EP1922412A2 - Conjugues d'anticorps humanises et procedes correspondants, essais biologiques, reactifs et trousses - Google Patents

Conjugues d'anticorps humanises et procedes correspondants, essais biologiques, reactifs et trousses

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Publication number
EP1922412A2
EP1922412A2 EP06787249A EP06787249A EP1922412A2 EP 1922412 A2 EP1922412 A2 EP 1922412A2 EP 06787249 A EP06787249 A EP 06787249A EP 06787249 A EP06787249 A EP 06787249A EP 1922412 A2 EP1922412 A2 EP 1922412A2
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EP
European Patent Office
Prior art keywords
human
fragment
humanized antibody
antibody conjugate
fab2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06787249A
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German (de)
English (en)
Other versions
EP1922412A4 (fr
Inventor
Linda-Marie Anderson-Mauser
Mary Margaret Foltz
Robert John Dwyer
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Siemens Healthcare Diagnostics Inc
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Siemens Medical Solutions Diagnostics Inc USA
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Publication of EP1922412A2 publication Critical patent/EP1922412A2/fr
Publication of EP1922412A4 publication Critical patent/EP1922412A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to synthetically and recombinantly-derived humanized antibody conjugates and related methods, analytical assays including, for example, diagnostic assays, reagents, and kits.
  • Analytical assays use as a positive control reagent sera samples taken from a donor who has experienced an immune reaction caused by exposure to the antigen targeted by the assay. It is often very difficult and expensive to obtain serum-based control reagents for assays which detect antibodies to certain antigens. For particular grass pollens and hepatitis viral strains, identifying willing human donors who have a suitable serum antibody titer and epitope specificity can entail a worldwide search. Using sera-derived control reagents also introduces complexities associated with batch-to-batch and donor-to-donor variability.
  • Recombinant humanized monoclonal antibodies can be designed both to target a specific epitope and to contain human immunoglobulin amino acid sequences which are recognized by assay reagents.
  • murine-derived Mabs the antibody comprises around five percent or less of murine protein. See Presta, et al, J. Immunol. 1993 Sep l;151(5):2623-32.
  • United States Patent No. 6,680,209 discloses detecting an analyte in a human sample containing human antibodies that specifically bind to antibodies from a nonhuman species, but does not describe the use of recombinant humanized antibodies as control reagents.
  • Biotechniques 35: 672-674 (October 2003) describes high-throughput isolation of recombinant antibodies against recombinant allergens, but does not describe the use of recombinant humanized antibodies as control reagents.
  • the invention provides humanized antibody conjugates comprising a human immunoglobulin fragment which is bound by a cross-linking functional group to a non-human antibody fragment (e.g., a non-human monoclonal antibody fragment) comprising an antigen-binding amino acid sequence.
  • Humanized antibody conjugates of the invention are chemically synthesized to exhibit antigen binding specificities which are recognized by reagents used in a given diagnostic assay.
  • humanized antibody conjugates of the invention are robust compositions which do not exhibit batch-to-batch or donor-to-donor variation.
  • humanized antibody conjugates of the invention can be made inexpensively and quickly and prove well-suited for use as control reagents or capture antibodies in competitive and sandwich immunoassays.
  • humanized antibody conjugates of the invention comprise a human antibody fragment (e.g., a Fc fragment of either human IgG or human IgE) bound by a cross-linking functional group to an antibody fragment (e.g., a Fab2 fragment) which is derived from a non-human antibody (e.g., a non-human Mab) and which contains an antigen-binding amino acid sequence.
  • the antigen may comprise, for example, an allergen, a virus, a bacteria, or a ligand associated with a disease.
  • humanized antibody conjugates of the invention comprise a Fc fragment of human IgG bound by a cross-linking functional group to a Fab2 fragment which is derived from a murine Mab and which contains an antigen-binding amino acid sequence.
