WO2011106272A1 - Nouveaux essais de liaison utiles dans l'identification d'anticorps ayant des demi-vies modifiées - Google Patents

Nouveaux essais de liaison utiles dans l'identification d'anticorps ayant des demi-vies modifiées Download PDF

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WO2011106272A1
WO2011106272A1 PCT/US2011/025580 US2011025580W WO2011106272A1 WO 2011106272 A1 WO2011106272 A1 WO 2011106272A1 US 2011025580 W US2011025580 W US 2011025580W WO 2011106272 A1 WO2011106272 A1 WO 2011106272A1
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antibody
fragment
fcrn
dissociation
target
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PCT/US2011/025580
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Weiron Wang
Ping Lu
Thomayant Prueksaritanont
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Merck Sharp & Dohme Corp.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the invention relates to the identification of target antibodies exhibiting desired in vivo characteristics useful in the prevention and treatment of various diseases.
  • 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.
  • mAbs monoclonal antibodies
  • 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.
  • immunoglobulins There are five types of immunoglobulins in humans. These groups are known as IgG,
  • IgM, igD, IgA, and IgE are distinguished based on the isotypes of the heavy chain gene ( ⁇ , ⁇ , ⁇ , a, and ⁇ 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).
  • VH variable domain
  • CHI constant heavy domain 1
  • CH2 constant heavy domain 2
  • CH3 constant heavy domain 3
  • 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).
  • 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.
  • CDRs complementarily- determining regions
  • 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.
  • FcyR Fc gamma receptors
  • FcRn neonatal Fc receptor
  • 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.
  • FcRn certain cellular receptors
  • 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 ⁇ 2 microglobulin (( ⁇ 2 ⁇ ) (4). Crystal structure has shown that FcRn binds to the Fc domain of IgG at the Cy2-Cy3 3 interface (6-7).
  • 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
  • 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 improved FcRn binding properties also show longer half life in vivo (10-12).
  • 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.
  • 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.
  • 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.
  • 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
  • PK pharmacokinetics
  • 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.
  • 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.
  • 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
  • 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
  • SPR response units can be replaced with enzyme linked immunosorbent assay (ELISA) units such as color change of the reaction solution at OD 4 50nm.
  • ELISA enzyme linked immunosorbent assay
  • 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
  • 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
  • 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.
  • assoc or "Ka”
  • Kdis or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of 3 ⁇ 4 to K a (i.e. 3 ⁇ 4/ ⁇ ⁇ ) and is expressed as a molar concentration (M).
  • 3 ⁇ 4 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.TM system.
  • K. 0ff as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • %bound antibody effectively describes antibodies exhibiting the desired neutral pH FcRn "dissociation pattern" meaning the level of slow dissociation fraction.
  • the aim of the invention is to identify target antibodies that have less slow dissociation fraction.
  • 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.
  • the characteristics include a low "slow dissociation fraction" and increased or longer half life.
  • 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 "arm 11 interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • 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.
  • SPR surface plasmon resonance
  • 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,
  • a BIAcore sensor chip e.g., CMS chip
  • the binding of mAbs to the immobilized FcRn is measured at a certain flow rate to obtain sensorgrams using BIAevaluation software.
  • 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.
  • 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 ⁇ -phase.
  • the mAb elimination phase terminal half lives are determined with non-compartmental model using data points in ⁇ - 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.
  • 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 k 2 /Q as it relates to in vivo terminal t ⁇ a in humans. DETAILED DESCRIPTION OF THE INVENTION
  • 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.
  • 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.
  • compositions of the invention can be used to achieve methods of the invention.
  • 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.
  • 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.
  • 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.
  • the term "about” refers to an approximation of a stated value within an acceptable range. Preferably the range is +/-5% of the stated value.
  • 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
  • substantially identical 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.
  • 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%.
  • 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.
  • 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.
  • the desired characteristics of the target antibody include a lower "slow
  • dissociation fraction at neutral pH (e.g. low %bound).
  • 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
  • RIA radioimmunoprecipitation
  • 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).
  • 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.
  • the capture receptor can be incubated with variant polypeptides (e.g., antibodies) diluted in buffers at pH 6.0 or pH 7.0.
  • variant polypeptides e.g., antibodies
  • a molecule specific for human antibodies is added (e.g., goat (Fab')i anti-human-Fab conjugated to an enzyme).
  • Fab' goat
  • 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.
  • 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.
  • 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.
  • the FcRn is incubated with an antibody of the present invention or a fragment thereof conjugated to a labeled compound (e.g., 3 H or I25 I) in the presence of increasing amounts of an unlabeled second antibody.
  • 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).
  • Example 1 Development of in vitro FcRn binding assay for determination of antibody in vivo half life.
  • Soluble human, rhesus macaque and hybrid human-mouse FcRn were engineered to be expressed using the Bac-to-BacTM 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 ⁇ -subunit. The corresponding full-length ⁇ chain ( ⁇ 2 ⁇ ) cDN A was co-expressed using a separate baculovirus expression vector.
  • Viruses containing FcRn a- and ⁇ -subunits (human a + ⁇ , rhesus a + ⁇ , or human a + mouse ⁇ ) 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 ⁇ 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 ⁇ 2 ⁇ . 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.
  • 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).
  • 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 NaP0 4 , 150 mM NaCl and 0.05% (v/v) Surfactant 20) with a flow rate of 30 ⁇ /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.
  • FcRn binding affinity dissociation constant, KD
  • 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 integrity maintenance of instrument are critical for the neutral pH dissociation assay. A series of report points during the dissociation phase were recorded.
  • 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 t a b mty U Binding (%) ⁇
  • the whole dissociation process was captured by a series of reporting points from 2 to 110 seconds following dissociation.
  • 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 A3 ⁇ 4 are the apparent first order rate constants for the faster and slower dissociation phases, and C is the RU at end of dissociation.
  • the inventors 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.
  • the heterozygous Tg276 human FcRn mice used in this study were obtained from Jackson Laboratory (Bar Harbor, ME). They are deficient in mouse FcRn- ⁇ chain and carry a human FcRn- ⁇ chain gene (16).
  • each animal (3-4/group) received a single intravenous injection of mAb at 10 mg/kg via tail vein.
  • a series of 10 ⁇ L ⁇ 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 ⁇ L 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).
  • IACUC Institutional Animal Care and Use Committee
  • 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
  • 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 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.
  • SPR Surface Plasmon Resonance
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Table II 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.
  • Kd/C as the most promising combination to predict in vivo PK.
  • Kd/C may also be referred to herein as "k2/B”.
  • Fig 4 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.
  • FcRn 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.
  • 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.
  • 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.

