WO2006071877A2 - Orally deliverable and anti-toxin antibodies and methods for making and using them - Google Patents
Orally deliverable and anti-toxin antibodies and methods for making and using them Download PDFInfo
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- WO2006071877A2 WO2006071877A2 PCT/US2005/047100 US2005047100W WO2006071877A2 WO 2006071877 A2 WO2006071877 A2 WO 2006071877A2 US 2005047100 W US2005047100 W US 2005047100W WO 2006071877 A2 WO2006071877 A2 WO 2006071877A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- 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
- C07K2317/522—CH1 domain
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/524—CH2 domain
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/53—Hinge
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/74—Inducing cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- This invention generally relates to medicine, infectious disease and the use of recombinant antibodies in the treatment of bacterial disease, e.g., those caused by enteric bacterial toxins.
- the invention provides antibodies modified for increased resistance to proteolysis and/or acidic conditions to improve therapeutic efficacy, e.g., for oral administration, and methods for making and using these antibodies.
- the invention provides combinations of monoclonal antibodies, e.g., "synthetic polyclonals,” that work synergistically to neutralize bacterial toxins, e.g., enteric bacterial toxins such as Clostridium difficile toxin A.
- Antibodies are ideal therapeutic agents for their specificity and flexibility.
- the antibody (Ab) targets a cell or an organism through its binding of a specific epitope on an antigen mediated as dictated by the variable region of the antibody molecule.
- the antibody's specificity is complemented by its ability to mediate and/or initiate a variety of biological activities.
- antibodies can modulate receptor-ligand interactions as agonists or antagonists.
- Antibody binding can initiate intracellular signalling to stimulate cell growth, cytokine production, or apoptosis.
- Antibodies deliver agents bound to the Fc region to specific sites.
- Antibodies also elicit antibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and phagocytosis.
- ADCC antibody-mediated cytotoxicity
- CDC complement-mediated cytotoxicity
- phagocytosis phagocytosis
- Antibodies are proteins and thus are susceptible to degradation by proteolytic enzymes present in, for example, the blood and digestive tract. Complete or partial degradation of the antibody prevents a therapeutically effective amount from reaching a distant target site when the antibody is administered systemically.
- the stomach manufactures pepsin and contains hydrochloric acid (pH range between 1.5 and 3).
- the low pH denatures the proteins, resulting in an increased vulnerability to pepsin degradation.
- the average person secretes about 400 mL of gastric fluid per meal, containing 50 to 300 ⁇ g pepsin/mL.
- the transit time in the stomach varies from 0.5 to 4.5 h.
- Protein digestion continues in the duodenum and jejunum, where proteolytic enzymes of the pancreas (trypsin, trypsinogen, chymo-trypsinogen, pro- carboxy-peptidase, and pro-elastase) attack the remaining breakdown products.
- proteolytic enzymes of the pancreas trypsin, trypsinogen, chymo-trypsinogen, pro- carboxy-peptidase, and pro-elastase
- the pH of the small intestine ranges from 6.3 to 7.5 with a transit time in the order of 1 to 4 h. hi the colon, the pH is between 7.5 and 8 with a transit time of 8-16 h, creating a harsh environment for any orally administered protein.
- antibodies are degraded after oral administration. Antibodies are initially degraded into F(ab') 2 , Fab and Fc fragments.
- the F(ab') 2 and Fab fragments retain some of their biological activity.
- degradation in these harsh conditions significantly limits the usefulness of orally administered antibodies.
- An application for orally administered therapeutic antibodies of the invention includes the prevention, treatment and/or diagnosis of gastrointestinal infections and diseases.
- gastrointestinal infections and diseases For example, one enteric pathogen, Clostridium difficile, a common gram-positive, spore-forming, anaerobic bacillus, is the leading cause of nosocomial diarrhea associated with antibiotic therapy.
- C. difficile infection results from a disruption of the normal bacterial flora of the colon, followed by colonization of C. difficile, and the release of destructive toxins that lead to mucosal damage and inflammation.
- Antibiotic therapy is the key factor that is responsible for altering the colonic flora and allowing C. difficile to flourish.
- the invention provides antibodies that are sequence modified, e.g., recombinantly engineered or sequence modified as synthetic proteins, to increase their stability in harsh conditions, e.g., conditions comprising acidic pH or the presence of proteases, and methods of making and using them.
- harsh conditions e.g., conditions comprising acidic pH or the presence of proteases
- antibodies of the invention are useful as therapeutic antibodies that retain biological activity systemically and at the target site, e.g., in the gut (e.g., stomach, intestine) even after oral delivery.
- antibodies of the invention can be delivered orally and retain biological activity in the presence of low pH and/or proteolytic enzymes for efficacy in a digestive tract environment.
- antibodies of the invention have protease resistance (e.g., greater protease resistance than unaltered, or wildtype antibody), increased thermotolerance and/or reduced sensitivity to the negative effects of extreme pH, such as those in the stomach environment, in comparison the a starting, or unaltered (e.g., wildtype) antibody sequence.
- the amount of protease resistance added to the antibody by practicing the invention can be complete or partial, or even only involve the modification of one site.
- antibodies of the invention are therapeutic antibodies retaining biological activity systemically and at a target site.
- the invention provides isolated or recombinant antibodies having resistance to proteolysis (e.g., an increased resistance to proteolysis in comparison the a starting, or unaltered (e.g., wildtype) antibody sequence) made by a method comprising: (a) providing an antibody having at least one protease cleavage site: and (b) engineering (e.g., genetic engineering a nucleic acid coding sequence) at least one amino acid residue modification in the antibody, wherein the at least one amino acid residue modification(s) results in a resistance to (e.g., an increased resistance to) proteolysis, and the at least one amino acid residue modification comprises: (i) at least one amino acid substitution at any one or more of amino acid positions Tl 55, L179, L235, F241, Y296, L309, Y349, L365, L398, F404, Y407 or Y436 of an IgG heavy chain;
- (x) at least one amino acid substitution selected from the group of amino acid substitutions of K133G and K274Q in a IgG heavy chain;
- any one or combination of modifications of steps (i) to (x) are in a variable antibody region, a constant antibody region, or in both the variable antibody region and the constant antibody region.
- the antibody comprises human antibody sequence in the constant region, human antibody sequence in the variable region or human antibody sequence in the constant and the variable region.
- the invention provides an isolated or recombinant antibody comprising a "variant portion" (e.g., a modified amino acid sequence, a modified motif, a modified protease cleavage site, and the like) comprising at least one amino acid modification, wherein said variant portion results in resistance to (e.g., an increased resistance to) proteolysis, and methods for making and using these modified antibodies.
- the modification is in a protease cleavage site or at a site flanking the protease cleavage site.
- the modification is at the Pl, Pl', P2, P3, P4, P2 ⁇ P3 ⁇ or P4 residue of the protease cleavage site.
- the modification to the amino acid sequence generates a protease resistance motif, rendering the protease cleavage site non-cleavable or less susceptible to protease cleavage.
- the modifications are in a variable antibody region, a constant antibody region, or in both the variable antibody region (e.g., a CDR region) and the constant antibody region.
- the variant portion comprises any number of modifications including one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more amino acid residue modifications, hi some embodiments, the modifications are made to the same protease cleavage motif throughout the antibody. In other embodiment, the modifications are made to different protease cleavage motifs.
- the variant portion of the antibody modified comprises any portion of the antibody including the heavy chain, a light chain, or both, or variable region or constant region or both, hi some embodiments, the variant portion (modified part of the antibody sequence) is the Fc region, the hinge region, the CH L domain, the CH 1 domain, the CH 2 domain, the CH 3 domain, the Fab region, or any combination thereof. In alternative embodiments, the variant portion is a V H or V L domain, provided the cleavage site does not have a negative effect on the desired antibody function.
- the modifications in an antibody of the invention comprise at least one mutation in the amino acid sequence of the antibody.
- the mutation can be introduced by modifications, additions or deletions to a nucleic acid encoding the antibody.
- the modifications, additions or deletions to a nucleic acid encoding the antibody can be introduced by any method, including for example error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
- GSSM Gene Site Saturation Mutagenesis
- SLR synthetic ligation reassembly
- the modifications, additions or deletions to a nucleic acid encoding the antibody can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
- the variant portion of an antibody of the invention comprises at least one amino acid substitution at any one or more of amino acid positions T155, L179, L235, F241, Y296, L309, Y349, L365, L398, F404, Y407, and Y436 of an IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the modified amino acid residues (i.e., the variant portion) is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions L234, L242, F243, F275, Y278, Y300, L306, W313, L314, Y319, L351, L368, Y391, F405, L406, L410, F423, L432, or Y436 of an IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions Fl 16, K126, R143, K169 or Kl 83 of a kappa (light) chain, e.g., SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- a kappa (light) chain e.g., SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions K133, K205, K210, K274, K326, K340, R355, K360 or K392 of an IgG heavy chain, e.g., SEQ ID NO: 1 , SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the modified amino acid residues (i.e., the variant portion) of an antibody of the invention comprises at least one amino acid substitution at the Pl or Pl' site of cleavage in a trypsin cleavage motif, wherein the substituted amino acid is K or R 3 whereby the amino acid substitution confers increased resistance to trypsin proteolysis.
- the variant portion comprises at least one amino acid substitution, at the Pl or Pl' site of cleavage in a pepsin cleavage motif, wherein the substituted amino acid is L, F, Y, W, I, or T, whereby the amino acid substitution confers increased resistance to pepsin proteolysis, hi some embodiments, the variant portion comprises at least one amino acid substitution at the Pl or Pl' site of cleavage in a chymotrypsin cleavage motif, wherein the substituted amino acid is F, Y, or W, whereby the amino acid substitution confers increased resistance to chymotrypsin proteolysis.
- antibodies of the invention comprise at least one amino acid substitution, or all of the combination of amino acid substitutions, as set forth in Tables 3 A or 3B (Example 1), Table 4 (Example 1), or Table 5 (Example 1), below.
- the modified amino acid residues (i.e., the variant portion) of an antibody of the invention comprises at least one amino acid substitution selected from the group of amino acid substitutions of L235P, L398Q, F404Y, Ll 791, and T155S in the IgGi heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution selected from the group of amino acid substitutions of Fl 16S and K126A in the kappa light chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution selected from the group of amino acid substitutions of K133G and K274Q in the IgG heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the resistance to proteolysis of an antibody of the invention, or an antibody used in a method of the invention comprises a greater resistance to proteolysis relative to a corresponding unmodified, or "wildtype," antibody.
- the increased resistance to proteolysis can be at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more than that of the unmodified antibody.
- the modified antibody can be partially or completely resistant to cleavage by more than one protease.
- an antibody of the invention, or an antibody used in a method of the invention comprises an IgG, IgM, IgD, IgE, or IgA antibody.
- the antibody is an IgG antibody of a particular isotype, e.g., an IgG 1 , IgG 2 , IgG 3 , or IgG 4 antibody.
- the antibody can be a human, murine, rat, rabbit, bovine, camel, llama, dromedary, or simian antibody.
- the antibody can be a chimeric antibody (e.g., a humanized antibody, for example, a mixture of mouse and human sequence, such as SEQ ID NO:1 and SEQ ID NO:2 as variable regions, with human sequence completing the sequence for a complete antibody), a bispecific antibody, a fusion protein, or a biologically active (e.g., antigen binding) fragment thereof.
- the humanized antibody comprises a variable region comprising a mouse sequence or a sequence derived from a mouse and a constant region comprising a human sequence.
- an antibody of the invention comprises the heavy chain variable region sequence encoded in SEQ ID NO: 1 and the light chain variable region sequence encoded in SEQ ID NO:2 and the remainder of the antibody (e.g., constant region) comprising human sequence, thus making a "humanized” chimeric antibody (similarly, in alternative aspects, the "humanized” chimeric antibody comprises the variable region sequence combinations SEQ ID NO:3 and SEQ ID NO:4, or, SEQ ID NO:5 and SEQ ID NO:6, or, SEQ ID NO:7 and SEQ ID NO:8).
- an antibody of the invention or an antibody used in a method of the invention can be modified in any suitable manner.
- the modification comprises the addition of a post-translational modification site, an N-glycosylation site, an O-glycosylation site, an alkyl chain, or a small molecule.
- the modification comprises covalent or non- covalent addition of a second molecule, e.g., to the Fc chain of the antibody.
- the second molecule comprises an antibody secretory component, a carbohydrate, a disulfide bond site, or a salt bridge site, m one aspect, the second molecule or addition sequence comprises a moiety that serves to "shuttle” the protein from the gut into a cell and/or into the bloodstream (e.g., acting as a "transport” or “carrier” moiety to shuttle an orally administered protein into the blood or plasma).
- a transferrin polypeptide moiety, a cell wall binding domain (CWB) domain of Clostridium difficile toxin A, or an equivalent protein serves as a "shuttle", "transport” or “carrier” moiety or domain to allow an antibody of the invention (or an antibody used in a method of the invention) enter cells (e.g., those lining the gut) or to allow an orally administered antibody of the invention enter into the bloodstream from the gut.
- the "shuttle", “transport” or “carrier” moiety or domain can be sequence spliced into an antibody sequence or be covalently or non-covalently joined to or linked to an antibody sequence.
- the antibody and the "shuttle” domain are linked by a cleavable domain that is cleaved after entry into a cell and/or the bloodstream (thus “liberating” the antibody from the "shuttle” domain).
- the Fc region of an antibody of the invention is further modified to alter an activity of the Fc region, e.g., to abrogate, diminish or enhance an Fc-mediated antibody-mediated cytotoxicity (ADCC), a complement-mediated cytotoxicity (CDC), complement activation, Fc receptor activation and/or binding or phagocytosis.
- ADCC antibody-mediated cytotoxicity
- CDC complement-mediated cytotoxicity
- the Fc region of the antibody can also be further modified to increase or decrease binding affinity to the Fc receptor (FcR).
- the antibody is further modified to have a) an antigen binding activity comparable to, less than, or superior to the unmodified antibody; b) a chemical stability comparable to, less than, or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to, less than, or superior to the unmodified antibody; d) a pH tolerance comparable to, less than, or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced or diminished dimerization of Fc regions; or k) any combination thereof.
- an antibody of the invention has a) an antigen binding activity comparable to, less than, or superior to the unmodified antibody; b) a chemical stability comparable to, less than, or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to, less than, or superior to the unmodified antibody; d) a pH tolerance comparable to, less than, or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced or diminished dimerization of Fc regions; or k) any combination thereof.
- the antibody of the invention maintains its native conformation at about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4 or pH 3 or more acidic (a lower pH) or is further modified to do so.
- the antibody retains biological activity (e.g., antigen binding) at about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4 or pH 3 or more acidic (a lower pH) or is further modified to do so.
- the antibody can further comprise additional mutations that render the antibody more, or less, resistant to pH dependent unfolding.
- the proteolysis inhibited by the antibody modifications of the invention includes digestion mediated by proteases from the gastrointestinal track, the blood, or the bile.
- the proteolysis is mediated by pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsinogen, carboxy-peptidase, pro-carboxy-peptidase, elastase, pro- elastase, or any combination thereof.
- the protease can be selected from a group of proteases released or produced by an exogenous organism or any organism within the digestive tract or released or produced within the digestive tract, e.g., by cells within the tract.
- the proteases inhibited by the antibody modifications of the invention include proteases released or produced by an abnormal, infected, cancerous or otherwise diseased tissue, hi some embodiments, an antibody of the invention, or an antibody used in the methods of the invention, specifically binds to a pathogen.
- the pathogen can be a bacterium, a virus and a fungus. In some cases, the pathogen is an intestinal pathogen, including but not limited to enterotoxigenic E.
- the pathogen is Streptococcus mutans.
- an antibody of the invention specifically binds to a toxin.
- the toxin can be a bacterial toxin, a chemical toxin or an environmental toxin.
- the bacterial toxin is a cholera toxin, an Escherichia coli toxin, a. Streptococcus toxin, a Bordetell ⁇ pertussis toxin, and a Clostridium toxin.
- the Clostridium toxin can comprise a botulinum toxin or a Clostridium difficile toxin.
- the botulinum toxin or Clostridium difficile toxin can comprise botulinum neurotoxin, C.
- An antibody of the invention, or an antibody used in the methods of the invention can specifically bind a virulence factor.
- the virulence factor can be an adherence factor, a coat protein, an invasion factor, a capsule, an exotoxin, or an endotoxin.
- An antibody of the invention can specifically bind to a dietary enzyme.
- the dietary enzyme can be a lipase, an esterase, a urease, a lyase, a protease, an isomerase, a ligase or a synthetase.
- the invention provides an isolated or recombinant nucleic acid comprising a sequence encoding an antibody of the invention, a vector comprising the encoding nucleic acid, and a cell comprising the encoding nucleic acid or the vector comprising the encoding nucleic acid.
- the invention provides a pharmaceutical composition comprising an antibody of the invention, or an antibody used in or made by a method of the invention, and a suitable excipient.
- the composition is formulated as a suspension, a liquid, a capsule, a tablet, a gel, a powder, a microsphere, a liposome, a multiparticulate core particle or a spray.
- the antibody comprises from about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more, or from about 50% to about 95% of the batch size (weight/weight) of the pharmaceutical composition.
- the composition is formulated for enteric delivery.
- the pharmaceutical composition further comprises an enteric coating.
- the invention provides a method of ameliorating, treating or preventing gastrointestinal infections or other disorders caused by a pathogen or a toxin comprising administering orally a pharmaceutically effective amount of the antibody of invention, or the pharmaceutical composition comprising an antibody of the invention, or an antibody used in or made by a method of the invention, to a subject in need thereof, whereby the infection or other disorders is treated or prevented.
- the invention provides a kit for ameliorating or preventing one or more symptoms of virulence factor-associated symptom or disease, comprising a) a pharmaceutical composition comprising an antibody of the invention, or an antibody used in or made by a method of the invention; and b) instruction for administering the pharmaceutical composition.
- the invention also provides a method of identifying a protease cleavage site in an antibody, which method comprises the steps of: a) determining putative sites of protease cleavage in the antibody; b) prioritizing the protease cleavage sites based on the likely exposure of the site to proteases; and c) identifying a site as the protease cleavage site as one whose position results in an exposure to proteases in the three-dimensional antibody structure.
- the putative sites of protease cleavage are determined in step (a) by identifying protease cleavage motifs using N-terminal sequencing, gel electrophoresis analysis, or mass spectral analysis of peptide fragments derived from an antibody digested by protease.
- the putative sites of protease cleavage can also be determined in step (a) by identifying known protease motifs, ⁇ .
- the protease cleavage sites are prioritized in step (b) based on the surface exposure on the folded form of the antibody solved by x-ray crystallography or NMR spectroscopy.
- the protease cleavage sites can also be prioritized in step (b) based on the surface exposure determined using a probe of 1.4 angstroms.
- the invention also provides a computer program product having embedded thereon code for a computer implemented method for identifying a protease cleavage site in an antibody; where in one aspect the method comprises the steps of: a) determining putative sites of protease cleavage in the antibody; b) prioritizing the protease cleavage sites based on the likely exposure of the site to proteases; and c) identifying a site as the protease cleavage site as one whose position results in an exposure to proteases in the three- dimensional antibody structure.
- an article e.g., a product of manufacture, e.g., a computer
- a machine-readable medium including machine-executable instructions, the instructions being operative to cause a machine to practice a method of this invention.
- a computer comprising this computer program product is also provided.
- the identified protease cleavage site has 20% surface area exposure to the probe, wherein the protease cleavage site comprises hydrophobic and aromatic amino acids. In other embodiments, the identified protease cleavage site has 35% surface area exposure to the probe, wherein the protease cleavage site comprises basic amino acids.