  • the invention provides humanized antibody conjugates in which: (1) an antibody fragment (e.g., a Fab2 fragment) which is derived from a non-human antibody and which contains an antigen-binding amino acid sequence is bound by a cross-linking functional group to a human antibody fragment (e.g., a Fc fragment of either human IgG or human IgE); and (2) the cross- linking functional group is formed by the reaction of: (a) a thiol (disulfide)-binding moiety which has been derivatized onto the human antibody fragment; and (b) a reduced thiol (disulfide) bond on the non-human antibody fragment.
  • an antibody fragment e.g., a Fab2 fragment
  • a cross-linking functional group e.g., a Fc fragment of either human IgG or human IgE
  • the cross- linking functional group is formed by the reaction of: (a) a thiol (disulfide)-binding moiety which has
  • the invention provides humanized antibody conjugates in which a murine Mab Fab2 fragment which contains an antigen- binding amino acid sequence is bound by a cross-linking functional group to a Fc fragment of human IgG, and wherein (1) the cross-linking functional group is formed by the reaction of: (a) a thiol (disulfide)-binding moiety which has been derivatized onto the Fc fragment by reaction of the Fc fragment with a hetero-bifunctional cross- linking agent; and (b) a reduced thiol (disulfide) bond on the Fab2 fragment; and (2) the Fab2 fragment is obtained by an aspartic proteinase digestion of the murine Mab which preserves the thiol (disulfide) bond on the Fab2 fragment.
  • the cross-linking functional group is formed by the reaction of: (a) a thiol (disulfide)-binding moiety which has been derivatized onto the Fc fragment by reaction of the Fc fragment
  • the invention also provides methods for synthesizing humanized antibody conjugates chemically, as well diagnostic assays, methods, reagents, and kits which use such chemically synthesized humanized antibody conjugates. [0015] In another embodiment, the invention provides diagnostic methods and kits which use recombinant, humanized Mabs as control reagents. [0016] These and other aspects of the invention are described further in the following detailed description.
  • FIG. 1 illustrates Novex electrophoresis gel analyses of humanized antibody conjugate Fab2 fragments, as determined in accordance with the experiment(s) of Example 1.
  • FIG. 2 illustrates Novex electrophoresis gel analysis of human IgG digested with ficin, as determined in accordance with the experiment(s) of Example 1.
  • FIG. 3 illustrates SDS polyacrylamide gel analysis of the digested humanized antibody conjugate Fc fragment, as determined in accordance with the experiment(s) of Example 1.
  • FIG. 4 illustrates the elution profile of the humanized NS5 conjugation reaction mixture from a chromatography column.
  • FIG. 5 illustrates SDS gradient gel analysis of humanized hepatitis C antibody conjugate fractions, as determined in accordance with the experiment(s) of Example 1 and illustrated in FIG. 4.
  • DETAILED DESCRIPTION OF THE INVENTION [0022] 1. Definitions.
  • An "allergen” means any substance that triggers an immune response.
  • An “analytical method” shall mean the detection, measurement, determination, characterization or other assessment of a substance or substances in, or the composition, state or other condition of, a test sample, including, without limitation, (a) testing performed for research or commercial purposes, such as medical, veterinary or industrial testing, and (b) in vitro or in vivo testing where the test sample is of human or animal origin, and including, without limitation, (a) qualitative and quantitative assays, (b) multiplexing assays, classification, sequencing and other characterization assays, (c) indexing, reflex and combination assays involving the detection, measurement, determination, characterization or other assessment of more than one substance in, or condition of, a test sample, and/or the calculation of an index or application of a mathematical or other algorithm, and (d) the performance of calibration, control, or other standardization steps.
  • analytical method includes those methods, reagents, kits, calibrators, controls that are used for the testing of human or animal specimens for the purpose of diagnosis, prognosis, or monitoring the progress of disease or monitoring the effect of treatment of disease, in the human or animal from which the specimens were taken.
