WO1991017177A1 - Anticorps de haute affinite contre des petits peptides - Google Patents

Anticorps de haute affinite contre des petits peptides Download PDF

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Publication number
WO1991017177A1
WO1991017177A1 PCT/US1991/003009 US9103009W WO9117177A1 WO 1991017177 A1 WO1991017177 A1 WO 1991017177A1 US 9103009 W US9103009 W US 9103009W WO 9117177 A1 WO9117177 A1 WO 9117177A1
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WO
WIPO (PCT)
Prior art keywords
antibody
amino acid
acid sequences
affinity
small peptide
Prior art date
Application number
PCT/US1991/003009
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English (en)
Inventor
Delia R. Bethell
Michael E. Jolley
Priscilla Wilkins Stevens
Original Assignee
Baxter Diagnostics Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Baxter Diagnostics Inc. filed Critical Baxter Diagnostics Inc.
Publication of WO1991017177A1 publication Critical patent/WO1991017177A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the antibodies of the present invention are obtained by restructuring the architecture of the variable regions of both heavy and light chains. Utilizing computerized molecular modelling techniques, the three dimensional structure of an antibody combining site of known sequence can be visualized, thereby readily identifying the functional groups pendant on amino acids which interact in the binding of antigen to the antibody. High energy interactions, as may occur, for example, from the close proximity of amino acid residues on antigen and antibody of like charge can be eliminated by substitution of an amino acid of opposite charge. Similarly, the compatibility of like hydrophobic residues on proximate antigen/antibody sites, or the juxtaposition of hydrogen bonding donors to hydrogen bonding acceptors can be exploited to increase the affinity of binding of antigen and to antibody.
  • an antibody of known amino acid sequence is computer modelled to display the three dimensional structure of antibody in binding engagement with its antigen, followed by visual identification of the affinity affecting chemical groups contained in the affinity domains of the antibody combining site.
  • Amino acids with ionic, hydrophobic, or hydrogen bonding properties more compatible with closely proximate chemical groups on a peptide antigen can be substituted in the antibody molecule.
  • the substituted structure is then remodelled, and the comparative binding energies are calculated.
  • An antibody structure is selected which has a lower energy in the antigen-bound state.
  • the antibody may then be expressed from genes modified to produce the substituted amino acid sequences.
  • small antigens such as peptides.
  • the portions of the antigen combining site which are too distant from the small peptide antigen to contribute to affinity can be restructured by insertion of functional groups which now interact with antigen.
  • a closely related problem in the diagnostic field involves the detection of small peptides cleaved enzy atically from zymogens.
  • F1.2 a peptide of 426 amino acids, known as F1.2
  • Antibodies raised to F1.2 cross-react with the uncleaved prothrombin, so that an immunological assay of desired specificity cannot be obtained.
  • Attempts to obtain greater specificity utilizing conjugated synthetic peptides spanning only a portion of the fragment peptide result either in antibodies which also cross-react with prothrombin, or are of low affinity.
  • Antibody molecules contain variable regions associated with binding specificity and affinity, and constant sequence portions associated with complement fixation, transport, etc. Within the variable regions, which are at the amino-terminal end of both the heavy and light chains comprising the antibody molecule, are found domains which are called the Complementarity Determining Regions (CDRs), and Framework Regions (FRs).
  • CDRs are responsible for binding affinity and specificity.
  • CDR3 of the heavy chain variable region (H3) is thought to be primarily responsible for specificity
  • CDR3 of the light chain (L3) is thought to play a major role in affinity (see Kabat, Sequences of Proteins of Immunological Interest, 4th Ed. 1987).
  • amino acid residues in other CDRs and FRs have been observed to contribute binding contacts with antigens.
  • Figure 1A, IB, and 1C give the amino acid sequences of a peptide, an immunigen containing the peptide, and a larger second peptide containing the first peptide.
  • Figures 2A, 2B, 2C, and 2D are amino acid sequences of the native and substituted light and heavy chain variable regions respectively.
  • Figure 3 is a stereoscopic visualization of an arginine residue substituted for the native glu98.
  • Figure 4 is a steroscopic representation of an unfavorable contact between glu57 of the heavy chain and an aspartic acid residue of the antigen.
  • antibodies to small peptides or other haptens may be obtained by immunizing test animals with peptide-carrier conjugates, either with or without an adjuvant. After a series of immunization boosts, antibody-producing lymphocytes are harvested, fused with myeloma cells, and grown out under conditions which select for stable hybridomas, according to conventional techniques.
  • the amino acid sequences of antibody heavy and light chains are compared to and aligned with sequences of antibody heavy and light chains for which X-ray crystallographic structural data are available.
  • a principal source of such alignments is Kabat,
  • Crystal structure data is generally obtained from the Protein Data Bank, Bernstein, et al., 0. Mol. Biol., 112: 535-542 (1977), or Abola, et al., Crystal!ographic Databases - Information Content, Software Systems, Scientific Applications, eds. Allen, et al., Data Commission of the Int'l Union of Crystallography, 1987..
  • An initial three dimensional model of the antibody structure is prepared by assigning spatial coordinates derived from residues in the reference antibodies to corresponding residues of the modelled antibody.
