissociation phase of said antibody or fragment and FcRn, using surface plasmon resonance, which function is described by: RU_t = A Q ^k + B e^{~fo t} + C, wherein t is time, RUt is surface plasmon resonance response units at time t, A and B are initial values at time zero for the two, faster and slower, dissociation phases respectively; k and k2, are the apparent first order rate constants for the faster and slower dissociation phases, respectively; and C is the surface plasmon resonance response units at end of dissociation.; and

(ii) comparing the value k2/B for the target antibody or fragment and the second antibody or fragment;

wherein the target antibody or fragment is determined to have an increased or longer in vivo half life than the second antibody or fragment if the k2/B for the target antibody or fragment is larger than the k2 B for the second antibody or fragment;

wherein the target antibody or fragment is determined to have a decreased or shorter in vivo half life than the second antibody or fragment if the k2/B for the target antibody or fragment is smaller than the k2/B for the second antibody or fragment; and/or

wherein the target antibody or fragment is determined to have a similar in vivo half life to the second antibody or fragment if the k2 B for the target antibody or fragment similar to the k2/B for the second antibody or fragment.

2. A method for evaluating the in vivo half life of a target antibody or antigen-binding fragment thereof comprising:

(i) separately contacting the target antibody or fragment and a second antibody or fragment with FcRn under conditions favorable to binding between the antibody and FcRn;

(ii) subjecting complexes between the target antibody or fragment and the FcRn ; and, the second antibody or fragment and the FcRn; to conditions favoring dissociation of the antibody or fragment from the FcRn;

(iii) determining the percentage of target antibody or fragment or second antibody or fragment bound the FcRn at a timepoint following an initial fast dissociation phase of antibody or fragment /FcRn dissociation; wherein the target antibody or fragment is determined to have an increased or longer in vivo half life than the second antibody or fragment if the percentage of target antibody or fragment bound to FcRn at the time point following the initial fast dissociation phase of the antibody or fragment /FcRn dissociation is smaller than that for the second antibody or fragment;

wherein the target antibody or fragment is determined to have a decreased or shorter in vivo half life than the second antibody or fragment if the percentage of target antibody or fragment bound to FcRn at the time point following the initial fast dissociation phase of the antibody or fragment /FcRn dissociation for the target antibody or fragment is larger than that for the second antibody or fragment; and/or

wherein the target antibody or fragment is determined to have a similar in vivo half life to the second antibody or fragment if the percentage of target antibody or fragment bound to FcRn at the time point following the initial fast dissociation phase of the antibody or fragment /FcRn dissociation for the target antibody or fragment similar to that for the second antibody or fragment,

3. The method of claim 2 wherein the conditions favoring dissociation is a pH of about 7,3.

4. The method of claim 2 wherein the conditions favoring binding is a pH of about 6.0.

5. The method of claim 2 wherein the timepoint following the initial fast dissociation phase of antibody/FcRn dissociation is about 5 seconds after exposure to conditions favoring dissociation.

6. The method of claim 2 wherein the percentage of antibody bound to FcRn at the time point following the initial fast dissociation phase is determined by dividing the quantity of antibody bound to FcRn about 5 seconds after exposure to conditions favoring dissociation by the quantity of antibody bound to FcRn about 2 seconds before exposure to conditions favoring dissociation.

7. The method of any of the preceding claims wherein the second antibody is adalimumab, basiliximab, bevacizumab, cetuximab and/or trastuzumab.

8. The method of claim 1 wherein the biexponential decline function and k2/B is calculated by a process comprising determining the quantity of antibody bound to FcRn over time.

9. The method of claim 1 wherein the quantity of antibody bound to FcRn is determined over about 2 seconds to about 110 seconds following exposure of the antbody and FcRn to conditions favoring dissociation.

10. The method of any of the preceding claims the antibody or fragment is a monoclonal antibody.

1 1. The method of any of the preceding claims wherein the target antibody and the second antibody comprising substantially similar Fc amino acid sequences.

12. The method of claim 11 wherein the Fc is an IgG.

13. The method of any of the preceding claims further comprising determining the in vivo half life of the antibody in the body of a mammal.

14. The method of claim 13 wherein the mammal is a transgenic mouse having human FcRn and lacking functional mouse FcRn; or a primate.