  • an "antibody” is an intact immunoglobulin molecule comprising two of each of immunoglobulin light and heavy chains. Unless otherwise noted, “antibody” includes polyclonal or monoclonal antibodies. Antibodies used in the invention can be prepared by techniques generally known in the art, and are typically generated as either an isolated, naturally occurring protein, as a recombinantly expressed protein, or as a synthetic peptide representing an antigenic portion of a natural protein. [0027] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) or lambda ( ⁇ ) based on the amino sequences of their constant domain.
  • K kappa
  • ⁇ lambda
  • antibodies can also be assigned to different classes. There are at least five (5) major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g IgG-I, IgG-2, IgG-3 and IgG4; IgA-I and IgA-2.
  • An "antibody-binding region" maintains the structure of the antibody that interacts with the antigen.
  • W light and heavy variable
  • antibody fragment refers to a portion of a full-length antibody, and generally refers to the antigen binding or variable region.
  • antibody fragments include the Fc, Fab, Fab 1 , F(ab') 2 (also referred to herein as the Fab2 fragment), and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments: (1) the Fab fragment, which has a single antigen binding site; and (2) a residual "Fc" fragment, which crystallizes readily. Pepsin digestion yields:
  • an F(ab')2 fragment that has two antigen binding fragments which are capable of cross-linking antigens; and (2) a residual fragment pFc'.
  • An "Fv" fragment is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the V H - V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab '-SH or Fab' fragments contain cysteine residue(s) of the constant domains and have a free thiol group.
  • F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab') 2 pepsin digestion product.
  • a “chimeric” antibody is an antibody homolog in which all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both, have been substituted for the corresponding regions from another immunoglobulin light chain or heavy chain.
  • “chimeric antibody” refers to an antibody in which the variable region is derived from a nonhuman antibody, e.g., a murine or rat antibody, and the constant region is derived from a human antibody.
  • An immortal cell line typically myeloma cells
  • lymphocytes typically splenocytes
  • lymphocytes typically splenocytes
  • Humanized monoclonal antibody homologs can be prepared, e.g., by using in vzYr ⁇ -primed human splenocytes. See Boerner et ah, 1991, J. Immunol. 147:86-95. Humanized monoclonal antibodies can also be prepared by repertoire cloning. See Persson et al, 1991, Proc. Nat. Acad. Sci. USA 88: 2432-2436; Huang, et al 1991, J. Immunol. Methods 141: 227-236.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, supra at pages 2.7.1-2.7.12 and 2.9.1-2.9.3. See also Baines et al., Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc. 1992).
  • control reagent is a solution which has a known concentration of an analyte (e.g., antibody) and which, when used in a serology immunoassay, provides a positive control which validates assay function.
  • analyte e.g., antibody
  • Cross-linking functional groups form molecular bridges that link functional groups of two different molecules.
  • Cross-linking functional groups contain two reactive groups, one of which usually reacts with a primary amine group (e.g., N- hydroxy succinimide) and the other of which usually reacts with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
  • a primary amine group e.g., N- hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, halogens, etc.
  • one antibody fragment may be derivatized with a cross-linking moiety through reaction with a hetero-bifunctional cross-linking agent, and an intrinsic cross-linker such as a thiol (disulfide) bond on the other fragment may be reduced for reaction with the cross-linking moiety by exposure to a reducing agent.
  • reaction of a human Fc fragment with hetero-bifunctional cross-linking agent will derivatize the human Fc fragment by adding a thiol (disulfide)- binding group (e.g., a maleimide group) to primary amines accessible on the protein surface.
  • a thiol (disulfide)- binding group e.g., a maleimide group
  • Reaction of a non-human Fab2 fragment with reducing agents will reduce Fab2 fragment disulfide bonds and the reduced Fab2 fragment disulfide bonds will bind to the Fc fragment thiol (disulfide)-binding group to form a cross-linking functional group which will bind the two antibody fragments.