  • Standard molecular modelling loop-fitting algorithms are used to adjust regions of extreme difference between the reference and modelled antibodies, particularly differences in CDR loops.
  • An initial three dimensional model of the peptide is prepared using chemical information in CDR-H3 to generate a crude orientation of the peptide in the antibody combining site.
  • Molecular dynamics modelling may be used to refine the fit of the peptide within the antibody combining site and to calculate the energy of the antibody-peptide complex. Alternatively, visual examination of the molecules in space can give approximations of groups important for affinity interactions. Examples of molecular modelling software packages are SYBYL, CHARM, or AMBER. SYBYL, available from Tripos Associates, Inc., St. Louis, Missouri, is preferred.
  • the binding of antigen to antibody involves certain intermolecular interactions.
  • Chemical groups that affect affinity of antibody-antigen binding include ionic, hydrophobic, and hydrogen donor and hydrogen accepting moieties.
  • Ionic groups include amino acids having ionic side-chains such as arginine, lysine, histidine, glutamate or aspartate. Ionic interactions may also include amino or carboxyl terminii of peptide chains.
  • Hydrophobic regions commonly identified in protein structures are associated with a sequence of one or more amino acids with uncharged aliphatic or aromatic sidechains. Groups often involved in hydrogen bonding include serine, threonine, asparagine, glutamine, tyrosine, as well as ionic amino acids, and amide or carbonyl groups of the peptide backbone.
  • affinity enhancing substitutions can be predicted.
  • a positively charged amino acid may be substituted on the antibody molecule at a position of proximity to a negatively charged amino acid contained in the peptide.
  • substitution includes small deletions or insertions of amino acids. Such modifications, as well as one base changes resulting in a single amino acid substitution are conveniently carried out by standard mutation strategies performed on cloned genomic or cDNA. Chemical analogs of naturally occurring amino acids may also be substituted.
  • the antibody-peptide complex After substitutions have been introduced into the antibody molecule, the antibody-peptide complex is remodelled, and the binding energies are calculated and compared to the original complex. Calculations involved in the dynamic modelling are greatly facilitated by the use of a supercomputer.
  • Expression of the antibody genes may be effected by cloning into a variety of suitable vectors. Particularly favored are expression vectors which contain leader sequences such as pelB allowing for secretion of the expression products into the surrounding medium in an active form. Expression is also enhanced if the structural genes are transcribed from a strong promoter such as araB.
  • antibody is raised to a small peptide, followed by modelling of the antibody-antigen complex, as described previously.
  • a larger peptide containing the small peptide sequence is employed in order to identify additional favorable or unfavorable contact residues.
  • antibodies are selected for their exclusive specificity to the peptide and lack of reactivity to the carrier protein, it is to be expected that many amino acid residues in the binding site are available for substitution by residues more compatible with the larger peptide. Substitutions in those residues leading to increased favorable interaction with the large peptide would therefore be expected to increase affinity. In many cases specific antibodies cannot be generated by use of such larger peptide since several epitopes may be present therein.
  • Antibody TA1 is a urine monoclonal antibody specific for the
  • glutamic acid at position 57 of the heavy chain has an unfavorable contact with an aspartic acid from the larger peptide. Substitution of this residue by amino acid residues noted above would be expected to enhance affinity.
  • affinities although assay dependent, are generally considered to be 10 8 L/M and above. Affinities below 10 6 L/M are generally considered to be unacceptable. TA1 with an affinity of 5.37 x 10 5 , although highly specific for fragment F1.2 is unsuitable for use in all assay systems.
  • figure 1 shows the amino acid sequence of a synthetic peptide corresponding to the seven amino acid segment of the C-terminus of the F1.2 cleavage fragment. Additional CYS and GLY have been placed at the N-terminus to facilitate coupling of the peptide to the carrier protein.
  • Figure IB gives the configuration of the conjugate immunogen.
  • Figure 1C gives the amino acid sequence of the larger peptide used in modelling.
  • Figure 2A shows the L3 portion of the kappa chain of the Tal antibody. The highlighted residue is glutamine 98.
  • Figure 2B is the mutated amino acid sequence with arginine substituted at position 98.
  • Figure 2C is the amino acid sequence of H3, the highlight indicating the glutamic acid at 57.
  • Figure 2D gives the same amino acid sequence with the asparagine substitution at position 57.
  • Figure 3 is a stereoscopic representation demonstrating the interactions of the substituted arginine 98 with the three residues on the larger peptide, aspartic acid 16 and 14 and glutamic acid 15,
  • Figure 4 is a steroscopic representation demonstrating the unfavorable interaction between glutamic acid 57 of the heavy chain and aspartic acid 11 of the larger peptide.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On prépare des anticorps de haute affinité contre des petits peptides ou des petits épitopes peptidiques se trouvant à l'intérieur de protéines par substitution d'acides aminés sélectionnés à l'intérieur du site de combinaison d'antigènes, lesquels améliorent l'affinité de liaison d'antigènes. On sélectionne les substitutions d'acides aminés à l'aide de techniques de modelage moléculaire mettant en ÷uvre une analyse dynamique des structures natives et substituées.
PCT/US1991/003009 1990-05-04 1991-05-02 Anticorps de haute affinite contre des petits peptides WO1991017177A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US51908190A 1990-05-04 1990-05-04
US519,081 1990-05-04