- protease sites can be identified by the method of the invention. In one aspect, at least one protease cleavage site is identified. In some embodiments, the protease cleavage sites comprise the same protease cleavage motif. In other embodiments, the protease cleavage sites comprise two or more different protease cleavage motifs.
- the protease cleavage sites can be identified in the Fc region, the Fab region, the hinge region, CL, CH 1 , CH 2 , CH 3 , V L , V H , or a combination thereof.
- protease cleavage motifs include, but are not limited to, a protease selected from the group consisting of pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsin, pro-carboxy-peptidase and pro-elastase.
- the invention provides a method of engineering a protease-resistant antibody, which method comprises the steps of: a) providing an antibody or an amino acid sequence of the antibody; b) identifying at least one protease cleavage site in the amino acid sequence of the antibody; and c) introducing at least one modification in the amino acid sequence of the antibody, whereby the modification results in a variant portion that has an increased resistance to proteolysis.
- the invention provides a method of generating an engineered antibody that is orally deliverable, which method comprises the steps of: a) providing a nucleic acid encoding a wildtype antibody; b) introducing at least one modification into the coding sequence of the wildtype antibody to generate a modified antibody coding sequence, wherein the modification of the coding sequence is in or proximate to the coding sequence of at least one protease cleavage site and the modification results in expression of an antibody that is partially or completely resistant to digestion by the protease; and c) expressing the modified antibody coding sequence of step b) to generate an engineered antibody, wherein the engineered antibody retains its ability to specifically bind to antigen in the digestive system following oral administration, thereby rendering the engineered antibody orally deliverable.
- the modification is in a protease cleavage site or at a site flanking the protease cleavage site. In alternative embodiments, the modification is at the Pl , Pl', P2, P3, P4, P2 ⁇ P3 ⁇ or P4 residue of the protease cleavage site.
- the modification to the amino acid sequence generates a protease resistance motif, rendering the protease cleavage site non-cleavable or less susceptible to protease cleavage.
- An engineered antibody of the invention can comprise any number of modifications, including but not limited to, two, three, four, five, six, seven, eight, nine, ten, eleven, or more amino acid modifications.
- the modifications can be in a protease cleavage site or at a site flanking the protease cleavage site.
- the modification can be made to the same protease cleavage motif within the antibody or to different protease cleavage motifs.
- the modification is made in a protease cleavage site that is not flanked by an amino acid residue known to inhibit or attenuate protease cleavage.
- amino acids include Pro, Lys, Arg and His.
- An engineered antibody of the invention can comprise an IgG, IgM, IgD, IgE, or IgA antibody.
- the antibody is an IgG antibody.
- the antibody can be an IgG 1 , IgG 2 , IgG 3 , or IgG 4 antibody.
- the antibody can be a human, murine, rat, rabbit, bovine, camel, llama, dromedary, or simian antibody.
- the antibody can be a humanized antibody, a chimeric antibody, a bispecific antibody, a fusion protein, or a biologically active (e.g., antigen binding) fragment thereof.
- An engineered antibody of the invention can be modified in any portion of the antibody including the heavy chain, a light chain, or both.
- the modified portion is the Fc region, the hinge region, the CH L domain, the CH 1 domain, the CH 2 domain, the CH 3 domain, the Fab region, or any combination thereof.
- the modified portion is a V H or V L domain, provided the cleavage site does not have a negative effect on the desired antibody function.
- the modifications in the antibody of the invention comprise at least one mutation in the amino acid sequence of the antibody.
- the mutation is introduced by modifications, additions or deletions to a nucleic acid encoding the antibody.
- the modifications, additions or deletions to a nucleic acid encoding the antibody can be introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
- GSSM Gene Site Saturation Mutagenesis
- SLR synthetic ligation reassembly
- the modifications, additions or deletions to a nucleic acid encoding the antibody can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction- purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
- an engineered antibody of the invention comprises at least one amino acid substitution at any one or more of amino acid positions Tl 55, L179, L235, F241, Y296, L309, Y349, L365, L398, F404, Y407, and Y436 of an IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions L234, L242, F243, F275, Y278, Y300, L306, W313, L314, Y319, L351, L368, Y391, F405, L406, L410, F423, L432, or Y436 of, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions Fl 16, K126, R143, Kl 69 or Kl 83 of a kappa chain, e.g., SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions K133, K205, K210, K274, K326, K340, R355, K360 or K392 of, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- an engineered antibody of the invention comprises at least one amino acid substitution at the Pl or Pl' site of cleavage in a trypsin cleavage motif, wherein the substituted amino acid is K or R 5 whereby the amino acid substitution confers increased resistance to trypsin proteolysis.
- an engineered antibody comprises at least one amino acid substitution, at the Pl or Pl' site of cleavage in a pepsin cleavage motif, wherein the substituted amino acid is L, F, Y, W, I, or T, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the engineered antibody comprises at least one amino acid substitution at the Pl or Pl ' site of cleavage in a chymotrypsin cleavage motif, wherein the substituted amino acid is F, Y, or W, whereby the amino acid substitution confers increased resistance to chymotrypsin proteolysis.
- an engineered antibody of the invention comprises at least one amino acid substitution selected from the group of amino acid substitutions of L235P, L398Q, F404Y, Ll 791, and Tl 55 S in an IgG 1 heavy chain, wherein the numbering of the residues in an engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution(s) confer increased resistance to pepsin proteolysis.
- an engineered antibody comprises at least one amino acid substitution selected from the group of amino acid substitutions of Fl 16S and K126A in a kappa light chain, wherein the numbering of the residues in an engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- an engineered antibody comprises at least one amino acid substitution selected from the group of amino acid substitutions of K133G and K274Q in an IgG heavy chain, wherein the numbering of the residues in the engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- an engineered antibody of the invention (including any antibody made by a method of the invention, in addition to those disclosed herein) has greater resistance to proteolysis relative to the wildtype antibody.
- the increased resistance to proteolysis is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more than that of the unmodified (e.g., unaltered, or "wildtype") antibody.
- An engineered antibody can be partially or completely resistant to cleavage by more than one protease.
- An engineered antibody of the invention (including any antibody made by a method of the invention, in addition to those disclosed herein) can be modified (including further modified) in any suitable manner.
- the modification comprises the addition of a post-translational modification site, an N-glycosylation site, an O-glycosylation site, an alkyl chain, or a small molecule.
- the modification comprises covalent or non-covalent addition of a second molecule to the Fc chain of the antibody.
- the second molecule comprises an antibody secretory component, a carbohydrate, a disulfide bond site, or a salt bridge site.
- the Fc region of an engineered antibody of the invention is further modified to enhance antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and/or phagocytosis.
- ADCC antibody-dependent cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- phagocytosis phagocytosis.
- the Fc region of the antibody can also be further modified to increase binding affinity to the Fc receptor (FcR).
- an engineered antibody is further modified to have a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- an antibody of the invention has a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- an engineered antibody of the invention is modified to maintain (is modified such that it maintains) its native, or a least a functional (antigen-binding), conformation at about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4 or pH 3 or more acidic (a lower pH).
- the antibody retains at least some biological activity (antigen-binding) at pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4 or pH 3 or more acidic conditions.
- the antibody can further comprise additional mutations that render the antibody more resistant to pH dependent unfolding.
- an engineered antibody of the invention is modified to maintain (is modified such that it maintains) its native, or a least a functional (antigen-binding), conformation at alkaline conditions, e.g., pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11.
- non-natural amino acids are incorporated into an antibody of the invention to further increase resistance to pH dependent unfolding or resistance to proteases, e.g., see U.S. Patent App. No. 20050260711.
- the proteolysis is the digestion mediated by proteases from the gastrointestinal tract, the blood, or the bile.
- the proteolysis is mediated by pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsinogen, carboxy-peptidase, pro-carboxy-peptidase, elastase, pro-elastase, or any combination thereof.
- the protease can be selected from a group of proteases released or produced by an exogenous organism or any organism within the digestive tract, or released or produced in the digestive tract.
- the protease can be selected from a group of proteases released by an abnormal, infected, cancerous or otherwise diseased tissue.
- an engineered antibody of the invention specifically binds to a pathogen.
- the pathogen can be a bacterium, a virus and a fungus.
- the pathogen is an intestinal pathogen, including but not limited to enterotoxigenic E. coli, rotavirus, Cryptosporidium parvum, Clostridium difficile, Shigella flexneri, Enterococcus faecalis, Enterococcus faecium, Campylobacter jejuni, Staphylococcus aureus, E.
- the pathogen is Streptococcus mutans.
- an engineered antibody of the invention specifically binds to a toxin.
- the toxin can be selected from the group consisting of a bacterial toxin, a chemical toxin and an environmental toxin.
- the bacterial toxin is a cholera toxin, an Escherichia coli toxin, a Streptococcus toxin, a B ordetella pertussis toxin, and a Clostridium toxin.
- the Clostridium toxin can comprise a botulinum toxin or a Clostridium difficile toxin.
- the botulinum toxin or Clostridium difficile toxin can comprise botulinum neurotoxin, C. difficile toxin A (see below), or C. difficile toxin B (see, e.g., U.S. Patent App. No. 20040028705).
- An engineered antibody of the invention can specifically bind a virulence factor.
- the virulence factor can be an adherence factor, a coat protein, an invasion factor, a capsule, an exotoxin, or an endotoxin.
- An engineered antibody of the invention can specifically bind to a dietary enzyme.
- the dietary enzyme can be a lipase, an esterase, a urease, a lyase, a protease, an isomerase, a ligase or a synthetase.
- the invention provides an isolated or recombinant nucleic acid comprising a sequence encoding an engineered antibody of the invention (including the antibodies disclosed herein), a vector comprising the encoding nucleic acid, and a cell comprising the encoding nucleic acid or the vector comprising the encoding nucleic acid.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an engineered antibody of the invention (including the antibodies disclosed herein, and antibodies used in or made by a method of the invention), and a suitable excipient.
- the composition is formulated as a suspension, a liquid, a capsule, a tablet, a gel, a microsphere, a liposome, a multiparticulate core particle or a spray.
- the antibody comprises from about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more, or from about 50% to about 95%, of the batch size (weight/weight) of the pharmaceutical composition.
- the composition is formulated for enteric delivery.
- the pharmaceutical composition further comprises an enteric coating.
- the invention provides a method of ameliorating, treating or preventing gastrointestinal infections or other disorders caused by a pathogen or a toxin comprising administering orally a pharmaceutically effective amount of an engineered antibody of invention, or antibodies made by a method of the invention, or the pharmaceutical composition comprising an antibody of the invention, to a subject in need thereof, whereby the infection or other disorders is treated or prevented.
- the invention provides a kit for ameliorating or preventing one or more symptoms of toxin-associated or virulence factor-associated symptom or disease, comprising a) the pharmaceutical composition comprising an engineered antibody of the invention (including antibodies made by a method of the invention, and the exemplary antibodies disclosed herein); and b) instruction for administering the pharmaceutical composition.
- the invention provides methods for ameliorating and/or preventing toxicity associated with Clostridium difficile, comprising administering to a subject in need thereof: a) a therapeutically effective amount of a first monoclonal antibody (or equivalent synthetic Abs), wherein the first monoclonal antibody comprises the heavy chain variable region sequence of SEQ ID NO.l and the light chain variable region sequence of SEQ ID NO:2; and in one aspect, the remainder of the antibody (e.g., the constant region) comprises human Ab sequence; and b) a therapeutically effective amount of a second monoclonal antibody (or equivalent synthetic Ab), wherein the second monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO:3 and the light chain variable region sequence of SEQ ID NO:4 and in one aspect, the remainder of the antibody (e.g., the constant region) comprises human Ab sequence, whereby these antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin A.
- a therapeutically effective amount of a first monoclonal antibody or equivalent synthetic Abs
- the method further comprises administering a third monoclonal antibody (or equivalent synthetic Ab), wherein the third antibody is a monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO.6, and in one aspect the remainder of the antibody (e.g., the constant region) comprises human Ab sequence, whereby the antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin B.
- a third monoclonal antibody or equivalent synthetic Ab
- the third antibody is a monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO.6, and in one aspect the remainder of the antibody (e.g., the constant region) comprises human Ab sequence, whereby the antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin B.
- the invention provides a method of ameliorating or preventing toxicity associated with Clostridium difficile, comprising administering to a subject in need thereof: a) a first antibody that partially or completely inhibits binding of a Clostridium difficile toxin A to a cell; and b) a second antibody that partially or completely inhibits intracellular internalization of the Clostridium difficile toxin A, wherein the first antibody and the second antibody bind to the Clostridium difficile toxin A at non-overlapping epitopes.
- the method further comprises administering a therapeutically effective amount of at least a third antibody that partially or completely neutralizes Clostridium difficile toxin B.
- the second antibody is not the monoclonal antibody PCG-4 (see discussion below; Lyerly (1986) Infect Immun. 54:70-76).
- the invention also provides pharmaceutical compositions comprising these combinations of antibodies which are, in one aspect, formulated for oral administration.
- the first and second antibodies synergize to neutralize the toxin
- the first monoclonal antibody and the second monoclonal antibody bind to a Clostridium difficile toxin A at ToxA: 1800-2710.
- the third antibody is a monoclonal antibody that binds to a Clostridium difficile toxin B at ToxB: 1807-2366.
- the first monoclonal antibody and the second monoclonal antibody do not bind Clostridium difficile toxin B, and the third monoclonal antibody does not bind Clostridium difficile toxin A.
- the invention provides methods for ameliorating or preventing toxicity associated with abacterial toxin, comprising administering to a subject in need thereof (a) a first antibody that partially or completely inhibits binding of the bacterial toxin to a cell; and (b) a second antibody that partially or completely inhibits intracellular internalization of the toxin, wherein the first antibody and the second antibody bind to the toxin at non-overlapping epitopes.
- the bacterial toxin comprises a Clostridium difficile toxin A or a Clostridium difficile toxin B.
- the first and the second antibodies are formulated together in a pharmaceutical composition. The first and the second antibodies can be formulated for oral administration.
- the invention provides pharmaceutical compositions comprising (a) a first antibody that partially or completely inhibits binding of the bacterial toxin to a cell; and (b) a second antibody that partially or completely inhibits intracellular internalization of the toxin, wherein the first antibody and the second antibody bind to the toxin at non-overlapping epitopes.
- the bacterial toxin comprises a Clostridium difficile toxin A or a Clostridium difficile toxin B.
- the first and the second antibodies are formulated together in a pharmaceutical composition.
- the first and the second antibodies are formulated for oral administration.
- the methods of the invention can be useful in the treatment of the Clostridium toxin- related toxicity in a subject, wherein the toxicity comprises Clostridium- cooperate ⁇ Qd diarrhea, colitis or a related condition, whereby one or more symptoms of the Clostridium-mduced diarrhea, colitis, or related condition are ameliorated or prevented following administration of a pharmaceutical composition of the invention, e.g., a composition comprising one or more monoclonal antibodies of the invention, or an antibody modified by a method of the invention.
- the methods of the invention employ monoclonal antibodies comprising recombinant or synthetic antibodies.
- One or more of the antibodies can be rendered partially or completely resistant to proteolysis and/or orally deliverable using the antibody engineering methods of the invention.
- the invention provides a monoclonal antibody, or a biologically active (e.g., antigen binding) fragment thereof (or equivalent synthetic Abs), that binds to Clostridium difficile toxin A 3 wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and/or SEQ ID NO:2; and/or SEQ ID NO:3 and/or SEQ ID NO:4 (and in alternative aspects, the remainder of the antibody - such as the constant region - comprises human Ab sequence).
- the invention also provides an isolated or recombinant nucleic acid comprising a sequence encoding the antibody, a vector comprising the nucleic acid, and a cell comprising the nucleic acid or the vector. Pharmaceutical compositions and kits comprising the antibody are also provided.
- the invention provides a monoclonal antibody, or a biologically active (e.g., antigen binding) fragment thereof (or equivalent synthetic Abs), that binds to Clostridium difficile toxin B, wherein the variable region sequences of the antibody comprise SEQ ID NO:5 and/or SEQ ID NO: 6 (and in alternative aspects, the remainder of the antibody - such as the constant region - comprises human Ab sequence).
- the invention also provides an isolated or recombinant nucleic acid comprising a sequence encoding the antibody, a vector comprising the nucleic acid, and a cell comprising the nucleic acid or the vector. Pharmaceutical compositions and kits comprising the antibody are also provided.
- the antibodies of the invention can comprise an IgG antibody or fragments thereof.
- the antibody comprises a human, murine, rat, rabbit, camel, bovine, llama, dromedary, or simian antibody.
- the antibody comprises a humanized antibody, chimeric antibody, bispecific antibody, fusion antibody, a minibody or nanobody, a bivalent scFv (i.e., a diabody, having two chains and two binding sites, and maybe monospecific or bispecific), a triabody (three single chain antibodies), scFv, or biologically active (e.g., antigen binding) fragments thereof (see, e.g., U.S. Patent App. Pub. No. 20050234225).
- An antibody can be modified to increase resistance to proteolysis using the methods of the invention.
- the antibody can be modified to be orally deliverable, using, for example, the methods of the invention.
- the antibody can be modified to abrogate, diminish or enhance antibody-mediated cytotoxicity (ADCC), a complement-mediated cytotoxicity (CDC), or phagocytosis.
- ADCC antibody-mediated cytotoxicity
- CDC complement-mediated cytotoxicity
- phagocytosis phagocytosis.
- the Fc region of the antibody is modified to abrogate, diminish or increase (enhance) binding affinity to the Fc receptor (FcR).
- the antibody is modified to have: a) an antigen binding activity comparable to, less than or superior to the unmodified antibody; b) a chemical stability comparable to, less than or superior to the unmodified antibody, c) a thermostability or thermotolerance comparable to, less than or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- the antibody has: a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- the invention provides a monoclonal antibody produced by or isolated from a hybridoma selected from the group consisting of ATCC Accession No. (Ab designated 227 or 3359), ATCC Accession No. (Ab designated 543 or 3358), ATCC Accession No. (Ab designated F85), ATCC Accession No. (Ab designated F2), and ATCC Accession No. (Ab designated F87) (or equivalent synthetic Abs).
- a synthetic, isolated or recombinant antibody wherein the antibody has the same antigen binding specificity (e.g., binds to the same epitope) as a monoclonal antibody of the invention, or antibodies made by a method of the invention, including, e.g., antibodies having the same antigen binding specificity as the Ab designated 227 or 3359, the Ab designated 543 or 3358, the Ab designated F85, the Ab designated F2 and/or the Ab designated F87, or, a chimeric (e.g., "humanized") antibody having the same sequence or binding specificity as an antibody comprising the (light/heavy) variable region pairs SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6.
- the antibody has the same antigen binding specificity (e.g., binds to the same epitope) as a monoclonal antibody of the invention, or antibodies made by a method of the invention, including
- the methods and compositions of the invention greatly increase the therapeutic efficacy of antibodies by increasing the stability of the antibody following administration to the patient.
- Figure 1 shows the pepsin digestion profile OfIgG 1 , IgG 2 , IgG 3 , and IgG 4 , as discussed in Examples 1 and 3, below.
- Figure 2 depicts the pepsin digestion profile of IgGi on a reducing gel, as discussed in Example 1 , below.
- Figure 3 shows the chimeric antibody rPBA3 after it was expressed in mammalian cells, purified, and dialyzed, as discussed in detail in Example 1 , below.
- Figure 4 depicts the pH dependence of IgG 1 structure demonstrated by Circular
- Figures 5A and 5B show the degradation of rPBA-3 by acid, pepsin and high temperature, as discussed in detail in Example 1 , below.