  • Hetero-bifunctional cross-linking agents include but are not limited to succinimidyl-4-(N maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”); 4-(N maleimidomethyl)cyclohexane- 1 -carboxylic 3-sulfo-n-hydroxysuccinimide ester (“sulfo-SMCC”); m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS); sulfo-MBS; N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP); N-succinimidyl(4 ⁇ iodoacteyl) aminobenzoate (“SIAB”); succinimidyl-4-(p-maleimidophenyl)butyrate (“SMBP”); N-( ⁇ -maleimidobutyryloxy)succinimide ester ("GMBS)
  • Reducing agents include but are not limited to mercaptoethylamine
  • MEA N-oxydiethylene-2-benzothiazolyl sulfonamide
  • tri-n-butylphosphine dithiothreitol
  • beta-mercaptoethanol BME
  • Cysteine (thiol) proteinases include but are not limited to papain, ficin, actinidin, bromelain, mammalian lysosomal cathepsins, the cytosolic calpains (calcium- activated), and parasitic proteases (e.g Trypanosoma, Schistosoma).
  • Aspartic proteinases include but are not limited to pepsin, chymosin, lysosomal cathepsins D, processing enzymes such as renin, and fungal proteases (e.g., penicillopepsin, rhizopuspepsin, endothiapepsin).
  • an “epitope” is a molecular region on the surface of an antigen which is capable of eliciting an immune response and which is also capable of binding to or combining with the specific antibody produced by such a response.
  • "FR" is an abbreviation for Framework Region, which comprises the portions of the variable regions of an antibody which are adjacent to, or flank, the CDRs. In general, these regions have a structural function that affects the conformation of the variable region and are less directly responsible for the specific binding of antigen to antibody.
  • Humanized antibodies are antibody molecules that bind to a specific antigen and have one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule. See, e.g., United States Patent No. 5,585,089; Riechmann et ah, Nature, 332:323 (1988).
  • murine or rat CDRs are transferred from heavy and light variable chains of mouse or rat antibodies into a variable region designed to contain a number of amino acid residues found within the FR region in human IgG.
  • General techniques for cloning murine immunoglobulin variable domains are described in Orlandi et ah, Proc. Nat'lAcad.
  • a "humanized recombinant antibody” is an antibody which is initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. References cited above disclose methods for synthesizing humanized recombinant antibody which are well-known to those of ordinary skill in the art.
  • "Humanized antibody conjugates" of the invention comprise a human immunoglobulin fragment which is bound by cross-linking functional groups to a ligand-binding region comprising a non-human antibody fragment, e.g., a non-human antibody fragment.
  • Immunoassays determine the presence of a patient diagnostic serum marker in a biological sample by reacting the sample with an antibody that binds to the serum marker, the reaction being carried out for a time and under conditions allowing the formation of an immunocomplex between the antibodies and the serum markers. The quantitative determination of such an immunocomplex is then performed.
  • the antibody used is an antibody generated by administering to a mammal (e.g., a rabbit, goat, mouse, pig, etc.) an immunogen that is a serum marker, an immunogenic fragment of a serum marker, or an anti-serum marker-binding idiotypic antibody.
  • kits for detecting serum marker-binding antibodies feature the use of serum marker-binding antibodies generally (regardless of whether they are raised to one of the immunogens described above).
  • a sandwich immunoassay format may be employed which uses a second antibody that also binds to a serum marker, one of the two antibodies being immobilized and the other being labeled.
  • Preferred immunoassays detect an immobilized complex between a serum marker and a serum marker-binding antibody using a second antibody that is labeled and binds to the first antibody.
  • the first version features a sandwich format in which the second antibody also binds a serum marker.
  • a serum marker -binding antibody can be a capture antibody attached to an insoluble material and the second a serum marker -binding antibody can be a labeling antibody.
  • the assays used in the invention can be used to determine a blood marker, e.g., a plasma or serum marker in samples including urine, plasma, serum, peritoneal fluid or lymphatic fluid.
  • Immunoassay kits for detecting a serum marker can also be used, and comprise a serum marker -binding antibody and the means for determining binding of the antibody to a serum marker in a biological sample.
  • the kit includes one of the second antibodies or the competing antigens described above.