Publications (1)

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WO1991017177A1 true WO1991017177A1 (fr) 1991-11-14

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AU (1) AU7796291A (fr)
WO (1) WO1991017177A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0551440A1 (fr) * 1990-09-14 1993-07-21 The Trustees Of The University Of Pennsylvania Production de peptides bioactifs sur la base de la structure des immunoglobulines
WO1998017792A1 (fr) * 1996-10-18 1998-04-30 Sunol Molecular Corporation Molecules de liaison et methodes assistees par ordinateur d'augmentation de leurs affinite de liaison
EP1097174A1 (fr) * 1998-07-10 2001-05-09 Gradipore Limited Anticorps anticoagulants lupiques diriges contre la region f1 de la prothrombine
US6372884B1 (en) 1987-07-16 2002-04-16 The Trustees Of The University Of Pennsylvania Biologically active compounds and methods of constructing and using the same
WO2010137020A1 (fr) 2009-05-28 2010-12-02 Yeda Research And Development Co. Ltd. Méthodes de traitement d'inflammation
EP2322214A1 (fr) 2003-04-17 2011-05-18 Altropus Gmbh Anticorps recombinant immunogène
US20130316353A1 (en) * 2006-05-19 2013-11-28 Alder Biopharmaceuticals, Inc. Culture method for obtaining a clonal population of antigen-specific b cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372884B1 (en) 1987-07-16 2002-04-16 The Trustees Of The University Of Pennsylvania Biologically active compounds and methods of constructing and using the same
EP0551440A1 (fr) * 1990-09-14 1993-07-21 The Trustees Of The University Of Pennsylvania Production de peptides bioactifs sur la base de la structure des immunoglobulines
EP0551440A4 (en) * 1990-09-14 1994-06-08 Univ Pennsylvania Design of bioactive peptides based on immunoglobulin structure
WO1998017792A1 (fr) * 1996-10-18 1998-04-30 Sunol Molecular Corporation Molecules de liaison et methodes assistees par ordinateur d'augmentation de leurs affinite de liaison
US6127524A (en) * 1996-10-18 2000-10-03 Dade Behring Inc. Binding molecules and computer-based methods of increasing the binding affinity thereof
EP1097174A1 (fr) * 1998-07-10 2001-05-09 Gradipore Limited Anticorps anticoagulants lupiques diriges contre la region f1 de la prothrombine
EP1097174A4 (fr) * 1998-07-10 2002-08-14 Gradipore Ltd Anticorps anticoagulants lupiques diriges contre la region f1 de la prothrombine
EP2322214A1 (fr) 2003-04-17 2011-05-18 Altropus Gmbh Anticorps recombinant immunogène
US20130316353A1 (en) * 2006-05-19 2013-11-28 Alder Biopharmaceuticals, Inc. Culture method for obtaining a clonal population of antigen-specific b cells
WO2010137020A1 (fr) 2009-05-28 2010-12-02 Yeda Research And Development Co. Ltd. Méthodes de traitement d'inflammation

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Publication number Publication date
AU7796291A (en) 1991-11-27

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