- Figures 6 A and 6B shows the pepsin digestion profile of wildtype (unaltered) and mutant antibodies at pH 1.2, as discussed in detail in Example 1, below.
- Figures 7 A and 7B illustrates pepsin digestion profile of wildtype and mutant antibodies at pH 3.0, as discussed in detail in Example 1, below.
- Figure 8 A illustrates the CLUSTALW alignment of repeat domains of several CWBs, as discussed in detail in Example 2, below.
- Figure 8B illustrates spectra of toxin A and toxin B repeat domains, as discussed in detail in Example 2, below.
- Figure 8C illustrates the temperature dependence of CWB-domain structure, as discussed in detail in Example 2, below.
- Figure 9 illustrates photographs of adherent CHO cells cultured in the absence (media only) and presence of 20 ng (100 ⁇ L total volume) toxin A with and without anti-toxin A antibodies present, as discussed in detail in Example 2, below.
- Figures 1OA, 1OB, 1OC, 1OD and 1OE illustrates the antibody competition for toxin binding sites using static concentrations of toxin and titrating the amount of antibody in solution, as discussed in detail in Example 2, below.
- Figure 1 IA illustrates the thermal denaturation of ToxA:2459-2710 in the absence and presence of CWB-binding ligands monitored by the CD signal of the protein at 230 nm, as illustrated in Figure 11 B-E, as discussed in detail in Example 2, below.
- Figure 12 depicts the attenuation of cell surface binding of ToxA: 2459-2710 by antitoxin A antibodies as determined by flow cytometry, as discussed in detail in Example 2, below.
- Figure 13 illustrates the ileal loop model, as discussed in detail in Examples 2 and 4, below.
- Figures 14A to D show the activity of anti-Clost ⁇ dium difficile toxin A antibodies in the ileal loop model, as discussed in detail in Examples 2 and 4, below.
- Figures 15A to F illustrate the histology of rat intestinal mucosa, as discussed in detail in Example 4, below.
- Figure 16 shows weight versus length measurement for rat ileal loops incubated with saline, 5 ⁇ g toxin A independently and in the presence of various concentrations of 3359 (or
- Figure 17(A) illustrates data showing the titration of antibody 3358 in the presence of fixed amounts of the 3359 antibody
- Figure 17(B) illustrates the titration of antibody 3359 in the presence of fixed amounts of the 3358 antibody, as discussed in detail in Example 5, below.
- Figures 18 A to D illustrate data for antibody competition for toxin binding site experiments, studied by surface plasmon resonance, as discussed in detail in Example 5, below.
- Figures 19A to D illustrate data showing CHO cell surface binding of ToxA:l IR, as determined by flow cytometry, as discussed in detail in Example 5, below.
- Figures 2OA to F illustrate SSC and fluorescence profile data showing the effect of antitoxin A antibodies on ToxA:l IR cell surface association;
- Fig. 2OA and 2OC illustrate data showing that both the 3358 and rPCG-4 antibodies significantly increased the amount of CWB- domain detected at the cell surface;
- Fig. 2OB illustrates data showing that the 3359 antibody inhibited cell surface association of ToxA:l IR;
- Fig. 2OD illustrates data showing that the combination of the 3359 and 3358 antibody inhibits ToxA: 1 IR binding the CHO cell surface similar to the behavior of 3359 alone; as discussed in detail in Example 5, below.
- Figures 21 A to F illustrate photomicrographs of the histology of rat intestinal mucosa after treatment with toxin A with or without anti-toxin A antibodies, as discussed in detail in Example 5, below.
- Figure 22 graphically illustrates data from a rat ileal loop assay showing that antibodies 3359 and 3358 prevent toxin A-induced intestinal fluid secretion in rat ileal loops, as discussed in detail in Example 5, below.
- Figure 23 graphically illustrates data showing the efficacy of systemic dosing with antitoxin A and anti-toxin B antibodies in C. difficile in hamsters, as discussed in detail in Example 5, below.
- Figures 24A to F illustrate photomicrographs of the histology of hamster intestinal mucosa after C. difficile challenge, as discussed in detail in Example 5, below.
- Figure 25 illustrates the results of the pepsin digestion profiles of IgGl, IgG2, IgG3 and IgG, as discussed in detail in Example 6, below.
- Figures 26A and 26B illustrate data showing that acidic conditions alone in the absence of pepsin led to decreases in functional antibody, as discussed in detail in Example 6, below.
- Figures 27A to D illustrate a graphic summary of data showing the pH dependence of
- IgGl structure demonstrated by Circular Dichroism (CD) experiments, as discussed in detail in Example 6, below.
- Figures 28A and B illustrate the pepsin digestibility of the wildtype antibody and the mutant combinations at acidic conditions where the molecule remained folded and the pepsin is still active; examples of pepsin digestions are shown in Fig. 28A; data summarized in Fig. 28B, as discussed in detail in Example 6, below.
- Figure 29 illustrates pictures of cells cultured in the presence or absence of toxin and toxin-neutralizing antibody after pepsin digestion, as discussed in detail in Example 6, below.
- Figure 30 illustrates pictures of gels showing pancreatin digestion profiles of IgGl, IgG2, IgG3 and IgG4, as discussed in detail in Example 6, below.
- Figure 31 in table form summarizes combinations of antibody mutations identified to confer resistance to pancreatin digestion, as discussed in detail in Example 6, below.
- Figure 32 illustrates data from a time course of IgG recovery from the stomach, cecum and distal colon after oral administration of antibody, as discussed in detail in Example 6, below.
- Figure 33 illustrates data from a time course of antibody recovery from mouse feces after oral administration of 1 mg of the optimized antibody and a control antibody, as discussed in detail in Example 6, below.
- Figure 34 depicts a time course of antibody recovery from mouse feces after oral administration of 2.5 mg of the optimized antibody and a control antibody, as discussed in detail in Example 6, below.
- Figure 35 illustrates a photograph of a Western blot analysis of samples described in
- the invention provides antibodies modified to improve their therapeutic efficacy upon administration to a subject, e.g., after oral administration, and methods for making and using them.
- amino acid residues in the antibody are modified to improve the stability of the antibody.
- an antibody of the invention is modified or engineered to increase protease resistance, thereby reducing or eliminating sensitivity to proteolysis.
- the antibodies are further modified to alter other characteristics, such as thermotolerance, pH stability, binding affinity, immunogenicity, half-life, host cell expression and stability in pharmaceutical formulations that also contribute to therapeutic efficacy.
- an antibody of the invention also can be modified to include post-translation modification sites, second molecules, disulfide bond sites, or salt bridges that enhance antibody stability upon administration, particularly by the oral route.
- the invention also provides methods for engineering such antibodies, e.g., by modifying the nucleic acid sequence that encodes the antibody.
- Antibodies of the invention including antibodies made by methods of the invention, and antibodies described herein, are useful in methods of ameliorating, treating or preventing a disease, infection, or other disorder caused by an abnormal cell, pathogen, or toxin comprising administering orally a pharmaceutically effective amount of the antibody.
- the antibody of the invention can be in the form of a pharmaceutical composition for administration to a subject in need thereof, e.g., to treat, prevent or ameliorate a disease, infection or other disorder.
- An antibody of the invention can be co-administered with at least one bioactive agent or drug that can include, but are not limited to an antibiotic, a second antibody, a radionuclide, a chemotherapeutic agent, or a biologically active (e.g., antigen binding or toxic) protein, hi some embodiments, the biologically active protein is a toxin-degrading or inactivating protease.
- a bioactive agent or drug can include, but are not limited to an antibiotic, a second antibody, a radionuclide, a chemotherapeutic agent, or a biologically active (e.g., antigen binding or toxic) protein, hi some embodiments, the biologically active protein is a toxin-degrading or inactivating protease.
- Treatment of certain infectious diseases, such as Clostridium difficile is particularly amenable to treatment with oral antibodies, including the antibodies of the invention, such as antibodies made by methods of the invention.
- an antibody of the invention is co-administered with an agent that facilitates protease resistance or stability to harsh conditions, e.g., extremes in pH, such as the acidic conditions of the stomach and/or the alkaline conditions of the intestine.
- harsh conditions e.g., extremes in pH, such as the acidic conditions of the stomach and/or the alkaline conditions of the intestine.
- Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs; natural killer (NK) cells, neutrophils, and macrophages can recognize bound antibody on a target cell and subsequently lyse the target cell. See e.g., Ravetch (1991) Annu. Rev. Immunol. 9:457-92.
- Amino acid or “amino acid sequence” include an oligopeptide, peptide, polypeptide, peptidomimetic or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules that encodes an antibody of the invention, or biologically active (e.g., antigen binding) fragment thereof.
- polypeptide and protein include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
- polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
- the peptides and polypeptides of the invention also include all "mimetic” and “peptidomimetic” forms.
- polypeptide also includes peptides and polypeptide comprising non-natural residues.
- engineered protease site refers to a protease site that has been modified from the naturally existing sequence by at least one amino acid substitution.
- protease site that has been modified from the naturally existing sequence to generate a motif that is less susceptible or resistant to protease cleavage.
- proteases refers to all polypeptides, e.g., enzymes, which catalyze the hydrolysis of peptide bonds. Protease activity includes hydrolyzing peptide bonds at high temperatures, low temperatures, alkaline pHs and at acidic pHs.
- the proteases can be naturally occurring, recombinantly generated, and/or synthetic. Exemplary proteases include pepsin, trypsin, trypsinogen, chymo-trypsin, pro-carboxy-peptidase, and pro-elastase.
- nucleic acids and nucleic acid sequences include oligonucleotides, nucleotides, polynucleotides or fragments of any of these, to e.g., DNA or RNA ⁇ e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded which encodes an antibody of the invention, or a biologically active (e.g., antigen binding) fragment thereof.
- the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides.
- the term also encompasses nucleic-acid- like structures with synthetic backbones.
- isolated includes a material (e.g., an antibody used to practice the invention) removed from its original environment, e.g., the natural environment if it is naturally occurring.
- a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides can be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
- Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
- the invention comprises isolated, recombinant or synthetic Ab light or variable region polypeptides that are "substantially identical" to an exemplary sequence of the invention, e.g., having a sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8 5 and having the same (or substantially the same) antigen binding specificity, as discussed below.
- a "substantially identical" amino acid sequence also can include a sequence that differs from a reference sequence (e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8,) by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
- a reference sequence e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO
- a conservative amino acid substitution substitutes one amino acid for another of the same class ⁇ e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
- One or more amino acids can be deleted, for example, from an antibody, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for antibody activity can be removed.
- a "substantially identical" amino acid sequence also can include a sequence that hybridizes under stringent conditions to a reference sequence (e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO.4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8), as discussed, below.
- a reference sequence e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO.4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8.
- the term “synergize” refers to the ability of one agent to increase the anti-pathogenic or neutralizing effect of a second agent. Synergistic activity, thus, includes but is not limited to an increased biological effect ⁇ e.g., more potent or longer lasting) using the two agents together that is not observed when the agents are used separately, a more effective biological effect, e.g., elimination of multiple types of toxicity not achievable with the administration of a single agent, or a reduction in the amount of agents necessary for administration to achieve the biological effect observed with a single agent.
- pathogen refers to any organism that induces or elicits a undesired symptom or disease state.
- a pathogen may be a bacteria, virus, or fungus.
- the pathogen can be an organism residing at a site that has gained antibiotic resistance or has overgrown other flora.
- the term "subject” embraces human as well as other animal species, such as, for example, canine, feline, bovine, porcine, rodent, and the like. It will be understood by the skilled practitioner that the subject having a pathogen or disease targeted by the antibody of the invention.
- the term “ameliorating, treating or preventing” include a postponement of one or more symptoms associated with the gastrointestinal infection or other disorder, a reduction in the severity of such symptoms that will or are expected to develop, or a complete elimination of such symptoms.
- These terms further include ameliorating existing pathogen- related symptoms, reducing duration of disease, preventing additional symptoms, ameliorating or preventing serious sequelae, preventing or reversing mortality, reducing or preventing fecal shedding, and reducing or preventing pathogen transmission .
- the terms denote that a beneficial result has been conferred on a subject with a pathogen, or with the potential of exposure to such a pathogen.
- the term "ameliorating, treating or preventing” further includes inhibiting the activity of a toxin which is associated with the development of a particular disease state or medical condition.
- the microbial toxin can be an endotoxin or exotoxin produced by a microorganism, such as a bacterium, a fungus or a protozoan.
- the toxin can be inhibited by any mechanism, including, but not limited to, binding of the toxin by the antibody.
- a “therapeutically effective amount” or a “pharmaceutically effective amount” is an amount sufficient to inhibit or prevent, partially or totally, tissue damage or other symptoms associated with the action of the virulence factor within or on the body of the subject or to prevent or reduce the further progression of such symptoms.
- a therapeutically effective dose refers to that ingredient alone.
- a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
- bioactive agent refers to any synthetic or naturally occurring compound that binds the antigen and/or enhances or mediates a desired biological effect.
- Bioactive agents include, for example, a pharmaceutical agent, such as a chemotherapeutic drug, a toxin, a cytokine, a ligand, another antibody, regulatory moieties such as zinc fingers and leucine zippers, or any combination thereof.
- the agent in an antitumor agent.
- the term "antitumor agent” refers to agent that inhibits tumor growth through the induction of an immune response, stasis, cell death, senescence, apoptosis, ankoisis (constitutive epithelial cell apoptosis resulting from detachment from basement membrane) or necrosis.
- Antibody compositions and related methods include, for example, a pharmaceutical agent, such as a chemotherapeutic drug, a toxin, a cytokine, a ligand, another antibody, regulatory moieties such as zinc fingers and leucine zippers, or any combination thereof.
- the agent in an antitumor agent.
- the invention provides antibodies with improved therapeutic efficacy.
- the invention provides isolated, recombinant or synthetic antibodies comprising a variant portion and/or a constant region comprising at least one amino acid modification, wherein said variant portion results in an increased resistance to proteolysis.
- an antibody of the invention comprises the heavy chain variable region sequence encoded in SEQ ID NO: 1 and the light chain variable region sequence encoded in SEQ ID NO.2, and the remainder of the antibody (e.g., constant region) comprises human sequence, thus making a "humanized" chimeric antibody.
- the "humanized" chimeric antibody comprises the variable region sequence combinations SEQ ID NO:3 and SEQ ID NO:4, or, SEQ ID NO:5 and SEQ ID NO:6, or, SEQ ID NO:7 and SEQ ID NO:8.
- the invention also provides novel combinations of monoclonal antibodies that when administered together have a synergistic antitoxin effect, as described herein.
- Any suitable method of generating an antibody to be modified using the methods of the invention can be employed.
- Methods of immunization, producing and isolating antibodies are known to those of skill in the art and described in the scientific and patent literature. See, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N. Y.
- Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom Trends Biotechnol. 15:62-70 (1997); Katz (1997) Annu. Rev. Biophys. Biomol Struct. 26:27-45 (1997).
- any isolated or recombinant antibody or biologically active (e.g., antigen binding) fragment thereof can be modified to increase the resistance to proteolysis.
- Antibodies can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. The antibodies can be recombinantly expressed in vitro or in vivo. The antibodies of the invention can be made and isolated using any method known in the art. Antibodies of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.
- antibody synthesis can be performed using various solid-phase techniques ⁇ see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
- Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, etal, MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition).
- the eliciting antigen may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art.
- the eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein).
- the antigen may be produced in a genetically modified cell.
- the DNA encoding the antigen may genomic or non-genomic ⁇ e.g., cDNA) and encodes at least one epitope in the extracellular domain of the antigen.
- Any genetic vectors suitable for transformation of the cells of interest may be employed, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids.
- the antibody of the methods and compositions herein specifically bind at least one epitope of the extracellular domain of the virulence factor of interest.
- antibody refers to any form of a peptide, polypeptide or peptidomimetic derived from, modeled after or substantially encoded by, an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g., FUNDAMENTAL IMMUNOLOGY, Fifth Edition, W. E. Paul, ed., Lipincott, Williams & Wilkins (2003); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
- antibody fragments are those that retain antigen-binding and include Fab, Fab', F(ab') 2 , Fd, and Fv fragments; diabodies; triabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; minibodies, nanobodies, minibodies and multispecific antibodies formed from antibody fragments.
- an Ab binding fragment or derivative retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of its biological activity.
- an antigen-binding fragment of the invention can include conservative amino acid substitutions or non-natural residues that do not substantially alter its binding and/or biologic activity.
- Antibodies of the invention also encompass monoclonal (including full length monoclonal antibodies), polyclonal, multispecific (e.g., bispecific), minibody, heteroconjugate, diabody, triabody, chimeric, humanized, human, murine, and synthetic antibodies as well as antibody fragments that specifically bind a desired antigen and exhibit a desired binding property and/or biological activity.
- variable portion refers to an amino acid sequence which differs from the native amino acid sequence of an antibody by virtue of at least one amino acid residue modification.
- a native (or “wildtype” or unaltered) amino acid sequence refers to the amino acid sequence of an antibody found in nature.
- a “variant portion” of the antibody includes any domain or region of the antibody that has an amino acid modification, or any subdomain or subregion thereof.
- Variant portions include, but are not limited to the Fc region, the Fab region, the CH 1 domain, the CH 2 domain, the CH 3 domain, the hinge region, the variable region, the constant region, the light chain and/or the heavy chain.
- the term "specific” can refer to the selective binding of the antibody to the target antigen epitope.
- Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. In one aspect, the antibody lacks significant binding to unrelated antigens.
- the term "antigen” refers to a molecule which is specifically recognized and bound by an antibody. An antigen which elicits an immune response in an organism, as evidenced by production of specific antibodies within the organism is termed an "immunogen.” The specific portion of the antigen or immunogen which is bound by the antibody is termed the "binding epitope" or "epitope.”
- amino acid modification refers to a change in the amino acid sequence of a predetermined amino acid sequence.
- exemplary modifications include an amino acid substitution, insertion and/or deletion, hi one aspect of the methods of the invention, an amino acid modification comprises an amino acid residue substitution.
- An amino acid modification at a specified position e.g. of the Fc region, refers to the substitution, deletion, or deletion of the specified residue where the numbering of the residues is that of the EU index in Kabat. See, e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242 (1991).
- a designation of Fl 16S indicates that the phenylalanine (F) at position 116 is substituted with a serine (S) at position 116.
- the term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity. See, e.g., U.S. Patent No.
- humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
- the humanized antibody will comprise substantially all of at least one, and in one aspect two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
- the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin.
- Fc immunoglobulin constant region
- an antibody of the invention or an antibody used in a method of the invention, comprises a heterologous moiety that serves as a "shuttle", “transport” or “carrier” moiety or domain to allow an antibody of the invention (or an antibody used in a method of the invention) enter cells (e.g., those lining the gut) or to allow an orally administered antibody of the invention enter into the bloodstream from the gut.
- a heterologous moiety that serves as a "shuttle", “transport” or “carrier” moiety or domain to allow an antibody of the invention (or an antibody used in a method of the invention) enter cells (e.g., those lining the gut) or to allow an orally administered antibody of the invention enter into the bloodstream from the gut.
- a "shuttle", "transport” or “carrier” moiety or domain comprises a transferrin polypeptide moiety (or active binding- internalization fragment thereof), Pseudomonas exotoxin (or active binding-internalization fragment thereof), a cell wall binding domain (CWB) domain of Clostridium difficile toxin A (or active binding-internalization fragment thereof), or an equivalent protein.
- CWB cell wall binding domain
- transferrin as a "shuttle", “transport” or “carrier” moiety, and that transferrin is a natural transport protein well known in the art; see also USPN 6,891,028; 6,825,037; 6,743,893; 6,361,779.