  • protein protein
  • polypeptide and peptide are used interchangeably herein to refer to a polymer of amino acids.
  • a protein may include more than one polypeptide chain.
  • a polypeptide can be expressed recombinantly or can be produced synthetically.
  • Solid phase means a non-aqueous matrix to which an antibody or humanized antibody conjugate can adhere.
  • solid phases include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles.
  • a non-human Mab such as a murine or rat Mab which binds to an antigen of interest is digested in an aspartic proteinase such as pepsin to separate the Mab Fab2 fragment from the Mab intact IgGl.
  • the non-human Mab is preferably digested: (1) in a buffered reaction mixture comprising excess antibody (e.g., around fifty to around two hundred times more antibody than aspartic proteinase on a weight basis); (2) at a pH of between about 3 to about 6 (most preferably at a pH of from about 4 to about 5); and (3) at temperature of around 30° C to around 40° C.
  • Aspartic proteinase digestion yields a Fab2 fragment which retains a thiol bond between the two cysteines present on each of the two heavy and light chain fragments.
  • An aspartic proteinase such as pepsin cuts behind the disulfide bond in the immunoglobulin ⁇ see Essential Immunology, Ivan Roitt (W. Clowes and Sons, 3 rd ed., 1977)).
  • Available thiol groups which are removed from the binding site, and which are proximate to the pepsin digestion site, are reduced gently with mercaptoethylamine.
  • Human IgG is digested enzymatically using a cysteine (thiol) proteinase such papain and ficin to generate the Fc fragment.
  • the Fc fragment is derivatized with a thiol (disulfide)-linking maleimide group by reaction with a hetero-bifunctional cross- linking agent such as SMCC.
  • Humanized antibody conjugates are formed by the reaction at room temperature of an excess of Fab2 fragments with Fc fragments (preferably in a weight ratio of Fab2:Fc of around 1.5:1 to 5.0:1, more preferably around 2:1 to 3:1); cross- linking functional groups join the Fab2 and Fc fragments through reaction of the reduced Fab2 thiol (disulfide) group and the Fab maleimide moiety.
  • Fc fragments preferably in a weight ratio of Fab2:Fc of around 1.5:1 to 5.0:1, more preferably around 2:1 to 3:1
  • cross- linking functional groups join the Fab2 and Fc fragments through reaction of the reduced Fab2 thiol (disulfide) group and the Fab maleimide moiety.
  • Anti-HepC NS5 Mab (Bayer Clone ML414-14A2.5A4 Anti HCV NS5), deposited under the Budapest Treaty on July 14, 2005 with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, ATCC Dep. No. PTA-6861, (hereinafter "NS5") was used to generate the antigen binding portion of the conjugate.
  • NS5 was digested with pepsin to produce a Fab2 fragment from intact IgGl as follows.
  • NS5 was placed into a 0.1 M citrate, pH 3.5 buffer using a NAP-5 column (Pharmacia).
  • the amount of pepsin (Sigma) used to digest NS5 was approximately 1/30 of NS5 weight and the reaction was incubated at 37° C.
  • Samples were removed at regular intervals and an optimal digestion time of 45 minutes was determined by examining the progress of the digestion on a No vex electrophoresis gel.
  • the Fab2 fragment prepared from the functioning capture portion of NS5 weighed approximately 100 KD.
  • the Optimization of Pepsin Digestion of anti-HCV clone NS5 is illustrated in Fig. 1. Note that the Lanes correspond to the Samples as follows: Lane # Sample
  • the resultant Fc preparation was predominantly 50 KD in weight; no single molecular weight could be determined due to the heterogeneous nature of the native protein mixture.
  • the digested, non- reactive medium was purified by passing it over a protein A column. This step served to remove the Fc fragment from the remaining digested portion of the IgGl molecule by allowing the intact Fc to bind to the protein A resin, while allowing the remainder of the digest reaction to flow through the column. The intact Fc fragment was then eluted and collected. SDS polyacrylamide gel analysis demonstrated that the resultant Fc fragment pool was recovered. The SDS Gel at Various Steps in Isolation of Human Fc Fragment is illustrated in Fig. 3. Note that the Lanes in Fig. 3 correspond to the Samples as follows:
  • Fab2 and Fc fragments were linked to form a humanized antibody conjugate as follows.