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Abstract

La présente invention a pour objet des procédés d'identification d'anticorps cibles ayant des caractéristiques souhaitées. De préférence, les anticorps identifiés selon les procédés selon l'invention présentent une demi-vie in vivo plus longue par rapport aux autres anticorps ayant une séquence d'acides aminés de la région Fc similaire ou identique.
PCT/US2011/025580 2010-02-23 2011-02-21 Nouveaux essais de liaison utiles dans l'identification d'anticorps ayant des demi-vies modifiées WO2011106272A1 (fr)

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JP2017207494A (ja) * 2012-02-15 2017-11-24 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｆｃ受容体に基づくアフィニティークロマトグラフィー
US10683368B2 (en) 2014-11-06 2020-06-16 Hoffmann-La Roche Inc. Fc-region variants with modified FcRn-binding and methods of use

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017207494A (ja) * 2012-02-15 2017-11-24 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｆｃ受容体に基づくアフィニティークロマトグラフィー
JP2016508223A (ja) * 2012-12-27 2016-03-17 ゼネラル・エレクトリック・カンパニイ 水の広いダイナミックレンジの導電率測定
US10683368B2 (en) 2014-11-06 2020-06-16 Hoffmann-La Roche Inc. Fc-region variants with modified FcRn-binding and methods of use
US11440971B2 (en) 2014-11-06 2022-09-13 Hoffmann-La Roche Inc. Fc-region variants with modified FcRn-binding and methods of use

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