- the antibodies of the invention can comprise a transferrin fragment (e.g., a human transferrin fragment), a peptide capable of binding to a transferrin receptor (e.g., a human transferrin receptor), thereby internalizing (into a cell); the sequences comprising HAIYPRH (SEQ ID NO:32) and THRPPMWSPVWP (SEQ ID NO:33); see, e.g., USPN 6,743,893.
- a transferrin fragment e.g., a human transferrin fragment
- a peptide capable of binding to a transferrin receptor e.g., a human transferrin receptor
- the fusion product is internalized in cells expressing a transferrin receptor (e.g., a human transferrin receptor (hTfR)).
- a transferrin receptor e.g., a human transferrin receptor (hTfR)
- the antibodies of the invention also comprise recombinant fusions or heteromolecules (the Ab does not have to be recombinant, as the "shuttle", “transport” or “carrier” moiety can be joined to the Ab by chemical or other means, too) with other known peptides or proteins that can effect the internalization of the chimeric polypeptide into a cell, e.g., examples of exemplary naturally occurring ligands that can be used as "shuttle", “transport” or “carrier” moieties with Abs of the invention (and the receptors to which they bind) include: bombesin, gastrin, low density lipoprotein (LDL), epidermal growth factor (EGF), tumor necrosis factor (TNF), tumor growth factor (TGF), catecholamines (beta adrenergic receptors), asialofetuin (asialoglycoprotein receptor), somatostatin, N-formyl peptide, insulin, angiotens
- LDL low density lipoprotein
- a "shuttle", “transport” or “carrier” moiety or domain comprises the translocation domain of a bacterial toxin, e.g., Clostridium difficile toxin A or toxin B,
- Pseudomonas exotoxin e.g., Pseudomonas exotoxin A (Trinity Biosystems, Menlo Park, CA), cholera toxin, ricin toxin or Shiga-like toxin, or active binding-internalization fragments thereof, or equivalent proteins, all of which are well known in the art; see, e.g., USPN 6,022,950; 5,328,984; 5,080,898; 4,675,382; 4,666,837; 4,594,336.
- the antibody provided herein is a human antibody.
- human antibody refers to an antibody in which essentially the entire, or substantially all of, sequences of the light chain and heavy chain sequences, including the complementary determining regions (CDRs), are derived from human genes.
- human antibodies of the invention can also include non-natural or synthetic residues or peptidomimetic residues.
- human monoclonal antibodies are prepared by the trioma technique, the human B-cell technique ⁇ see, e.g., Kozbor, et al, Immunol. Today 4: 72 (1983) , EBV transformation technique (see, e.g., Cole et al.
- the human antibody is generated in a transgenic mouse.
- Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used.
- fully human antibody sequences are made in a transgenic mouse engineered to express human heavy and light chain antibody genes.
- An exemplary description of preparing transgenic mice that produce human antibodies found in Application No. WO 02/43478. B cells from transgenic mice that produce the desired antibody can then be fused to make hybridoma cell lines for continuous production of the antibody.
- bispecific antibody refers to an antibody, or a monoclonal antibody, having binding specificities for at least two different antigenic epitopes.
- the epitopes are from the same antigen.
- the epitopes are from two different antigens.
- Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al, Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan, et al, Science 229:81 (1985).
- Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger, etal, Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al, J. Immunol. 152:5368 (1994).
- heteroconjugate antibody refers to two covalently joined antibodies. Such antibodies can be prepared using known methods in synthetic protein chemistry, including using crosslinking agents. See, e.g., U.S. Patent No. 4,676,980.
- single-chain Fv or "scFv” antibody refers to antibody fragments comprising the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide can further comprises a polypeptide linker between the V H and V L domains, e.g., in one aspect this enables the sFv to form the desired structure for antigen binding.
- Designing and making scFVs and Fvs are well known in the art, see, e.g., Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp.
- the term “diabodies” can refer to antibody fragments (e.g., small antibody fragments) with two antigen-binding sites; the fragments can comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
- V H heavy chain variable domain
- V L light chain variable domain
- the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
- Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger (1993) Proc. Natl. Acad. Sci. USA 90:6444-48.
- the term “triabodies” refers to antibody fragments with three antigen-binding sites.
- minibody refers to a scFv joined to a CH3 domain may also be made using an antibody of the invention. See, e.g., U.S. 5,837,821 ; Hu et al, Cancer Res. 56:3055-61 (1996).
- nanobody refers to a single variable region (VHH) domain, originally characterized in camels and llamas can also be employed. See, e.g., Davies et al, BioTechnology 13:475-79 (1995); Cortez-Retamozo, et al, Cancer Res. 64:2853-57 (2004).
- fusion protein refers to an antibody or an antigen binding fragment thereof that is fused to a heterologous protein or protein fragment.
- fusion proteins include N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
- Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts, histidine-tryptophan modules, FLAG tags, cleavable linker sequences ⁇ e.g., Factor Xa or enterokinase). See, e.g., Williams, Biochemistry 34:1787-97 (1995); Dobeli, Protein Expr. Purif. 12:404-14 (1998); Kroll, DNA Cell Biol. 12:441-53 (1993); PROTEIN PURIFICATION: A PRACTICAL APPROACH (Roe, ed.,
- MONOCLONAL ANTIBODIES A PRACTICAL APPROACH (Shephard et al., eds., Oxford University Press 2000).
- biologically active refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or indirectly exerting a biologic effect.
- Direct effects include, but are not limited to the modulation of a growth signal, the modulation of an anti-apoptotic signal, the modulation of an apoptotic or necrotic signal, the modulation of the ADCC cascade, the modulation of the CDC cascade, inhibition of ligand-receptor interactions, modulation of internalization, and eliciting phagocytosis.
- Modulation of an activity can include the inhibition or stimulation of a particular activity.
- Indirect effects include, but are not limited to toxicity due to conjugate delivery ⁇ e.g., radionuclide) or sensitization to secondary agents ⁇ e.g., phototoxic agent).
- protease cleavage site refers to residues on the antibody sequence recognized and cleaved by a particular protease when accessible to the protease.
- protease recognizes and binds a region of the polypeptide that brackets the scissile peptide bond, i.e., the bond that is to be cleaved.
- Most proteases bind several amino acid residues in their active sites. Using the nomenclature of Schechter and Berger ⁇ Biochem. Biophys. Res. Commun.
- the bond to be hydrolyzed is formed between the Pl residue (N-terminal side of the cleaved bond) and the Pl' residue (C-terminal side of the cleaved bond) of the substrate.
- the residues adjacent to Pl on the N-terminal side of the sessile bond are labeled P2 - Pw, and the residue adjacent to the P V site on the C-terminal side are labeled P2' - Pra'.
- the protease has corresponding "subsites" where the residues of the substrate fit, identified as Sl, Sl', etc.
- the protease cleavage sites of the invention can consist on two, three, four, five, six, or more residues.
- an antibody of the invention specifically binds to a pathogen, a virulence factor, a dietary enzyme or a toxin, such as a bacterial toxin, e.g., Clostridium difficile toxin.
- a pathogen e.g., a virulence factor
- a dietary enzyme e.g., a bacterial toxin
- a toxin such as a bacterial toxin, e.g., Clostridium difficile toxin.
- modifications in an antibody of the invention comprise at least one mutation in the amino acid sequence of the antibody.
- the variant portion in the antibody sequence can comprise any number of modifications including two, three, four, five, six, seven, eight, nine, ten, eleven, or more amino acid modifications.
- the modification of the antibody is in a protease cleavage site or at a site flanking the protease cleavage site.
- a protease cleavage site can be identified by any suitable method. In some embodiments, sites of protease cleavage are identified using known protease cleavage motifs. In other embodiments, sites of protease cleavage are identified by characterizing the fragments that result from protease digestion. Such methods include, but are not limited to well known methods that characterize sequences following protease digestion, e.g., N-terminal sequencing, gel electrophoresis analysis, mass spectral analysis, and crystallographic studies.
- the modification is at the P 1 , P 1 ' , P2, P3 , P4, P2 ' , P3 ' , or
- protease cleavage site P4 residue of the protease cleavage site.
- the modification to the amino acid sequence generates a protease resistance motif, rendering the protease cleavage site non-cleavable or less susceptible to protease cleavage.
- the modifications are made to the same protease cleavage motif throughout the antibody. In other embodiment, the modifications are made to different protease cleavage motifs.
- the modifications can be made in a protease cleavage site that is not flanked by an amino acid residue known to inhibit or attenuate protease cleavage. Such amino acids include Pro, Lys, Arg and His.
- An inhibitory or attenuating residue is any residue that interferes with, the formation of the catalytic triad or two catalytic diads that acts as a proton shuttle or reduces the availability of the catalytic site.
- the variant portion of the antibody can include any portion of the antibody, e.g., including the heavy chain, a light chain, or both.
- the amino acid residue modifications are in (the variant portion is in) the Fc region, the hinge region, the CH L domain, the CH 1 domain, the CH 2 domain, the CH 3 domain, the Fab region, or any combination thereof.
- the variant portion is a V H or V L domain, provided the cleavage site does not have a negative effect on the desired antibody function.
- a mutation does not have a negative impact on antibody function if the antibody at least retains some of its ability to specifically bind its antigen (e.g., in some aspects, with less specific binding affinity).
- the antibody retains at least one of its biological activities (e.g., Fc receptor function) in addition to its ability to specifically bind the antigen.
- the mutation is introduced by modifications, additions or deletions to a nucleic acid encoding the antibody.
- a nucleic acid encoding the antibody modified by the method of the invention can be altered by any suitable means. For example, site-directed mutagenesis may be employed. See, e.g., Ling et al. (1997) Anal Biochem. 254(2): 157-178; Dale et al. (1996) Methods MoI. Biol. 57:369-374; Smith (1985) Ann. Rev. Genet.
- Protocols that can be used to practice the invention e.g., to modify antibody sequences to generate protease resistant Abs for oral administration
- various diversity generating methods are described, e.g., in U.S. patent application Ser. no. (U.S. Ser. No.) 09/407,800, filed Sep. 28, 1999; U.S. Pat. No. 6,379,964; U.S. Pat. Nos. 6,319,714; 6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCTVUS00/01203; U.S. Pat. No. 6,436,675; PCT/USOO/01202, filed Jan. 18, 2000, and, e.g. U.S. Ser. No.
- Non-stochastic, or "directed evolution,” methods useful in generating an antibody of the invention include, e.g., "gene site saturation mutagenesis” (GSSM) or “saturation mutagenesis", synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate antibodies with new or altered properties ⁇ e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like).
- Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for proteolytic or other activity. Any testing modality or protocol can be used, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.
- nucleic acid encoding the antibody can be introduced by any suitable method including, but not limited to error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
- GSSM Gene Site Saturation Mutagenesis
- SLR synthetic ligation reassembly
- the modifications, additions or deletions to a nucleic acid encoding the antibody can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction- purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
- Antibodies of the invention can be generated by non-stochastic mutation of the nucleic acids that encode them by, e.g., Gene Site Saturation Mutagenesis, or, GSSM, as described, e.g., in U.S. Patents No. 6,171,820, No. 6,562,594, 6,764,835, and U.S. Patent Publication No. 2004 0018607.
- codon primers containing a degenerate N 5 N 5 GAT sequence are used to introduce point mutations into a polynucleotide, e.g., an antibody of the invention, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified.
- oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N 5 N 5 GZT sequence, and, optionally, a second homologous sequence.
- the downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N 5 N 3 GAT sequence includes codons for all 20 amino acids.
- one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a Ml range of codon substitutions.
- At least two degenerate cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
- more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.
- This plurality of N 5 N, G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
- oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
- simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence.
- degenerate cassettes having less degeneracy than the N,N,G/T sequence are used.
- degenerate N,N,N triplet sequence it may be desirable in some instances to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
- use of degenerate triplets e.g., N 5 N, G/T triplets
- the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated.
- an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet 32 individual sequences can code for all 20 possible natural amino acids.
- a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides.
- the use of a non-degenerate oligonucleotide in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.
- Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.
- each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules (e.g., anti-toxin antibodies of the invention) such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).
- the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.
- an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased glucan hydrolysis activity under alkaline or acidic conditions), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.
- favorable amino acid changes may be identified at more than one amino acid position.
- One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if two specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i.e. 2 at each of three positions) and no change at any position.
- site-saturation mutagenesis can be used together with shuffling, chimerization, recombination and other mutagenizing processes, along with screening.
- This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
- the iterative use of any mutagenizing process(es) is used in combination with screening.
- the GSSM comprises use of codon primers (containing a degenerate
- N,N,N sequence to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position.
- the oligos used are comprised contiguously of a first homologous sequence, a degenerate N 5 N 5 N sequence and in one aspect but not necessarily a second homologous sequence.
- the downstream progeny translational products from the use of such oligos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N 5 N 9 N sequence includes codons for all 20 amino acids, hi one aspect, one such degenerate oligo (comprised of one degenerate N 5 N 5 N cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions. In another aspect, at least two degenerate N 5 N 5 N cassettes are used - either in the same oligo or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
- N 5 N 5 N sequence can be contained in one oligo to introduce amino acid mutations at more than one site.
- This plurality of N 5 N 5 N sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
- oligos serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N 5 N 5 N sequence, to introduce any combination or permutation of amino acid additions, deletions and/or substitutions.
- the invention provides for the use of degenerate cassettes having less degeneracy than the N 5 N 5 N sequence.
- degenerate cassettes having less degeneracy than the N 5 N 5 N sequence.
- N 5 N 9 N triplet sequence N 9 N 9 GyT 9 or an N,N,G/C triplet sequence.
- a degenerate triplet such as N 9 N 5 GAT or an
- N 5 N 9 G/C triplet sequence as disclosed in the instant invention is advantageous for several reasons.
- this invention provides a means to systematically and fairly easily generate the substitution of the full range of possible amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide.
- the invention provides a way to systematically and fairly easily generate 2000 distinct species (i.e., 20 possible amino acids per position times 100 amino acid positions). It is appreciated that there is provided, through the use of an oligo containing a degenerate N 5 N 5 GAT or an N 5 N, G/C triplet sequence, 32 individual sequences that code for 20 possible amino acids.
- This invention also provides for the use of nondegenerate oligos, which can optionally be used in combination with degenerate primers disclosed. It is appreciated that in some situations, it is advantageous to use nondegenerate oligos to generate specific point mutations in a working polynucleotide. This provides a means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.
- each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide.
- the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g., cloned into a suitable E. coli host using an expression vector) and subjected to expression screening.
- clonal amplification e.g., cloned into a suitable E. coli host using an expression vector
- an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.
- favorable amino acid changes may be identified at more than one amino acid position.
- One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid and each of two favorable changes) and 3 positions.
- this invention provides for the use of saturation mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.
- mutagenesis can be use to replace each of any number of bases in a polynucleotide sequence, wherein the number of bases to be mutagenized is in one aspect every integer from 15 to 100,000.
- the number of bases to be mutagenized is in one aspect every integer from 15 to 100,000.
- a separate nucleotide is used for mutagenizing each position or group of positions along a polynucleotide sequence.
- a group of 3 positions to be mutagenized may be a codon.
- the mutations can be introduced using a mutagenic primer, containing a heterologous cassette, or a mutagenic cassette.
- Exemplary cassettes can have from 1 to 500 bases.
- Each nucleotide position in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E can be referred to as a designer oligo).
- Saturation mutagenesis can comprise mutagenizing a complete set of mutagenic cassettes (wherein each cassette is in one aspect about 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is in one aspect from about 15 to 100,000 bases in length).
- a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized.
- a grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of saturation mutagenesis.
- Such groupings are exemplified by deletions, additions, groupings of particular codons and groupings of particular nucleotide cassettes.
- sequences to be mutagenized include a whole gene, pathway, cDNA, an entire open reading frame (ORF) and entire promoter, enhancer, repressor/transactivator, origin of replication, intron, operator, or any polynucleotide functional group.
- a "defined sequences" for this purpose may be any polynucleotide that a 15 base-polynucleotide sequence and polynucleotide sequences of lengths between 15 bases and 15,000 bases (this invention specifically names every integer in between). Considerations in choosing groupings of codons include types of amino acids encoded by a degenerate mutagenic cassette.
- this invention specifically provides for degenerate codon substitutions (using degenerate oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acids at each position and a library of polypeptides encoded thereby.
- SLR synthetic ligation reassembly
- SLR synthetic ligation reassembly
- SLR directed evolution process
- SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non- stochastically.
- SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.
- SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged.
- this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10 100 different chimeras.
- SLR can be used to generate libraries comprised of over 1O 1000 different progeny chimeras.
- aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
- the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
- the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
- the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.
- a ligase e.g. T4 DNA ligase
- the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates (e.g., anti-toxin antibodies) that serve as a basis for producing a progeny set of finalized chimeric polynucleotides (e.g., nucleic acids encoding protease resistant Abs).
- progenitor nucleic acid sequence templates e.g., anti-toxin antibodies
- finalized chimeric polynucleotides e.g., nucleic acids encoding protease resistant Abs.
- the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.
- the demarcation points can be located at an area of homology, and are comprised of one or more nucleotides. These demarcation points are in one aspect shared by at least two of the progenitor templates.
- the demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.
- the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.
- a demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.
- a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences.
- a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences.
- a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
- a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.
- all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
- the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
- the assembly order is by design (or non-stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.
- the ligation reassembly method is performed systematically.
- the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one.
- this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.
- the progeny molecules generated in one aspect comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
- the saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species.
- the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template.
- a codon can be altered such that the coding for an originally amino acid is altered.
- This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecular homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
- the present invention provides a non-stochastic method termed synthetic gene reassembly, that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or chimerized randomly, but rather are assembled non-stochastically.
- the synthetic gene reassembly method does not depend on the presence of a high level of homology between polynucleotides to be shuffled.
- the invention can be used to non- stochastically generate libraries (or sets) of progeny molecules comprised of over 10 100 different chimeras. Conceivably, synthetic gene reassembly can even be used to generate libraries comprised of over 10 1000 different progeny chimeras.
- the invention provides a non-stochastic method of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
- the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules.
- progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i.e. chimerized or shuffled.
- Optimized Directed Evolution System A non-stochastic gene modification system termed "optimized directed evolution system" can also be used to generate antibodies of the invention, or used in methods of the invention to modify antibody-encoding sequences, e.g., to generate protease resistant Abs.
- Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.
- Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
- a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
- One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence.
- Each oligonucleotide in one aspect includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. Additional information can also be found, e.g., in U.S. Patent Nos. 6,537,776, and 6,361,974.
- In vivo shuffling of nucleic acids can also be used to generate antibodies of the invention, or used in methods of the invention to modify antibody-encoding sequences, e.g., to generate protease resistant Abs.
- In vivo shuffling can be performed utilizing the natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
- recombination In vivo reassortment is focused on "inter-molecular” processes collectively referred to as “recombination” which in bacteria, is generally viewed as a “RecA-dependent” phenomenon.
- the invention can use recombination processes of a host cell to recombine and re-assort (e.g., antibody) sequences, or the cells' ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion.
- This process of "reductive reassortment” occurs by an "intra-molecular", RecA-independent process.
- novel polynucleotides can be generated by the process of reductive reassortment.
- the method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector and their subsequent introduction into an appropriate host cell.
- the reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units.
- the reassortment process recombines and/or reduces the complexity and extent of the repeated sequences and results in the production of novel molecular species.
- Various treatments may be applied to enhance the rate of reassortment. These can include treatment with ultra-violet light, or DNA damaging chemicals and/or the use of host cell lines displaying enhanced levels of "genetic instability".