  • the Fab2 fragment was obtained by digestion of NS5 in pepsin as described above, the thiol (di-sulfide) bond between the two cysteines on the heavy and light chain fragments of NS5 was preserved. (Pepsin digestion cut the NS5 Fab2 fragment from intact IgGl at a location which preserved a thiol (disulfide) bond between the Fab2 fragment heavy and light chains
  • This thiol (disulfide) bond on the Fab2 fragment was activated by reaction of the Fab2 fragment with 5OmM mercaptoethylamine (MEA) at approximately 37° C for a period of around 90 minutes. Excess MEA was removed by desalting in a PBS 2mM EDTA reaction buffer using a NAP25 column (Pharmacia).
  • MEA 5OmM mercaptoethylamine
  • the Fc fragment was derivatized by adding a maleimide group to primary amines accessible on the protein surface through reaction of the Fc fragment with SMCC. Specifically, Fc fragment was added to an approximately twenty-five fold molar excess of SMCC (Pierce) and the resultant reaction mixture was allowed to stand for 25 minutes at room temperature. Excess SMCC was quenched by the addition of glycine to the reaction mixture and excess SMCC was removed by desalting the protein mixture over a NAP25 column equilibrated in pH 7.4
  • the Fab and Fc fragments were combined to form a reaction mixture comprising an approximately 2.4 fold molar excess of Fab to Fc and were allowed to react for approximately three hours at room temperature to form humanized Hep C antibody conjugate. Thereafter, the reaction mixture was stored overnight at 2° C - 8° C and excess maleimide remaining in the reaction mixture was quenched by the addition of twelve-fold molar excess MEA.
  • ADVIA Centaur® is a registered trademark of Bayer HealthCare LLC, Tarrytown, New York U. S. A.
  • Humanized Hep C Antibody Conjugate as a Control Reagent
  • Humanized Hepatitis C antibody conjugates made in the experiment(s) of Example 1 were used as control reagents in diagnostic kits employed with the Bayer ADVIA Centaur® hepatitis C assay.
  • a sample was incubated with Solid Phase containing recombinant peptide HCV antigens. Antigen-antibody complexes will form if anti-HCV antibody is present in the sample. Lite Reagent containing monoclonal anti-human IgG labeled with acridinium ester was used to detect human anti-HCV IgG in the sample.
  • the system automatically performed the following steps: (1) it dispensed 10 uL of sample into a cuvette; (2) it dispensed 100 ul of a sample diluent and incubated the test sample for 5 minutes at 37 0 C; (3) it dispensed 100 uL of Solid Phase reagent and incubated the reagent for 18 minutes at 37 0 C; (4) it separated the Solid Phase from the mixture and aspirated the unbound reagent; (5) it washed the cuvette with Wash 1; (6) it dispensed 50 uL of Lite Reagent, (7) it incubated the mixture for 18 minutes at 37°C; (8) it washed the cuvette with Wash 1; and (9) it dispensed 300 uL each of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction.
  • An assay which assists in the diagnosis of whether an individual is allergic to a particular antigen requires a positive control to determine whether the assay is functioning properly and is being used correctly.
  • a humanized antibody conjugate of the invention can be used as a control reagent in a Kentucky Bluegrass pollen diagnostic assay.
  • the methodologies described in this example apply to assays which assist in the diagnoses of allergies to any allergen.
  • a murine anti-pollen Mab such as Mab 27 described in
  • Mab 27 is placed into 0. IM citrate, pH 3.5 buffer using a
  • NAP-5 column (Pharmacia).
  • the amount of pepsin (Sigma) added is about 1/30 that of the antibody (w/w) and the reaction is allowed to incubate at 37°. Samples are removed at intervals; an optimal time is determined by examining the progress of the reaction on a Novex electrophoresis gel. Typically, a removal time would occur between around 15 to around 120 minutes, although digestion times of up to 4 hours are possible.
  • the size (approximately lOOKD) and the identity of the Fab2 fragment prepared from the functioning pollen-binding portion of Mab 27 are thereby determined.
  • the digestion reaction is performed as optimized and then quenched with IM Tris prior to the removal of all non-essential protein fragments by exhaustive dialysis against PBS/2MM EDTA using a 3OK MW membrane.
  • Human IgGl (Fitzgerald catalog #30-AI17) or another polyclonal human IgG or IgE preparation is digested with either papain and ficin enzymes (Sigma) to generate the Fc fragment.
  • the antibody protein is exchanged into a 5OmM Tris, 2mM EDTA pH 7.0 buffer using NAP-5 (Pharmacia) columns so as to facilitate protein digestion by the enzyme. Either or both of the enzymes are tested at approximately 1/30 (w/w) proportions relative to the antibody. Both enzyme and cysteine are added to the protein and the resultant digestion mixture is allowed to incubate at approximately 37 0 C.
  • Fc preparation should be predominantly 50 KD in weight, although a single molecular weight will not be assigned to the preparation due to the heterogeneous nature of the native human IgG.
  • the digestion reaction product is purified by passing the reaction product over a protein A column; this removes the Fc region fragment from the remaining digested portions of the IgGl molecule by allowing the intact Fc to bind to the protein A. The remainder of the digest reaction then flows through the column without binding. After washing the column, the intact Fc fragment is eluted with pH and/or salt conditions and collected. SDS polyacrylamide gel analysis demonstrates that the resultant Fc fragment was correctly recovered.
  • the Kentucky bluegrass binding Mab 27 Fab2 is reduced with mercaptoethylamine to generate Fab molecules which have an available thiol near the hinge region.
  • the Fc fragment derived from human IgG is derivatized with maleimide to facilitate reaction with the available thiol on the Fab.
  • the Fab2 fragment has a thiol bond between the two cysteines present on each of the two heavy and light chain fragments.
  • Available thiols at locations which are away from the binding site and near the pepsin digestion site are activated by gently reducing the disulfide bond with mercaptoethylamine.
  • a 5OmM MEA Fab2 fragment solution is incubated at 37° C for 90 minutes. This creates Fab fragments with an intact thiol group and bound heavy and light chains.
  • Excess MEA is removed by desalting the protein mixture on NAP25 columns (Pharmacia) containing a PBS 2mM EDTA reaction buffer.
  • the Fc fragment is derivatized by addition of a maleimide group to primary amines accessible on the surface of the molecule through reaction with twenty- five fold molar excess SMCC (Pierce) for 25 minutes at room temperature. Excess SMCC is quenched by the addition of glycine and excess SMCC is removed by desalting the protein mixture over a NAP25 column equilibrated in pH 7.4 PBS. [0097] Reduced Fab is added to the activated Fc mixture at a ratio of around 2.4 fold molar excess Fab': Fc and is allowed to react for around three hours at room temperature. The resultant reaction mixture is stored at between about 2-8°C for at least 8 hours.
  • the conjugation mixture is chromatographed on a 1.6 x 60 Superdex 200 column (Pharmacia). A conjugate mixture of approximately 1 mL volume containing 5-10 mg of protein is applied to the column and fractionated by size by running in PBS pH 7.4 buffer at 0.2 ml/min to achieve optimal size separation. Fractions are collected and analyzed by SDS gradient gel (4-20%) for correct size.
  • a single Fab'rFc conjugate weighs approximately lOOKD, but multimers may be formed in which more than one Fab' fragment conjugates to a Fc fragment. Additional peaks of greater than IOOKD may therefore be obtained. Pools of conjugate with varying molecular weights are made and tested for performance in an actual assay.