- the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution. Kabat Index and Numbering Scheme
- the invention provides antibodies having modified sequences based on the Kabat numbering system, i.e., based on the EU index as in Kabat (the Kabat numbering scheme is a widely adopted standard for numbering the residues in an antibody in a consistent manner).
- EU index in Kabat is not mentioned, a specific position or mutation may refer to the absolute position (residue number) in an antibody sequence or subsequence, as will be clear from context, for example in Tables 2, 3A, 3B, 4, 5, and 9 (Examples 1 to 3), and Example 6, Tables 1 and 2. Equivalents between absolute positions and Kabat/EU designations are given in Example 1, Table 1 and Example 3, Table 8.
- the variant portion of the antibody of the invention comprises at least one amino acid substitution at any one or more of amino acid positions T155, L179, L235, F241, Y296, L309, Y349, L365, L398, F404, Y407, and Y436 of an IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions L234, L242, F243, F275, Y278, Y300, L306, W313, L314, Y319, L351, L368, Y391, F405, L406, L410, F423, L432, or Y436 of a IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO: 5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- Example 1 describes, inter alia, an exemplary method using the Kabat numbering system
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions F116, K126, R143, Kl 69 or Kl 83 of a kappa (light) chain, e.g., SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- a kappa (light) chain e.g., SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions K133, K205, K210, K274, K326, K340, R355, K360 or K392 of a IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the variant portion of an antibody of the invention comprises at least one amino acid substitution at the Pl or P 1 ' site of cleavage in a trypsin cleavage motif, wherein the substituted amino acid is K or R, whereby the amino acid substitution confers increased resistance to trypsin proteolysis.
- the variant portion comprises at least one amino acid substitution, at the Pl or Pl' site of cleavage in a pepsin cleavage motif, wherein the substituted amino acid is L, F, Y, W, I 3 or T, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at the Pl or P 1 ' site of cleavage in a chymotrypsin cleavage motif, wherein the substituted amino acid is F, Y, or W, whereby the amino acid substitution confers increased resistance to chymotrypsin proteolysis.
- the variant portion of an antibody of the invention comprises at least one amino acid substitution selected from the group of amino acid substitutions of L235P, L398Q, F404Y, L179I, and T155S, in an IgG 1 heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution selected from the group of amino acid substitutions of Fl 16S and K126A in a kappa light chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution selected from the group of amino acid substitutions of K133G and K274Q in a IgG heavy chain, e.g., SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:5, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the Kabat numbering scheme is a widely adopted standard for numbering the residues in an antibody in a consistent manner.
- the Kabat numbering systems and database of aligned sequences are well known in the art, see, e.g., Kabat, et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition. NIH Publication No. 91-3242. See also, e.g., Johnson (1997) Genetics 145:777-786; Johnson (1997) Immunol. Cell Biol. 75:580-583; Johnson (2000) Nucleic Acids Res. 28: 214-218; Johnson (2001) Nucleic Acids Res. 29(l):205-206; Johnson (2004) Methods MoI. Biol.
- the Kabat Database of aligned sequences of proteins of immunological interest provides useful correlations between structure and function for, e.g., immunoglobulin nucleotide and amino acid sequences and their tertiary structures.
- the Kabat Database initially started in 1970 to determine the combining site of antibodies based on the available amino acid sequences, allows precise delineation of complementarity determining regions (CDR) of both light and heavy chains, and can be used to align sequences to derive structural and functional information, and to construct artificial antibodies with prescribed specificities.
- Antibody sequences can be compared to and tested against the Kabat sequence database.
- an antibody of the invention e.g., a wildtype antibody modified using the Kabat database according to the methods of the invention, has greater resistance to proteolysis relative to its comparable unaltered or "wildtype" antibody form.
- the increased resistance to proteolysis can be at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more than that of the unmodified antibody.
- the modified antibody can be partially or completely resistant to cleavage by more than one protease. Any suitable to determine sensitivity to proteolysis may be employed. See, e.g., PROTEOLYTIC ENZYMES: A PRACTICAL APPROACH 2ND ED.
- an antibody of the invention or an antibody modified by a method of the invention, can be an IgG, IgM, IgD, IgE, or IgA antibody. In some embodiments, the antibody is an IgG antibody.
- the antibody can be an IgG 1 , IgG 2 , IgG 3 , or IgG 4 antibody.
- Any suitable source can be used for the antibody.
- the antibody can be a human, murine, rat, rabbit, bovine, camel, llama, dromedary, or simian antibody.
- the antibody can be a humanized antibody, a chimeric antibody, a bispecific antibody, a fusion protein, or a biologically active fragment thereof.
- the antibody (or biologically active fragment thereof) is a fusion protein.
- the fusion protein can encompass additional peptide sequence that simplifies purification or production.
- Fusion proteins also may include domains and/or whole polypeptides that are biologically active in a manner that complements the activity of the antibody.
- the antibody can be fused to a cytokine, ligand, adhesion molecule, peptide, receptors, enzymes, therapeutic proteins, dyes, small organic molecules, or any biologically active portion thereof.
- the proteolysis is the digestion mediated by proteases from the , gastrointestinal tract, the blood, or the bile. In alternative embodiments, the proteolysis is mediated by pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsinogen, carboxy-peptidase, pro-carboxy-peptidase, elastase, pro-elastase, or any combination thereof.
- the protease can be one selected from a group of proteases released by an exogenous organism or any organism within the digestive tract, or released or produced in the digestive tract. In some embodiments, the protease can be selected from a group of proteases released or produced by an abnormal, infected, cancerous or otherwise diseased tissue.
- an antibody of the invention can be modified in any suitable manner to confer or enhance a desirable effector function or physical characteristic.
- the Fc region of an antibody of the invention is further modified to enhance ADCC, CDC, or phagocytosis.
- the Fc region of the antibody can also be further modified to increase binding affinity to the Fc receptor (FcR). See, e.g., U.S. Patent No. 6,737,056; and US 2004/0132101.
- the antibody is further modified to have a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- an antibody of the invention has a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- the modification of the antibody comprises one or more additions of post-translational modification sites.
- An antibody of the invention can also be glycosylated.
- the modifications can also comprise the addition of one or more N-glycosylation site or an O-glycosylation site, an alkyl chain or a small molecule, an addition of a disulfide bond site or a salt bridge site, and/or a covalent or non-covalent addition of a second molecule to the Fc chain of the antibody.
- the glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
- the glycosylation can be O-linked or N-linked.
- the second molecule comprises an antibody secretory component.
- the invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing ⁇ e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides.
- Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton,
- any suitable means can be used to determine the binding affinity of an antibody of the invention.
- the affinity is determined by surface plasmon resonance (Biacore).
- An antibody of the invention can also be modified further to increase binding affinity using methods known in the art. See, e.g., U.S. Patent No. 6,350,861.
- the modified antibody is more thermostable or thermotolerant than the wildtype antibody. Any suitable means may be employed to assess the thermostability of an antibody of the invention.
- the modified antibody retains at least its binding activity under conditions comprising a temperature range of between about 37 0 C to about 95°C; between about 55°C to about 85°C, between about 70 0 C to about 95 0 C, or, between about 9O 0 C to about 95°C.
- the increased thermostability confers a significant advantage for long-term storage for the antibodies of the invention, particularly when the antibodies require storage in places with little or no ability to control storage temperature, e.g., remote regions of third world countries.
- an antibody of the invention retains its binding activity as well as at least one desirable biological activity.
- An antibody of the invention can also be further modified to reduce the immunogenicity of the antibody upon administration to the subject.
- the immunogenicity of the antibody includes the elimination of one or several antigenic epitopes within the antibody (e.g., through amino acid substitution) as well as residues and/or motifs that triggers non-specific xenogenic or innate responses that interfere with or reduce the antibody's therapeutic efficacy. See, e.g., Schellekans, Clin. Ther. 24:1720-40 (2002); Graddis et al, Curr. Pharm. Biotech. 3:285-97 (2002).
- an antibody of the invention results in reduced aggregation with itself or other antibodies or can be further modified to reduce such aggregation. It is desirable to reduce the aggregation of the antibodies as this property can result in increased immunogenicity and/or increase clearance, i.e., reduced half-life, for the antibody. Any suitable methods can be used to determine the amount of aggregation for the antibody. See, e.g., Graddis et al., Curr. Pharm. Biotech. 3 :285-97 (2002). Modifications can then be made using the molecular biology methods known in the art including those disclosed herein.
- the determination of the half-life of the antibody can be determined by any suitable means.
- the antibody half-life can be determined by detection of the presence of the antibody (TyX examining the biological activity half-life, or any combination thereof.
- the modified or engineered antibody has an increased tolerance to acidic pH conditions (e.g., pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4 or pH 3 or more acidic conditions) relative to wildtype antibody.
- the engineered antibody has increased tolerance to alkaline pH conditions (e.g., pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more) relative to wildtype antibody. Any suitable method can be employed to determine pH tolerance.
- the engineered antibody is identified as pH tolerant when the antibody maintains sufficient native conformation at about pH 3 and above to maintain some biological activity. In some embodiments, the antibody maintains all its native conformation.
- the engineered antibody is identified as having greater protease resistance when the digestibility of the engineered antibody by the protease is increased at pH 3 relative to that of the wildtype antibody.
- the protease can be pepsin, trypsin, trypsinogen, chymo-trypsinogen, pro-carboxy-peptidase and/or pro-elastase.
- an engineered antibody it selected to retains biological activity in (or survives structurally, e.g., being "tolerant to", or is resistant to pH dependent unfolding) conditions comprising the conditions of the stomach, which approximate an acidity of at least pH 3.
- the present method can further comprise introducing additional mutations into a "wildtype" amino acid sequence to render an antibody more resistant to pH dependent unfolding.
- the antibodies of the invention are dimerized or trimerized (e.g., diabody or triabody Abs).
- dimerized or trimerized e.g., diabody or triabody Abs.
- Enhanced dimerization or other multimerization of Fc regions of antibodies can result in a greater biological efficacy for some targets.
- Such increased dimerization can be determined using any suitable means employing methods known in the art.
- the antibody is modified to improve solubility, e.g., improving solubility under conditions of alkaline or acidic conditions, e.g., as those in the stomach. Solubility of proteins can be determined using routine methods in the art.
- An antibody of the invention also can contain amino acid modifications that increase expression of the antibody in the host cell.
- the modified or engineered antibody can be expressed in vitro or in vivo.
- Any suitable host cell can be employed including, but not limited to, prokaryotic cells and eukaryotic cells such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include E. coli,
- the modifications permit or enhance antibody expression in a mammalian expression system or in a plant expression system.
- the modifications include mutation of the nucleic acid sequence encoding the antibody to provide codons in a nucleic acid to increase or decrease its expression in a host cell.
- any suitable method can be used to identify the mutations that permit or enhance host cell expression of the recombinant antibody.
- the method can comprise identifying a "non-preferred” or a "less preferred” codon in antibody-encoding nucleic acid and replacing one or more of these non-preferred or less preferred codons with a "preferred codon” encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid.
- a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell.
- Plant includes whole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seed, and plant cells and progeny of same.
- the class of plants which can be used to produce the antibodies of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploidy, diploid, or haploid cells.
- the antibodies and biologically active fragments thereof, of the invention include all "mimetic” and “peptidomimetic” forms.
- the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention.
- the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
- the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
- Antibodies of the invention can partially or completely comprise polypeptide mimetics, and can contain any combination of non-natural structural components.
- antibody mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
- a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
- Individual peptidomimetic residues in antibodies of the invention can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, NjN'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropyl- carbodiimide (DIC).
- glutaraldehyde N-hydroxysuccinimide esters
- bifunctional maleimides NjN'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropyl- carbodiimide (DIC).
- Antibodies of the invention can also be characterized as mimetics and can contain (comprise) all or some non-natural residues in place of naturally occurring amino acid residues.
- Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
- Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-I, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluorometliyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro- phenylalanine; D- or L-p-biphenylphenylalanine; D- or L- ⁇ -methoxy-biphenyl
- Aromatic rings of a non-natural amino acids in antibodies of the invention include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
- Mimetics of acidic amino acids in antibodies of the invention can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
- Carboxyl side groups e.g., aspartyl or glutamyl
- Carboxyl side groups can also be selectively modified by reaction with carbodiimides (R'- N--C--N--R') such as, e.g., 1- cyclohexyl-3(2-mo ⁇ holin- yl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide.
- Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
- Mimetics of basic amino acids in antibodies of the invention can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
- Nitrile derivative e.g., containing the CN-moiety in place of COOH
- Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
- Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, in one aspect, under alkaline conditions.
- one or more conventional reagents including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, in one aspect, under alkaline conditions.
- Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane.
- N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
- Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
- alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
- Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta- (5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; ⁇ -chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
- cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta- (5-imidozoyl) propionic acid
- chloroacetyl phosphate N-alkylmaleimides
- 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
- Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha- amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g. , methionine sulfoxide.
- Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
- Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para- bromophenacyl bromide.
- Other mimetics include, e.g.
- amino acid substitution in antibodies of the invention can also include the substitution of an amino acid (or peptidomimetic residue) of the opposite chirality.
- any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but also can be referred to as the R- or S-form.
- an antibody of the invention specifically binds to a pathogen.
- Any pathogen can be targeted by an antibody of the invention.
- the pathogen is selected from the group consisting of a bacteria, a virus and a fungus. More specifically, the pathogen can be an intestinal pathogen. In specific embodiments, the intestinal pathogen is selected from the group consisting of enterotoxigenic E. coli, rotavirus,
- the pathogen is Streptococcus mutans.
- the bacteria is a Helicobacter pylori, an Escherichia sp., a Cryptosporidium sp., a Clostridium sp. or a Shigella sp.
- the fungal pathogen is Candida albicans or Aspergillus fumigatus.
- the viral pathogen is a species in the genera of rotavirus, hepatitis, astrovirus, picornavirus, adenovirus, or parvovirus.
- the anti-pathogenic effect of an antibody of the invention, or an antibody used in a method of the invention can result from the specific binding of the antibody to a virulence factor.
- the ability of proteins in a biological sample to bind to the antibody may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding maybe determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of an antibody to the sample may be detected using a secondary antibody having such a detectable label thereon.
- Alternative assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.
- These monoclonal antibodies can bind with at least a Ka of about 1 ⁇ M, or at least about 300 nM, or at least about 30 nM, or in one aspect, at least about 10 nM, in one aspect, at least about 3 nM or better, usually determined by ELISA. Any suitable method may be employed to determine the biological activity of an antibody in the presence of the virulence factor.
- Such assays include binding assays, in vitro assays assessing morphology, viability, phagocytosis, cytotoxicity ⁇ e.g., ADCC and CDC), and/or proliferation, and in vivo models such as the ileal loop models and passive immunotherapy models. See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition); Rafael et al., ADVANCED CURRENT PROTOCOLS IN CELLULAR IMMUNOLOGY (CRC Press 2000); and Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
- an antibody has anti-virulence factor activity if the antibody reduces the pathogenicity of the organism and/or the toxicity of the virulence factor by at least 20%, at least 50%, 60%, 70%, 80%, 90%, or 100%.
- an antibody of the invention has anti- virulence activity if the antibody reduces the pathogenicity of the organism and/or the toxicity of the virulence factor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or 100% in presence of one or more other anti-virulence factors.
- a cell can be contacted with the antibody and the virulence factor, pathogenic organism, or cell in any suitable manner for any suitable length of time.
- the cells can be contacted with the antibody more than once during incubation or treatment.
- the dose required is in the range of about 1 ⁇ g/ml to 1000 ⁇ g/ml, or in the range of 100 ⁇ g/ml to 800 ⁇ g/ml.
- the exact dose can be readily determined from in vitro cultures of the cells and exposure of the cell to varying dosages of the antibody.
- the length of time the cell is contacted with the antibody is 1 hour to 3 days, or for 24 hours.
- an antibody of the invention specifically binds to a toxin.
- the toxin can be selected from the group consisting of a bacterial toxin, a chemical toxin and an environmental toxin.
- the bacterial toxin is a cholera toxin, an Escherichia coli toxin, a Streptococcus toxin, a Bordetella pertussis toxin, and a Clostridium toxin.
- the Clostridium toxin can comprise a botulinum toxin or a Clostridium difficile toxin.
- the botulinum toxin or Clostridium difficile toxin can comprise botulinum neurotoxin, C. difficile toxin A, or C. dijf ⁇ cile toxin B.
- the toxin is Ricin toxin.
- the anti-pathogenic effect of the antibody can result from the antibody binding the toxin, clearance of the toxin, inactivation of the toxin, and the like.
- An antibody of the invention, or an antibody used in method of the invention can specifically bind a virulence factor.
- the virulence factor can be an adherence factor, a coat protein, an invasion factor, a capsule, an exotoxin, or an endotoxin.
- the anti-pathogenic effect of the antibody can result from the antibody binding a virulence factor, clearance of the factor, inactivation of the factor, and the like.
- Exemplary adherence factors include those found in Bordetella pertussis, Campylobacter jejuni, Corynebacterium diphthe ⁇ ae, Eikenella histolytica, Escherichia coli, Helicobacter pylori, Salmonella enteriditis, Staphylococcus pyogenes, Streptococcus pyogenes, Vibrio cholerae, and Streptococcus viridans.
- antiphagocytic components Streptococcus pyogenes, Vibrio vunificans
- the toxins found in, e.g., Caniplobacter jejuni, Cornebacterium diptheriae, Legionella pneumophila, Pseudomonas aeruginosa, Shigella dysenterie, Trichomonas vaginalis, Staphylococcus aureus, Bartonella spp., Francisella tularensis, Proteus mirabilis, Salmonella spp., Yersinia spp., and Bacillus cereus.
- Exemplary capsule components include those found in Bacillus anthracis, Bordetella pertussis, Escherichia coli, Neisseria meningitides, Pasturella multocida, Staphylcoccus epidermis, and Yersinia pestis .
- Invasion factors include those associated with Clostridium spp., Leptospira interrogans, Staphylococcus aureus, and Vibrio spp.
- An antibody of the invention e.g., Abs made by the methods of the invention, or described herein
- the antibody can bind a dietary enzyme, and thus modulate its activity.
- the dietary enzyme can be a lipase, an esterase, a urease, a lyase, a protease, an isomerase, a ligase or a synthetase. See, e.g., US 2004/0002583.
- the invention provides isolated, recombinant and synthetic nucleic acids comprising a sequence encoding an antibody of the invention, a vector comprising the encoding nucleic acid, and/or a cell comprising the encoding nucleic acid or the vector comprising the encoding nucleic acid.
- the vector comprises the antibody-encoding nucleic acid operably linked to a promoter suitable for expression in the designated host cell.
- Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids.
- Exemplary host cells include gram negative bacteria, such as Escherichia coli and Pseudomonas fluorescens; gram positive bacteria, such as Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, and Bacillus subtilis.
- Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces spp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines, and insect cells and cell lines.
- yeast such as Saccharomyces spp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines, and insect cells and cell lines.
- the invention also includes antibodies and their encoding nucleic acids optimized for expression in these organisms and species. See, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.
- substantially identical in the context of two nucleic acids or polypeptides, can refer to two or more sequences that have, e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or more nucleotide or amino acid residue (sequence) identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, or by visual inspection.