  • This vector then is used to produce the Bayer humanized antibody in a suitable host, either eukaryotic or prokaryotic.
  • a suitable host either eukaryotic or prokaryotic.
  • the resultant recombinant Bayer protein molecule has the capability to recognize the Rubella El viral epitope and also has the necessary human IgG sequence so that it acts in the Rubella serology assay just as a native, positive patient sample would.
  • anti-El was used to generate the antigen binding portion of the conjugate.
  • anti-El was digested with pepsin to produce a Fab2 fragment from intact IgGl as follows.
  • Human IgM (Chemicon catalog#AG722) was digested with ficin to generate a humanized antibody conjugate Fc fragment. Since it was not known how the polyclonal human IgM preparation would tolerate digestion, experiments were conducted to identify the best way to prepare the Fc portion of the molecule. [00105] The enzyme ficin (Sigma) was tested with a highly pure human IgM preparation. The antibody protein was exchanged to facilitate digestion into 5OmM Tris, 2mM EDTA pH 7.0 buffer using NAP-5 (Pharmacia) columns. The enzyme was tested at 1/30 (w/w) proportion relative to the antibody. Both enzyme and cysteine were added to the protein and digested at 37° C.
  • the resultant Fc preparation was predominantly 50 KD in weight; no single molecular weight could be determined due to the heterogeneous nature of the native protein mixture.
  • the digested, non- reactive medium was purified by passing it over a protein A column. This step served to remove the Fc fragment from the remaining digested portion of the IgM molecule by allowing the intact Fc to bind to the protein A resin, while allowing the remainder of the digest reaction to flow through the column. The intact Fc fragment was then eluted and collected. SDS polyacrylamide gel analysis demonstrated that the resultant Fc fragment pool was recovered. See Fig. 3.
  • the Fab2 fragment was obtained by digestion of anti-El in pepsin as described above, the thiol (di-sulfide) bond between the two cysteines on the heavy and light chain fragments of anti-El was preserved. (Pepsin digestion cut the anti-El Fab2 fragment from intact IgGl at a location which preserved a thiol (disulfide) bond between the Fab2 fragment heavy and light chains
  • Pool I represents the highest molecular weight conjugate while pool III represents the lowest molecular weight, with pool II intermediate between the two. Pools were concentrated to approx. 1 mg/mL using an ultrafiltration cell equipped with a molecular weight cutoff membrane of 30 kD. The concentration of each pool was measured by A280 using an extinction coefficient of 1.36. As a preservative, Bovine Serum Albumin and
  • Sodium Azide were added to 0.5% and 0.1% respectively. After addition of preservative, the solution was filtered using a 0.2 um filter.

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Abstract

L'invention concerne des conjugués d'anticorps dérivés synthétiquement et par voie recombinante et les procédés correspondants, de dosages diagnostics, réactifs et trousses. Dans un mode de réalisation, l'invention concerne des conjugués d'anticorps humanisés contenant un fragment d'immunoglobuline humain qui est lié par un groupe fonctionnel de réticulation à un fragment d'anticorps non-humain (par ex., un fragment d'anticorps monoclonal non-humain) contenant une séquence d'acide aminé à liaison d'antigène.
EP06787249A 2005-07-15 2006-07-14 Conjugues d'anticorps humanises et procedes correspondants, essais biologiques, reactifs et trousses Withdrawn EP1922412A4 (fr)

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US7858752B2 (en) 2006-12-05 2010-12-28 Abbott Laboratories Recombinant antibodies against hepatitis C virus and methods of obtaining and using same
US20100143376A1 (en) * 2007-03-06 2010-06-10 Ribovax Biotechnologies S.A. Antibodies Specific for Rubella Virus
CN103383393B (zh) * 2013-06-28 2015-11-25 英科隆生物技术（杭州）有限公司 一种替代病人阳性血液的质控品
CN108445224A (zh) * 2018-02-26 2018-08-24 王贤俊 一种心型脂肪酸结合蛋白抗体片段复合胶乳颗粒及其制备方法

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