- the invention comprises isolated, recombinant or synthetic Ab light or variable region polypeptides having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8, and having the same (or substantially the same) antigen binding specificities as SEQ ID NO:1, SEQ ID NO:1,
- the invention provides nucleic acid and polypeptide sequences having substantial sequence identity to an exemplary sequence of the invention, e.g., SEQ ID NO:26 is the full length of the heavy chain of the Ab designated 227, or 3359; the full length of the light chain of the Ab designated 227, or 3359 (SEQ ID NO:27); the full length of the heavy chain of the Ab designated 543, or 3358 (SEQ ID NO:28); the full length of the light chain of the Ab designated 543, or 3358 (SEQ ID NO:29); the full length of the heavy chain of the Ab designated F87 (SEQ ID NO:30); the full length of the light chain of the Ab designated F87 (SEQ ID NO:31), or any of these sequences over a region of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues, or a region
- a "substantially identical" amino acid sequence also can include a sequence that hybridizes under stringent conditions to a reference sequence (e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, , SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8).
- a reference sequence e.g., an exemplary sequence of the invention, e.g., an Ab sequence of the invention comprising the variable regions SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, , SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8.
- Hybridization includes the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at
- Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
- stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below.
- nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
- hybridization under stringent conditions comprises hybridization in a buffer (solution) comprising about 50% formamide at about 37°C to 42°C; or, hybridization under stringent conditions can occur at conditions comprising about 35% to 25% formamide at about 30°C to 35°C, or, under conditions comprising about 42°C in 50% formamide, 5X SSPE, 0.3% SDS and 200 n/ml sheared and denatured salmon sperm DNA.
- the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.
- wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50 0 C or about 55°C to about 6O 0 C; or, a salt concentration of about 0.15 M NaCl at 72 0 C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50 0 C or about 55 0 C to about 6O 0 C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions. See Sambrook, Tij
- nucleic acids can include nucleic acids (e.g., nucleic acids used to practice the invention) adjacent to a "backbone” nucleic acid to which it is not adjacent in its natural environment.
- nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
- Backbone molecules include nucleic acids such as expression vectors, self- replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
- the enriched nucleic acids represent 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
- Recombinant polypeptides or proteins can refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
- synthetic polypeptides or protein are those prepared by chemical synthesis, also are described, below.
- expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene ⁇ i.e., a protein coding sequence, such as an antibody of the invention) in a host compatible with such sequences.
- Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.
- expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
- operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. In one aspect, it refers to the functional relationship of transcriptional regulatory sequence to a transcribed sequence.
- a promoter is operably linked to a coding sequence, such as a nucleic acid of the invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
- promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
- some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
- a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
- the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
- Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
- Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids.
- RNA autonomous self-replicating circular or linear DNA or RNA
- plasmids viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879
- plasmids e.g., viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879
- a recombinant microorganism or cell culture is described as hosting an "expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
- the vector may either be stably replicated by the cells during mitosis as an autonomous structure
- promoter includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a mammalian or plant cell.
- promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
- a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
- These cis-acting sequences can interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
- “Constitutive” promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
- “Inducible” or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
- Plasmids can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
- a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
- the invention provides a monoclonal antibody, or a biologically active fragment thereof, that binds to Clostridium difficile toxin A, wherein the variable region sequences of the antibody comprise SEQ ID NO:1 and SEQ ID NO:2; or SEQ ID NO:3 and SEQ ID NO:4.
- the invention also provides an isolated or recombinant nucleic acid comprising a sequence encoding the antibody, a vector comprising the nucleic acid, and a cell comprising the nucleic acid or the vector.
- Pharmaceutical compositions and kits comprising the antibody are also provided.
- the invention provides a monoclonal antibody, or a biologically active fragment thereof, that binds to Clostridium difficile toxin B, wherein the variable region sequences of the antibody comprise SEQ ID NO: 5 and SEQ ID NO:6.
- the invention also provides an isolated or recombinant nucleic acid comprising a sequence encoding the antibody, a vector comprising the nucleic acid, and a cell comprising the nucleic acid or the vector.
- Pharmaceutical compositions and kits comprising the antibody are also provided.
- the invention provides a monoclonal antibody produced by or isolated from a hybridoma selected from the group consisting of ATCC Accession No. (Ab designated 227 or 3359), ATCC Accession No. (Ab designated 543 or 3358), ATCC
- the invention also provides hybridomas comprising ATCC Accession No. (Ab designated 227 or 3359), ATCC Accession No. (Ab designated 543 or 3358), ATCC Accession No. (Ab designated F85), ATCC
- the invention provides isolated or recombinant Abs having the same antigen binding specificity as a monoclonal antibody of the invention, and the nucleic acids that encode them.
- the invention provides isolated or recombinant polypeptides having a sequence identity (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) to a sequence of an antibody of the invention, e.g., an Ab produced by a hybridoma of the invention.
- the identity can be over the full length of the polypeptide, or, the identity can be over a region of at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.
- a sequence that hybridizes to the disclosed sequences under high stringency conditions is also provided by the invention.
- Antibodies of the invention can also be shorter than the full length of exemplary antibodies.
- the invention provides antibodies (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., as an antibody; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary antibody of the invention, where the antibody fragment at least retains the ability to bind the antigen of interest.
- an isolated or recombinant peptide comprising an epitope bound by a monoclonal antibody of the invention, or a monoclonal antibody generated by a hybridoma of the invention, e.g., a hybridoma selected from the group consisting of ATCC Accession No. (Ab designated 227 or 3359), ATCC Accession No. (Ab designated 543 or 3358),
- the invention also provides hybridomas comprising ATCC Accession No. (Ab designated 227 or 3359), ATCC
- the invention provides methods of identifying a protease cleavage site in an antibody, which method comprises the steps of: a) determining putative sites of protease cleavage in the antibody; b) prioritizing the protease cleavage sites based on the likely exposure of the site to proteases; and c) identifying a site as the protease cleavage site as one whose position results in an exposure to proteases in the three-dimensional antibody structure.
- the putative sites of protease cleavage are determined in step (a) by identifying protease cleavage motifs using N-terminal sequencing, gel electrophoresis analysis, or mass spectral analysis of peptide fragments derived from an antibody digested by protease.
- the putative sites of protease cleavage can also be determined in step (a) by identifying known protease motifs.
- the protease cleavage sites are prioritized based on the physical location in the antibody, e.g., the hinge region, and the relative exposure to proteases, e.g., sites available after solvent treatment. In some embodiments, the protease cleavage sites are prioritized based on the anticipated protease profile of the target microenvironment. In some embodiments, the protease cleavage sites are prioritized in step (b) based on the surface exposure on the folded fo ⁇ n of the antibody solved by x-ray crystallography or NMR spectroscopy. The protease cleavage sites can also be prioritized in step (b) based on the surface exposure determined using a probe of 1.4 angstroms.
- the antibodies can be prioritized using any suitable amount of surface exposure. For example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 and/or 100% exposure can be used as standards for prioritization.
- the identified protease cleavage site has 20% surface area exposure to the probe, wherein the protease cleavage site comprises hydrophobic and aromatic amino acids, hi other embodiments, the identified protease cleavage site has 35% surface area exposure to the probe, wherein the protease cleavage site comprises basic amino acids.
- Measurements of solvent accessibility can also be used to prioritize the protease cleavage motifs using the exposed van der waals surface or surface residues extrapolated from B-values (PRINCIPLES OF PROTEIN X-RAY CRYSTALLOGRAPHY, (Drenth, ed., Springer Verlag 1994)) or order parameters (Lipari et al., J. Amer. Chem. Soc. 104: 4546 (1982)) from X-ray or NMR structure determination methodologies, respectively.
- the 'exposed residue' cutoffs are comparable, even if they do not numerically provide the exact surface area in angstroms squared.
- the putative protease cleavage sites within an antibody are prioritized by their surface exposure within the context of the folded form of the antibody.
- the contact surface area of every residue is calculated from the antibody structure using probe of 1.4 Angstroms (the approximate radius of a water molecule).
- the surface exposure of an IgG antibody was calculated from the deposited crystal structures of the human IgGl Fc constant domain (Sonderman et al, Nature 406: 267 (2000)) and the Fab domain (Cho, et al, Nature 421 : 756 (2003)) using the program MolMol (Koradi, et al, J. MoI. Graph. 14: 51 (1996)).
- a probe of 1.4 Angstroms is used to scan the surface of a protein and the area in square Angstroms that the probe is able to contact is defined as the solvent accessible surface area.
- a cutoff of 20% surface area exposure to solvent i.e. the probe
- hydrophobic and aromatic amino acids i.e. L, M, I, V, F, Y and W
- a cutoff of 35% was used for basic residues (K, R and H) to classify them as highly exposed and susceptible to potential proteolysis by proteases such as pepsin and those found in pancreatin.
- Any number of protease sites can be identified by the method of the invention. In one aspect, at least one protease cleavage site is identified.
- the protease cleavage sites comprise the same protease cleavage motif. In other embodiments, the protease cleavage sites comprise two or more different protease cleavage motifs.
- the protease cleavage sites can be identified in the Fc region, the Fab region, the hinge region, C L , CH I , CH 2 , CH 3 , V L , V H , or a combination thereof.
- protease cleavage motifs include, but are not limited to, a protease selected from the group consisting of pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsin, pro-carboxy-peptidase and pro-elastase.
- the invention provides a method of engineering a protease-resistant antibody, which method comprises the steps of: a) providing an antibody or an amino acid sequence of the antibody; b) identifying at least one protease cleavage site in the amino acid sequence of the antibody; and c) introducing at least one modification in the amino acid sequence of the antibody, whereby the modification results in a variant portion that has an increased resistance to proteolysis.
- the invention provides a method of generating an engineered antibody that is orally deliverable, which method comprises the steps of: a) providing a nucleic acid encoding a wildtype antibody; b) introducing at least one modification into the coding sequence of the wildtype antibody to generate a modified antibody coding sequence, wherein the modification of the coding sequence is in or proximate to the coding sequence of at least one protease cleavage site and the modification results in expression of an antibody that is partially or completely resistant to digestion by the protease; and c) expressing the modified antibody coding sequence of step b) to generate an engineered antibody, wherein an engineered antibody retains its ability to specifically bind to antigen in the digestive system following oral administration, thereby rendering the engineered antibody orally deliverable.
- the modification is in a protease cleavage site or at a site flanking the protease cleavage site. In alternative embodiments, the modification is at the Pl, Pl ', P2, P3, P4, P2', P3', or P4 residue of the protease cleavage site.
- One type of modification, therefore, that can be made is to modify a residue that is know to naturally occur within antibody sequences such as IgG, IgA, IgM, IgD, IgE, etc. Such substitutions can identifies through database analysis. See, e.g., Demerast et al., J. MoL Biol. 335:41-48 (2004).
- the modification to the amino acid sequence generates a protease resistance motif, rendering the protease cleavage site non-cleavable or less susceptible to protease cleavage.
- An engineered antibody of the invention, or an Ab used in a method the invention can comprise any number of modifications, including but not limited to, two, three, four, five, six, seven, eight, nine, ten, eleven, or more amino acid modifications.
- the modifications can be in a protease cleavage site or at a site flanking the protease cleavage site.
- the modification can be made to the same protease cleavage motif within the antibody or to different protease cleavage motifs.
- the modification is made in a protease cleavage site that is not flanked by an amino acid residue known to inhibit or attenuate protease cleavage.
- Such amino acids include Pro, Lys, Arg and His.
- An engineered antibody of the invention, or an Ab used in a method the invention can be an IgG, IgM, IgD, IgE, or IgA antibody.
- the antibody is an IgG antibody.
- An antibody can be an IgG 1 , IgG 2 , IgG 3 , or IgG 4 antibody.
- the antibody can be a human, murine, rat, rabbit, bovine, camel, llama, dromedary, or simian antibody.
- the antibody can be a humanized antibody, a chimeric antibody, a bispecific antibody, a fusion protein, or a biologically active fragment thereof.
- an engineered antibody of the invention, or an Ab used in a method the invention can be modified in any portion of the antibody including the heavy chain, a light chain, or both.
- the modified portion is the Fc region, the hinge region, the CH L domain, the CHi domain, the CH 2 domain, the CH 3 domain, the Fab region, or any combination thereof.
- the modified portion is a V H or V L domain, provided the cleavage site does not have a negative effect on the desired antibody function.
- modifications in the antibody of the invention, or an Ab used in a method the invention comprise at least one mutation in the amino acid sequence of the antibody.
- the mutation is introduced by modifications, additions or deletions to a nucleic acid encoding the antibody.
- the modifications, additions or deletions to a nucleic acid encoding the antibody can be introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
- GSSM Gene Site Saturation Mutagenesis
- SLR synthetic ligation reassembly
- the modifications, additions or deletions to a nucleic acid encoding the antibody can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
- an engineered antibody of the invention comprises at least one amino acid substitution at any one or more of amino acid positions T155, L179, L235, F241, Y296, L309, Y349, L365, L398, F404, Y407, and Y436 of a IgG heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions L234, L242, F243, F275, Y278, Y300, L306, W313, L314, Y319, L351, L368, Y391, F405, L406, L410, F423, L432, orY436 of a IgG heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions Fl 16, K126, R143, Kl 69 or Kl 83 of a kappa chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- the variant portion comprises at least one amino acid substitution at any one or more of amino acid positions K133, K205, K210, K274, K326, K340, R355, K360 or K392 of a IgG heavy chain, wherein the numbering of the residues in the variant portion is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pancreatin proteolysis.
- an engineered antibody of the invention comprises at least one amino acid substitution at the Pl or Pl' site of cleavage in a trypsin cleavage motif, wherein the substituted amino acid is K or R, whereby the amino acid substitution confers increased resistance to trypsin proteolysis.
- an engineered antibody comprises at least one amino acid substitution, at the Pl or Pl ' site of cleavage in a pepsin cleavage motif, wherein the substituted amino acid is L, F, Y, W, I, or T, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the engineered antibody comprises at least one amino acid substitution at the Pl or Pl' site of cleavage in a chymotrypsin cleavage motif, wherein the substituted amino acid is F, Y, or W, whereby the amino acid substitution confers increased resistance to chymotrypsin proteolysis.
- an engineered antibody of the invention comprises at least one amino acid substitution selected from the group of amino acid substitutions of L235P, L398Q, F404Y, Ll 791, and T155S in an IgG 1 heavy chain, wherein the numbering of the residues in the engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis,
- the engineered antibody comprises at least one amino acid substitution selected from the group of amino acid substitutions of Fl 16S and K126A in a kappa light chain, wherein the numbering of the residues in the engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- the engineered antibody comprises at least one amino acid substitution selected from the group of amino acid substitutions of K133G and K274Q in an IgG heavy chain, wherein the numbering of the residues in the engineered antibody is that of the EU index as in Kabat, whereby the amino acid substitution confers increased resistance to pepsin proteolysis.
- an engineered antibody of the invention, or an Ab used in a method the invention has greater resistance to proteolysis relative to the wildtype antibody. The increased resistance to proteolysis is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more than that of the unmodified antibody.
- An engineered antibody can be partially or completely resistant to cleavage by more than one protease.
- An engineered antibody of the invention can be modified in any suitable manner.
- the modification comprises the addition of a post-translational modification site, an N-glycosylation site, an O-glycosylation site, an alkyl chain, or a small molecule.
- the modification comprises covalent or non-covalent addition of a second molecule to the Fc chain of the antibody.
- the second molecule comprises an antibody secretory component, a carbohydrate, a disulfide bond site, or a salt bridge site.
- the Fc region of an engineered antibody of the invention, or an Ab used in a method the invention is further modified to enhance ADCC, CDC, or phagocytosis.
- the Fc region of the antibody can also be further modified to increase binding affinity to the Fc receptor (FcR).
- an engineered antibody is further modified to have a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced immunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- an antibody of the invention has a) an antigen binding activity comparable to or superior to the unmodified antibody; b) a chemical stability comparable to or superior to the unmodified antibody; c) a thermostability or thermotolerance comparable to or superior to the unmodified antibody; d) a pH tolerance comparable to or superior to the unmodified antibody; e) a reduced iinmunogenicity; f) a reduced aggregation; g) an increased half-life relative to the unmodified antibody; h) an increased expression in a host cell; i) a stability in pharmaceutical formulation comparable or superior to that of the unmodified antibody; j) an enhanced dimerization of Fc regions; or k) any combination thereof.
- an engineered antibody of the invention maintains its native conformation at about pH 3 and above or is modified to do so.
- the antibody retains biological activity at pH 3 or is further modified to do so.
- the antibody further comprises additional mutations that render the antibody more resistant to pH dependent unfolding.
- the proteolysis is the digestion mediated by proteases from the gastrointestinal track, the blood, or the bile.
- the proteolysis is mediated by pepsin, pancreatin, trypsin, trypsinogen, chymo-trypsinogen, carboxy-peptidase, pro-carboxy-peptidase, elastase, pro-elastase, or any combination thereof.
- the protease can be selected from a group of proteases released by an exogenous organism or any organism within the digestive tract, or released or produced in the digestive tract.
- the protease can be selected from a group of proteases released or produced by an abnormal, infected, cancerous or otherwise diseased tissue.
- an engineered antibody of the invention specifically binds to a pathogen.
- the pathogen can be a bacteria, a virus and a fungus.
- the pathogen is an intestinal pathogen, including but not limited to enterotoxigenic E. coli, rotavirus, Cryptosporidium parvum, Clostridium difficile, Shigella flexneri, Enterococcus faecalis, Enterococcus faecium, Campylobacter jejuni, Staphylococcus aureus, E.
- an engineered antibody of the invention specifically bind to a toxin.
- the toxin can be selected from the group consisting of a bacterial toxin, a chemical toxin and an environmental toxin.
- the bacterial toxin is a cholera toxin, an Escherichia coli toxin, a Streptococcus toxin, a Bordetella pertussis toxin, and a Clostridium toxin.
- the Clostridium toxin can comprise a botulinum toxin or a Clostridium difficile toxin.
- the botulinum toxin or Clostridium difficile toxin can comprise botulinum neurotoxin, C. difficile toxin A, or C. difficile toxin B.
- An engineered antibody of the invention, or an Ab used in a method the invention can specifically bind a virulence factor.
- the virulence factor can be an adherence factor, a coat protein, an invasion factor, a capsule, an exotoxin, or an endotoxin.
- An engineered antibody of the invention, or an Ab used in a method the invention can specifically binds to a dietary enzyme.
- the dietary enzyme can be a lipase, an esterase, a urease, a lyase, a protease, an isomerase, a ligase or a synthetase.
- the invention provides an isolated or recombinant nucleic acid comprising a sequence encoding an engineered antibody of the invention, a vector comprising the encoding nucleic acid, and a cell comprising the encoding nucleic acid or the vector comprising the encoding nucleic acid.
- the invention provides a method of stabilizing antibody activity in the presence of a protease comprising introducing at least one mutation into the amino acid sequence of the antibody that reduces or eliminates the loss of antibody structural integrity after protease digestion, thereby stabilizing antibody activity.
- the stabilized antibody maintains at least a portion of its biological activity.
- the invention provides a method of stabilizing antibody activity in the presence of a protease comprising crosslinking linking one or more antibodies, wherein the antibody comprises an antibody made by a method of the invention or an antibody described herein, and crosslinking reduces or eliminates the loss of antibody structural integrity after protease digestion, thereby stabilizing antibody activity.
- the invention provides a method of stabilizing antibody activity in the presence of a protease comprising introducing at least one mutation into the amino acid sequence of the antibody, wherein the mutation permits association of the antibody with a secretory component, wherein the association with the secretory component
- the invention provides methods of ameliorating, treating or preventing disease, infection, or other disorder caused by an abnormal cell, pathogen or toxin comprising administering orally a pharmaceutically effective amount of the antibody of invention, or the pharmaceutical composition comprising the antibody, to a subject in need thereof, whereby the disease, infection or other disorder is treated or prevented.
- Any subject can be treated using the antibody of the invention where the disease, infection, or disorder suggests the desirability of such treatment.
- any mammal can be treated, including but not limited to humans, cattle, horses, hogs, dogs, cats, and the like.
- An antibody of the invention can target any abnormal cell, e.g., a cancer cell.
- the antibody will bind at least one antigen expressed on the cell surface of the cell.
- the cancer treated by this method can be an adenocarcinoma, squamous carcinoma, leukemia, lymphoma, melanoma, sarcoma, or teratocarcinoma.
- the tumor is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, head and neck, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus.
- the cancer is colon cancer or gastrointestinal cancer.
- the abnormal cell targeted by an antibody of the invention, or an antibody used in a method of the invention can be an inflammatory cell or a chronically activated cell. Such cells often result in chronic inflammation, inflammatory sequelae, or autoimmunity.
- the antibody can also target a virulence factor on a pathogen, including a toxin such as those disclosed herein.
- the invention provides a method of ameliorating, treating or preventing gastrointestinal infections or other disorders caused by a pathogen or a toxin comprising administering orally a pharmaceutically effective amount of an engineered antibody of invention, or the pharmaceutical composition comprising the antibody, to a subject in need thereof, whereby the infection or other disorders is treated or prevented.
- the present method further comprises the co-administration of at least one anti- infectious agent or drug.
- Any suitable anti-infectious agent or drug can be used.
- the anti-infectious agent or drug is selected from the group consisting of an antibiotic, a second antibody, and a biologically active protein.
- Any suitable antibiotic can be used in the methods of the invention.
- Exemplary antibiotics include beta-lactams, aminoglycosides, vancomycin, linezolid, chloramphenicol, macrolide antibiotics, trimethoprim/sulfamethazole, clindamycin, metronidazole, rifampin, mucopirin, fluoroquinolones, as well as generational derivatives of known classes of antibiotics.
- the second antibody comprises a second orally deliverable antibody produced by the method provided herein, wherein the second antibody is directed to a different target epitope or protein than the first antibody.
- the second antibody can also be targeted to a different virulence factor.
- the invention provides a method to ameliorate or prevent toxicity associated with Clostridium difficile, comprising administering to a subject in need thereof: a) a therapeutically effective amount of a first monoclonal antibody, wherein the first monoclonal antibody comprises the heavy chain variable region sequence of SEQ ID NO:1 and the light chain variable region sequence of SEQ ID NO:2; and b) a therapeutically effective amount of a second monoclonal antibody, wherein the second monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO.3 and the light chain variable region sequence of SEQ ID NO:4, whereby the antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin A.
- the method further comprises administering a third monoclonal antibody, wherein the third antibody is a monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO:6, whereby the antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin B.
- the third antibody is a monoclonal antibody comprising the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO:6, whereby the antibodies ameliorate or prevent the toxicity associated with Clostridium difficile toxin B.
- the invention provides a method of ameliorating or preventing toxicity associated with Clostridium difficile, comprising administering to a subject in need thereof: a) a first antibody that partially or completely inhibits binding of a Clostridium difficile toxin A to a cell; and b) a second antibody that partially or completely inhibits intracellular internalization of the Clostridium difficile toxin A, wherein the first antibody and the second antibody bind to the Clostridium difficile toxin A at non-overlapping epitopes, hi one embodiment, the method further comprises administering a therapeutically effective amount of a third antibody that partially or completely neutralizes Clostridium difficile toxin B.
- the second antibody is not the monoclonal antibody PCG-4.
- the first and second antibodies synergize to neutralize the virulence factor at an antibody concentration lower than the antibody concentration necessary to observe partial neutralization by each antibody alone.
- the first monoclonal antibody and the second monoclonal antibody bind to a Clostridium difficile toxin A at ToxA: 1800-2710.
- the third antibody is a monoclonal antibody that binds to a Clostridium difficile toxin B at ToxB: 1807-2366.
- the first monoclonal antibody and the second monoclonal antibody do not bind Clostridium difficile toxin B, and the third monoclonal antibody does not bind Clostridium difficile toxin A.
- the methods of the invention employ monoclonal antibodies comprising recombinant or synthetic antibodies.
- One or more of the antibodies can be rendered partially or completely resistant to proteolysis and/or orally deliverable using the antibody engineering methods of the invention.
- the antibodies and methods of the invention can be useful in the treatment of the Clostridium toxin-related toxicity in a subject, wherein the toxicity comprises Clostridium- associated diarrhea, colitis or a related condition, whereby one or more symptoms of the Clostridium-induced diarrhea, colitis, or related condition are ameliorated or prevented following administration of the monoclonal antibodies.
- these antibodies can be an IgG antibody.
- the antibody is a human, murine, rat, rabbit, bovine, camel, llama, dromedary, or simian antibody.
- the antibody of the invention, or the Ab used in a method of the invention is a humanized antibody, chimeric antibody, bispecific antibody, fusion antibody, nanobody, diabody, scFv, or biologically active fragment thereof.
- An antibody of the invention, or an Ab used in a method of the invention can be modified to increase resistance to proteolysis.
- the antibody can be modified to be orally deliverable, using, for example, when practicing the methods of the invention.
- the biologically active protein is a toxin-degrading or toxin- inactivating protease.
- the protease is capable of partially or completely degrading or inactivating the targeted toxin.
- the toxin can come from any source, including but limited to a bacterial toxin, a chemical toxin and an environmental toxin.
- the antibody provided herein may be administered either simultaneously with the biologically active agent(s), or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the invention in combination with the biologically active agent(s).
- compositions and formulations comprising an antibody of the invention, or the novel combination of antibodies of the invention, or an antibody made by method of the invention (e.g., an antibody modified to be resistant, completely or partially, to a protease).
- the invention provides pharmaceutical compositions comprising an Ab of the invention, or the novel combination of antibodies of the invention, or an antibody used in or made by a method of the invention, and a suitable excipient (e.g., a pharmaceutically acceptable excipient).
- the invention provides combinations of monoclonal and/or synthetic antibodies, e.g., "synthetic polyclonals,” that work synergistically to neutralize bacterial toxins, e.g., enteric bacterial toxins such as Clostridium difficile toxin A.
- the pharmaceutical composition is formulated as a suspension, a liquid, a capsule, a tablet, a gel, a microsphere, a liposome, a multiparticulate core particle or a spray.
- the antibody comprises 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more, or from about 50% to about 95%, of the batch size (weight/weight) of the pharmaceutical composition.
- the pharmaceutical composition is formulated for enteric or oral delivery.
- the pharmaceutical composition further comprises an enteric coating or any coating for oral delivery, e.g., as gelatin capsules, liposomes or formulated as a pre-liposome formulation and then put into a capsule.
- the antibodies of the invention may serve as diagnostic tools.
- antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
- labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
- the antibodies provided herein can be useful as the antigen-binding component of fiuorobodies. See e.g., Zeytun et al, Nat. Biotechnol. 21 : 1473-79 (2003).
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an engineered antibody of the invention, or an antibody used in or made by a method of the invention, and a suitable excipient.
- the composition is formulated as a suspension, a liquid, a capsule, a tablet, a gel, a microsphere, a liposome, a multiparticulate core particle or a spray.
- the antibody comprises from about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more, or from about 50% to about 95%, of the batch size (weight/weight) of the pharmaceutical composition.
- the composition is formulated for enteric or oral delivery.
- the pharmaceutical composition further comprises an enteric coating or any coating for oral delivery, e.g., as gelatin capsules, liposomes or formulated as a pre-liposome formulation and then put into a capsule.
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 and EDs 0 .
- Antibodies exhibiting high therapeutic indices are used to practice the invention, e.g., are the antibodies modified by the methods of the invention.
- the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
- the dosage of such compounds lies in one aspect within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl. et ah, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1 (latest edition).
- Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the desired therapeutic effects, or minimal effective concentration (MEC).
- MEC minimal effective concentration
- the MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration necessary to achieve 50% neutralization of the targeted virulence factor activity.
- the subject is pretreated to lower the pH of the intestine (make more acidic) or increase (make more basic) the pH of the stomach. Such methods are well known in the art.
- the subject is pretreated or co-treated with at least one antibiotic.
- the disorder is an ulcer.
- any enteric pathogen can be treated using an antibody of the invention, or using the methods of the invention.
- the enteric pathogen is Clostridium difficile.
- the invention provides a method of ameliorating or preventing toxicity associated with a first virulence factor in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of at least two monoclonal antibodies, wherein the antibodies synergize to neutralize the effects of the first virulence factor, thereby ameliorating or preventing the toxicity associated with the first virulence factor.
- the first virulence factor is a toxin, alternatively a Clostridium sp. toxin, a toxin A or toxin B.
- the antibodies administered comprise a first antibody that partially or completely inhibits binding of a Clostridium difficile toxin to a cell; and a second antibody that partially or completely inhibits intracellular internalization of the Clostridium toxin, wherein the first antibody and the second antibody bind to the Clostridium toxin at non- overlapping epitopes.
- the monoclonal antibodies neutralize the first virulence factor to the same degree or greater than a polyclonal antiserum.
- the Clostridium difficile toxin is a Clostridium difficile toxin A or a Clostridium difficile toxin B.
- the monoclonal antibodies of the invention, or an Ab used in a method of the invention can be recombinant or synthetic antibodies as described above.
- a method of the invention comprises administering a therapeutically effective amount of a third antibody that partially or completely neutralizes a second virulence factor.
- the second virulence factor is Clostridium difficile toxin B and the first virulence factor is Clostridium difficile toxin A.
- the Clostridium toxin- related toxicity in the subject treated by the methods of the invention comprises Clostridium- associated diarrhea, colitis or a related condition, and whereby one or more symptoms of the Clostridium-mdxxced diarrhea, colitis, or related condition are ameliorated or prevented following administration of the monoclonal antibodies, hi alternative embodiments, at least one of the antibodies produced by the method of invention is partially or completely protease- resistant. In one aspect, an Fc portion of the antibody is partially or completely protease- resistant.
- the first and second antibodies synergize to neutralize the virulence factor at an antibody concentration lower than the antibody concentration necessary to observe partial neutralization by each antibody alone.
- the virulence factor is Clostridium difficile toxin A.
- the first monoclonal antibody and the second monoclonal antibody bind to a Clostridium difficile toxin A at ToxA:l 800-2710.
- the second monoclonal antibody is not PCG-4.
- the third antibody is a monoclonal antibody that binds to a Clostridium difficile toxin B at ToxB: 1807-2366.
- the first monoclonal antibody and the second monoclonal antibody do not bind Clostridium difficile toxin B, and the third monoclonal antibody does not bind Clostridium difficile toxin A.
- the antibodies each bind different virulence factors of the pathogen.
- the therapeutic methods of the invention can further comprise administering a therapeutically effective amount of a protease that partially or completely neutralizes the toxin.
- the protease is administered in the same formulation as the first antibody, the same formulation as the second antibody, or in the same formulation as the first and the second antibody.
- the antibodies and any other bioactive agent are administered in an enteral (enteric) formulation.
- enteral any suitable enteral formulation may be employed. See e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (latest edition).
- enteral formulation Any suitable enteral formulation may be employed. See e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (latest edition).
- the antibodies can be formulated as a suspension, a liquid, a capsule, a tablet, a gel, a plant matrix material, a microsphere, a liposome, a multiparticulate core particle or a spray.
- the invention provides a method of ameliorating or preventing toxicity associated with a virulence factor in a cell, comprising administering to the cell a therapeutically effective amount of at least two monoclonal antibodies, wherein the antibodies synergize to neutralize the effects of the virulence factor, thereby ameliorating or preventing the toxicity associated with the virulence factor.
- the invention provides a method of ameliorating or preventing toxicity associated with a virulence factor in a cell, comprising administering to the cell a) a first antibody that partially or completely inhibits binding of a Clostridium difficile toxin to a cell; and b)a second antibody that partially or completely inhibits intracellular internalization of the Clostridium difficile toxin, wherein the first antibody and the second antibody bind to the Clostridium difficile toxin at non-overlapping epitopes, and with the proviso that the second antibody is not the monoclonal Ab PCG-4.
- any suitable biologically active agent may be co-administered with an antibody of the invention.
- the antibodies or antigen binding fragments thereof provided herein may be conjugated to a bioactive agent.
- the Ab or antibodies can be co-administered with at least one bioactive agent.
- these antibodies and/or agents can be administered sequentially or simultaneously.
- Coadministered agents include but are not limited to agents such as cytokines, such as IL-2, IL- 12, interferon (IFN), Tumor Necrosis Factor (TNF); photosensitizers (for use in photodynamic therapy), including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as indium-Ill ( 111 In), iodine-131 ( 131 I), yttrium-90 ( 90 Y), bismuth-212 ( 212 Bi), bismuth-213 ( 213 Bi), technetium-99m ( 99m Tc), rhenium-186 ( 186 Re), and rhenium- 188 ( Re); antibiotics, such as doxorubicin, daunorubicin, methotrexate, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria to
- ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing antitumor agents ⁇ e.g., antisense oligonucleotides, siRNA, plasmids encoding toxins, methotrexate, etc.); other antibodies or antibody fragments, such as F(ab); anti-angiogenic agents including protamine, heparin, steroids, thalidomide, TNP-470, carboxy
- the invention further provides antibodies engineered by the methods of the invention and useful in the methods of treatment of pathogen-induced symptoms and diseases as described above.
- the first monoclonal antibody is produced by the hybridoma ATCC Accession No. (Ab designated 227 or 3359).
- the second monoclonal antibody is produced by ATCC Accession No. (Ab designated 543 or 3358).
- the third antibody comprises a monoclonal antibody produced by a hybridoma selected from the group consisting of ATCC Accession No. XXXX (Ab designated F85), ATCC Accession No. XXXX (Ab designated F2), and ATCC Accession No. XXXX (Ab designated F87).
- the invention provides pharmaceutical compositions comprising at least one antibody of the invention, e.g., a monoclonal antibody (Mab) or a novel combination of Mabs of the invention, and a suitable excipient.
- Formulations and excipients useful in the pharmaceutical compositions are those well known in the art.
- An antibody of the invention, or any antibody used in the methods of the invention may be administered to a subject in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. See e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (latest edition).
- Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
- pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).
- the characteristics of the carrier will depend on the route of administration.
- the pharmaceutical composition of the invention may also contain other anti-pathogen or anti-tumor agents such cytokines or chemotherapeutic agents as is desirable.
- the precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g., whole antibody, fragment, diabody or triabody), and the nature of any other molecule attached to the antibody.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Goodman et al, eds., McGraw-Hill Professionals, 9th Ed. 1996). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the desired therapeutic effects, or minimal effective concentration (MEC).
- the antibody dose is in the range of between about 0.1, 0.5, 1.0, 5.0 or 10.0 ⁇ g to 50, 60, 70, 80, 90 or 100 ⁇ g, or alternatively, 100, 200, 300, 400 or 500 ⁇ g to about 600, 700, 800, 900 or 1,000 ⁇ g (1 mg), or from about 1, 5, 10, 50 100, 200, 300, 400 or 500 mg to about 600, 700, 800, 900 or 1,000 mg (1 gm) or more for oral applications.
- the antibody is a whole antibody, in one aspect an IgG isotype, e.g., the IgG 1 isotype.
- a dose for a single treatment of an adult patient is proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight.
- Treatments may be repeated at, e.g., hourly, every 2, 4, 6, or 12 hours, daily, twice- weekly, weekly, every 21 days, every 28 days, or monthly intervals, at the discretion of the physician.
- treatment is periodic, and the period between administrations is, e.g., hourly, every 2, 4, 6, or 12 hours daily (e.g., b.i.d., t.i.d.), or weekly, or about two weeks or more, or about three weeks or more, or about four weeks or more, or about once a month.
- compositions for use in practicing the methods of the invention, or for formulating Abs of the invention can be formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically.
- These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating (e.g., to make a smooth, fine powder or paste, as by grinding when moist), emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
- a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils
- the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
- the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the invention, and in one aspect from about 1 to 50% protein of the invention.
- Antibodies of the invention, or antibodies used in the methods of the invention can be encapsulated into gelatin capsules, liposomes or formulated as a pre-liposome formulation and then put into a capsule.
- the capsule can be a soft gel capsule capable of tolerating a certain amount of water, a two piece capsule capable of tolerating a certain amount of water or a two piece capsule where the liposomes are preformed then dehydrated.
- the liposomes used to practice the invention can comprise any bilayer forming lipid, e.g., phospholipids, sphingolipids, glycosphingolipids, and ceramides.
- a soft gel capsule When using gelatin capsules, e.g., a soft gel capsule can be 10% on the interior, or, the concentration of water in a liposome formulation can range from 5% to 90% water.
- Capsulation can protect the liposome-antibody complex from the low pH of the stomach, emulsification from bile salts and degradation by digestive enzymes. This protection can be further enhanced when the outer shell of the capsule is coated with a polymer like hydroxyethylmethyl cellulose propyl ethyl acetate or hydroxypropylmethylcellulose propylethyl thallate. See, e.g., U.S. Patent No. 6,726,924.
- the bile acid transport system is manipulated to provide sustained systemic concentrations of orally delivered antibody formulations of the invention, Kits
- kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds or plants or plant parts, polypeptides (e.g., antibodies) and/or antibodies of the invention.
- the kits also can contain instructional material teaching the methodologies and industrial uses of the invention, as described herein.
- the invention provides a kit for ameliorating or preventing one or more symptoms of virulence factor-associated symptom or disease, comprising a) a pharmaceutical composition comprising the monoclonal antibodies disclosed herein and a suitable excipient; and b) instruction for administering the pharmaceutical composition.
- the pharmaceutical composition comprises a) a first monoclonal antibody that partially or completely inhibits binding of Clostridium difficile toxin A to a cell; b) a second monoclonal antibody that inhibits Clostridium difficile toxin A intracellular internalization, wherein the first monoclonal antibody and the second monoclonal antibody bind toxin A at non-overlapping epitopes, and with the proviso that the second monoclonal antibody is not PCG-4; c) a third monoclonal antibody that partially or completely neutralizes Clostridium difficile toxin B; d) an ax ⁇ i-Clos iridium difficile toxin protease; e) a suitable excipient; and instructions for administering the pharmaceutical composition.
- a compartment kit comprising one or more containers, wherein a first container comprises one or more antibodies engineered by the methods of the invention, and one or more other containers comprising one or more of the following: wash reagents, reagents necessary for administration of the antibody or capable of detecting presence of a bound antibody.
- the containers can be glass, plastic, or strips of plastic or paper.
- Types of detection agents include labeled secondary antibodies, other labeled secondary binding agents, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents that are capable of reacting with the labeled antibody.
- Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention.
- Example 1 Engineering antibodies resistant to intestinal fluids
- the invention provides antibodies for oral delivery, and methods for the development of antibodies that are stable in the digestive-tract environment.
- This example describes an exemplary method of the invention for developing antibodies that are stable in the digestive-tract environment, i.e., antibodies for oral delivery, and exemplary antibodies of the invention made by these methods.
- This example describes an exemplary method of the invention using the Kabat numbering system to design/ make an antibody with the scope of the invention (see, e.g., Table 1, below).
- IgG 2 and IgG 4 appeared to undergo extensive proteolysis at very early time points. However, IgG 1 and IgG 3 seemed to display superior "resistance" to pepsin digestion. On a reducing gel, IgG 1 exhibited a higher proportion of light chain maintained throughout time when compared to IgG 3 , see Figure 2. In Fig. 2, IgG 1 (10 ⁇ g/lane) was digested for 0, 2, 5, 10, 20 and 30 min with pepsin. Molecular weight marker (MW- kDa) is indicated.
- the antibody molecule exhibits a pH dependent unfolding event (see Figure 4) leading to some irreversible degradation/aggregation.
- Fig. 4 the spectra OfIgG 1 at pH values of 3 and above were highly indicative of ⁇ -sheet-like structure with a single minimum at 217 nm.
- the spectra changed radically to spectra highly indicative of random coil (unfolded) with the characteristic minimum at 197 nm.
- Determination of cleavage sites to mutate To identify residues that should be mutated to engineer an antibody molecule with increased resistance to pepsin, two approaches were employed: a proteomic approach and the calculation of surface exposure of potential pepsin cleavage sites within the antibody.
- Approach 1 Digested IgGl was assessed by mass spectral analysis to identify the pepsin cleavage sites. Because antibody fragments were still too large for analysis by tandem mass spectrometry (MS/MS), trypsin was used to generate smaller peptides in the presence of a 1 : 1 mixture of 16O/1 8 O, so that peptides produced with pepsin should have a normal isotopic distribution (singlet) and peptides produced from trypsin should have a modified distribution (doublet). Four pepsin cleavage sites were identified using this approach.
- Approach 2 The human IgG Fc structure was analyzed for exposed pepsin cleavage motifs as previously described in Delano, et al.
- Table 1 shows the position of mutations engineered in the human IgGl heavy chain. In red (or only bolded) are the highest priority based on surface exposure, sequence and proteomic analysis. In blue (or underlined) are the other potential pepsin cleavage sites based on sequence analysis only. All prioritized residues were >20% exposed to solvent based on an available crystal structure (Delano et al., 2000) and all de-prioritized residues were ⁇ 20% exposed unless otherwise indicated.
- a Proteomically determined cleavage site proteolytic enhancing flanking motif; proteolytic inhibitory flanking motif; d Carbohydrate interacting residue; e Greater than 20% exposed, de-prioritized; f Less than 20% exposed, prioritized; g Mutation derived from IgA sequence comparison; h Mutation derived from kappa chain sequence comparison; 'Residue within the hinge region. Mutations engineered in an exemplary human IgGl heavy chain f SEQ ID NO:9).
- + Expression was greater than wildtype; : Equivalent expression compared to wildtype; -: Less material was expressed than the wildtype; -: No expression.
- Each antibody variant was given a thermotolerance score according to the following criteria: +: A greater percentage of folded protein remaining at 75°C and/or 80°C compared to wildtype; : Equivalent percentage of folded protein remaining at each temperature point compared to wildtype; -: A lesser percentage of folded protein remaining at 75°C than wildtype; -: Thermal unfolding observed at 70°C. Detection: ⁇ Detected using anti Fab-AP. Otherwise detected with anti Fc-AP.
- Tables 3A and 3B shows the ELISA results after pepsin digestion at pH 3, pH 2 and pH 2.5 of the wildtype and the mutated antibody molecules.
- the parent antibody molecule as well as the mutants were expressed in mammalian cells, purified, and dialyzed. 1 ⁇ g was digested for the time indicated with pepsin (X0.005) at 37°C at pH 2, pH 2.5, and pH 3. Two separate tests were performed: one to detect the remaining constant domain and a test to assess the remaining binding activity of the antibody molecule. In all cases, antibody degradation was determined by measuring by ELISA the amount of antibody remaining after digestion. Mutations are listed below. Table 3 A shows the ELISA results after pepsin digestion of the wildtype and mutants.
- Table 3B shows the ELISA results after pepsin digestion at pH 2, 2.5 and 3 of the mutant combination. The percentage of toxin A binding activity remaining after digestion is reported after 0.5 h, 1 h, and 4 h digestion with pepsin.
- Fig. 5(A) illustrates ELISA detection of the remaining quantity of rPBA3 after incubation at pH 1, 2 and 3 using solutions containing hydrochloric and detected with AP-labeled anti-Fc.
- hi Fig. 5 (B) rPBA3 was digested by 0.005X SGF at 37 0 C for various incubation periods. Two separate detection antibodies were used, AP-labeled anti-Fc and AP-labeled anti-Fab 2 , in order to discriminate between Fc degradation and hinge clipping. Thermotolerance of rPBA3 incubated at various temperatures for both 10 and 30 minutes, hi all cases, antibody degradation was determined by measuring by an ELISA the amount of rPBA3 remaining after digestion.
- thermotolerance screening was performed for every member of the library to determine whether mutation at each pepsin-labile position was tolerated. The majority of the library members were also tested for tolerance at low pH. DNAs derived from the 72 variants were transfected into mammalian cells and the resulting supernatants were screened for thermotolerance, pH and pepsin-resistance. AU antibodies demonstrated a similar pH tolerance compared to wildtype. However, many mutants demonstrated inferior the ⁇ notolerance and/or expression compared to the wildtype molecule. Approximately 46% of the database selected mutants were destabilizing while 64% of the Alanine mutations were destabilizing (Table 4). Interestingly, this mutational strategy (based on an IgG sequence database developed for this invention) provided a significantly greater proportion of tolerable residue replacements relative to alanine scanning. Destabilizing mutations were eliminated from the mutant combinations described below.
- Table 4 shows the ELISA results after pepsin digestion at pH 3 the single mutants. The percentage of Fc remaining after digestion as well as the percentage of antibody binding to toxin A are reported after 0.5 h, 1 h, and 4 h digestion with pepsin at pH 3. Table 4
- FIG. 6 illustrates the pepsin digestion profile of wildtype and mutant antibodies at pH 1.2.
- the mutants carried either 4 mutations in the heavy chain constant domain (L258P, L332Q, F427Y, F264Y) or 6 mutations in the heavy chain constant domain (L258P, L332Q, F427Y, F264Y, L202I, L421Q).
- the antibodies were expressed in mammalian cells, purified, and dialyzed.
- Pepsin digestion time points were 0, 2, 5, 10 and 20 min (X0.005 SGF) at pH 1.2.
- FIG. 6(A) illustrates an SDS-Page Analysis of IgG digests with pepsin at pH 1.2. DTT was added to the samples prior to loading each 800 ng time point to wells in a 4-12% Bis-Tris gel. Gels were silver-stained.
- Fig. 6(B) illustrates an ELISA detection of each recombinant IgG after digestion.
- Figure 7 illustrates the pepsin digestion profile of wildtype and mutant antibodies at pH 3.0.
- Fig. 7(A) illustrates an SDS-Page/Silver stain analysis of pH 3, pepsin digestion.
- Time points included (Lanes 5-12, respectively) are 0, 2, 5, 10, 20, 30, 60 and 120 minutes.
- DTT was added to the samples prior to loading each 800 ng timepoint to wells in a 10% Bis-Tris gel. Gels were silver-stained.
- Lane 1 is the SEEBLUEPLUS2TM standard
- Lane 2 is reduced Fab fragment standard
- Lane 3 is reduced Fc fragment standard
- Lane 4 is each recombinant protein loaded at 1 ⁇ g.
- Fig. 7(B) illustrates an ELISA analysis of various recombinant IgGs digested by pepsin at pH 3.
- Digestion of the wildtype protein begins to generate Fc at 5 minutes.
- a second, lower molecular weight band ( ⁇ 10-12 kDa) also begins to form due to heavy chain degradation at the 10 mn time point.
- BD13964 and BD14079 were completely resistant to digestion for 2 hours at pH 3, 37°C.
- an accurate antibody fold sequence alignment based on the available crystal structures of the immunoglobulin subclasses and cell surface receptors is built. Most antibody folds whose structures are known will be structurally aligned to the lowest root-mean- squared deviation to create optimal sequence alignment. Sequences without crystal structures are aligned to the next most homologous antibody fold sequence whose structure is solved. The resulting sequence alignment is analyzed for co-varying pairs as described by Davidson and co workers (Larson et ah, 2000). This co- variation analysis identifies adequate residue replacements for salt bridges and aspartic acid and glutamic acid residues within IgGi that lead to the denaturation of the molecule at pH values below 3. Material and Methods
- IgG subclasses in gastric fluid All antibodies purchased from Calbiochem were isolated from human myelomas: IgGl with kappa light chain (CalBiochem Cat #400120), IgG2 with kappa light chain (CalBiochem Cat#400122), IgG3 with lambda light chain (CalBiochem Cat#400124), and IgG4 with lambda light chain (CalBiochem Cat#400126).
- Simulated gastric fluid (SGF) was prepared fresh daily as described in the United States Pharmacopoeia.
- IX SGF buffer comprised 3.2 mg/mL pepsin (Sigma Chemical Co., St. Louis, MO), NaCl (2 mg/mL) at pH 1.2.
- Dilutions were prepared in the same buffer.
- a master tube was prepared in a 1.5 mL microcentrifuge tube containing 60 ⁇ g of antibody and 120 ⁇ L 0.00 IX SGF in a final volume of 180 ⁇ L. The reaction was incubated at 37°C. At intervals of 0, 2, 5, 10, 20, and 30 min, aliquots of 30 ⁇ L containing 10 ⁇ g of antibody were removed from the master tube and added immediately to 7 ⁇ L 4X NUP AGETM LDS sample buffer (Invitrogen) and heated for 5 min at 100 0 C. Samples were subjected to SDS-PAGE using precast 4-12% Bis-Tris NUP AGETM gels (Invitrogen, Carlsbad, CA).
- Hybridoma culture Hybridoma cell line PBA3 expressing a Clostridium difficile antitoxin A recognizing antibody was obtained from ATCC. Cell lines were grown in DMEM (Dulbecco's Minimal Essential Medium with high glucose, Gibco, Invitrogen, Carlsbad, CA), 10% FBS (Sterile Fetal Bovine Serum, Sigma Chemical, St.
- Primers used for the amplification of the variable region from both the light chain and the heavy chains were designed as described previously (Coloma et ah, 1992; Dattamajumdar et al., 1996).
- Primers MLALT5 and 33615 were used for amplification of the variable region from the light chain (MLALT5: 5'-CACCATGAAGTTGCCTGTTAGGCTGTTG-S' (SEQ ID NO:10); 33615: 5'-GAAGATCTAGACTTACTATGCAGCATCAGC-S') (SEQ ID NO:11).
- Primers MVGlR and MHl were used for the amplification of the heavy chain variable region (MHl: 5'- ATATCCACCATGGRATGSAGCTGKGTMATSCTCTT-3 ' (SEQ ID NO: 12); MVGlR:
- Sense primers (based on the FRl region) and antisense primers (based on the 5 '-end of the constant region) were then designed for both chains following sequencing of the PCR products. PCR products obtained using these primers were cloned into the modified mammalian expression vector pCEP4 (Invitrogen, Carlsbad, CA).
- the modified vector either contained the signal peptide and the constant domain region of the heavy chain or the signal peptide and the constant domain of the light chain.
- the constant domain of the human IgGl was constructed by subcloning the appropriate heavy chain and light chain domains into pCEP4 from a human spleen cDNA library.
- the plasmid containing the light chain variable domain and its constant domain was designated BD 12585.
- the plasmid containing the variable domain and the constant domain of heavy chain was designated BD 12584. Both plasmids were sequenced.
- the chimeric antibody protein is referred as rPB A3 in the text.
- IgGl mutagenesis Site-directed mutagenesis on IgGl was used to generate IgGl variants in which all solvent-exposed residues in the CHl, CH2, and CH3 domains were individually altered to Ala or another residue (as specified in the list). All mutants were confirmed by DNA sequencing.
- Transfection ofrPBA3 library into 293F mammalian cell expression host All mutant plasmids were transformed into XLl -blue bacteria and stocked in glycerol. Plasmid DNA from every mutant was prepared as described by the manufacturer (Qiagen, endotoxin-free MaxiPrep kit Cat#12362).
- Plasmids were transfected into the adeno virus-transformed human embryonic kidney cell line 293 F using 293fectin in 12-well microtiter plates and using 293F-FreeStyle Media for culture.
- Light and heavy chain plasmids were transfected at 0.5 ⁇ g/mL for each plasmid and using a 1 : 1 light chain plasmid versus heavy chain plasmid ratio.
- Supernatants were collected 7 days after transfection. Expression levels varied from ⁇ 0.25-1.5 ⁇ g/mL. For larger transfections, the cells were spun down after 3 days and 1/2 the media was replenished with fresh media. Cell density upon transfection was generally 10 6 cells/mL. Supernatants were then spun down at 1200 rpm for 8 minutes at room temperature.
- each IgGl stock solution was determined by Bradford analysis (Bio-Rad Protein Assay, Hercules, CA cat# 500-0006) using a commercial myeloma IgGl stock solution (2 mg/ml - Calbiochem, cat# 400120) as a standard and by UV-absorbance at 280 nm using the method of Pace and coworkers (1995).
- SGF digestion stability assay for the mutants Simulated gastric fluid (SGF) was prepared fresh daily as described (Privalle et al, 2000) using 0.1X SGF buffer at pH 1.2 or pH 3 (3.2 mg/ml pepsin, 2 mg/ml NaCl; Sigma Chemical Co., St. Louis, MO).
- All recombinant antibodies were dialyzed into PBS and stored at 4°C.
- a master tube was prepared containing 1 ⁇ g/mL recombinant antibody and 0.0025X SGF at pH 1.2 and 0.005X SGF at pH 3.0.
- the pH of each reaction was monitored by first making appropriate dilutions of PBS with SGF and measuring the pH before and after neutralization with TrisHCl, pH 9.
- Antibodies were incubated at 37°C for intervals of 0, 2, 5, 10 and 20 min at pH 1.2 or at intervals of 0, 2, 5, 10, 20, 30, 60 and 120 min at pH 3.0. The reaction was neutralized before aliquots were taken either for ELISA analysis or for SDS-Page/Silver staining.
- Protein G (Sigma, cat# P-4689) was biotinylated using the EZ-LINK-BIOTIN- LC-ASATM kit (PIERCE catalog # 29982). Briefly, EZ-LINK-B IOTIN-LC- AS ATM was dissolved in DMSO and added individually to protein G at a 5:1 molar ratio. Protein G/biotin conjugation was induced for 20 minutes under a UV lamp in a PBS buffer. Conjugated protein G was removed from unreacted biotin by application of the reaction mixture to a desalting column (PIERCE D-SaIt Dextran Plastic Desalting Columns, catalog # 43230). 500 ⁇ L fractions from the desalting procedure were tested for protein absorption at 280 nm to detect the presence of biotinylated protein G.
- Microtiter Streptavidin plates (Sigma Chemical, St. Louis, MO, catalog #M5432) were coated with 200 ng per well of biotinylated protein G diluted into PBS buffer and incubated at 4°C overnight. The plates were then washed 3 times with TBST buffer. All samples were diluted in Tris buffer, pH 8.0 TBST buffer (Sigma, cat#T9039). Aliquots of 100 ⁇ L of each diluted sample were transferred to the protein G-coated plates and incubated for 1-2 hours at room temperature.
- the wildtype antibody began to unfold when heated to 75 0 C for 10 minutes and is completely unfolded when subjected to 8O 0 C for the same time period (Figure 5c).
- the unfolding was irreversible as cooling for any length of time did not result in the regeneration of signal in this ELISA format.
- the thermotolerance of each member of the constant domain mutant library was compared to the wildtype molecule by heating (side-by-side with the wildtype protein) to 70°C, 75°C and 80°C for 10 minutes.
- the amount of folded antibody remaining after heating was tested by ELISA (Table 3). Each antibody variant was given a thermotolerance score (see table 3).
- Clostridium difficile toxin A is well described in the art, see, e.g., Wren, et al., (1990) "Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction," FEMS Microbiol. Lett. 70:1-6 (1990) (NCBI accession no. A37052): 1 msliskeeli klaysirpre neyktiltnl deynklttnn nenkylqlkk lnesidvfmn
- Example 2 Antibody treatment of Clostridium difficile induced toxicity This example demonstrates that antibodies made by methods of the invention can bind and neutralize Clostridium difficile toxin.
- Antibodies raised against the C-terminal domains of toxins A and B were tested for their ability to bind and neutralize the affect of the toxins. Toxin neutralization in cell assays, antibody affinity measurements, epitope mapping and antibody competition experiments were all used to characterize and prioritize antibody candidates.
- One of the most neutralizing antibody against toxin A denoted 3358 or 543, demonstrated the ability to bind the full-length CWB-domain at approximately fourteen high affinity sites.
- the PCG-4 antibody (Lyerly (1986) Infect Immun. 54:70-76) was shown to contain between four and six high affinity binding sites. No single monoclonal antibody was fully neutralizing using the in vitro toxin neutralization assays.
- Toxins A and B were cloned directly from genomic DNA preparations of a control strain of C. difficile (ATCC # 51695).
- C. difficile was cultured at 37°C in a thioglycollate anaerobic broth (BBL#273127) for 48-72 hours. Cultures were pelleted and genomic DNA was extracted using a RNA/DNA Maxi Prep Kit (Qiagen, Cat#14162). DNA inserts were generated for the toxin A and toxin B C-terminal repeat regions using primers designed to match the NCBI deposited sequences (Toxin B ATCC accession #BCAA8O815.1; toxin A #AAA23283.1).
- Inserts were cloned into the pSE420 plasmid (Invitrogen, Cat#V4020) at the Ncol/Bglll using a Bsal cloning strategy (New England BioLabs, Cat#R0535S).
- the plasmid was modified to include a C-terminal, thrombin-cleavable hexa-histidine tag.
- the cloned toxin B sequences matched the NCBI reference sequence perfectly.
- the toxin A clones had 4 amino acid mutations (N1939D, L2080W, D2426H and A2427N) which were consistently amplified and can be strain specific.
- the plasmids containing the toxin inserts were given the following designations: ToxA: 1800-2710, BD11822; ToxA: 1800-1945, BD15049; ToxA:2078-2234, BD15050; ToxA:2459-2710, BD11711; ToxB:1808-2366, BD11713; ToxB:2207-2366, BD11712.
- Each toxin containing plasmid was transformed into BL21(DE3) (Invitrogen, cat#C60000-03) or recAl deficient XLl-blue (Stratagene, cat#200236) for improving the insert stability of the repeat domains. Protein expression was performed following standard protocols (1). Transformed cells were cultured in 2-6 L Luria Broth with 100 ⁇ g/mL carbenicillin at 37°C until cell densities of 0.7-0.9 were reached. At this point, expression was induced with 1 mM IPTG and cultures were grown for 12 hours at 25 0 C before harvesting. Cells were pelleted at 400Og and stored at -2O 0 C.
- a hybridoma cell line PBA-3 (ATCC# HB-8713) was purchased from the ATCC. The cell line was grown in DMEM (Dulbecco's Minimal Essential Medium with high glucose (Gibco/Invitrogen, Carlsbad, CA), 10% FBS (Sterile Fetal Bovine Serum, Sigma Chemical, St. Louis, MO), and IX glutamine/Penicillin / Streptomycin (Gibco/Invitrogen, Carlsbad, CA) and cryopreserved. Total RNA was isolated from 10 7 hybridoma cells using a procedure based on the RNeasy Mini kit (Qiagen, Hilden Germany).
- the poly-A+ RNA fraction was purified using an Oligotex mRNA mini kit (Qiagen) and used to generate first strand cDNA (Clontech cDNA synthesis kit, Clontech Laboratories, Inc., Palo Alto, CA). Primers used for the amplification of the variable region from both the light chain and the heavy chains were designed as described previously (58,59). Primers MLALT5 and 33615 were used for amplification of the variable region from the light chain (MLALT5: 5'-CACCATGAAGTTGCCTGTTAGGCTGTTG-S' (SEQ ID NO:10); 33615:
- the modified vector either contained the signal peptide and the constant domain region of the heavy chain or the signal peptide and the constant domain of the light chain.
- the constant domain of the human IgG 1 was constructed by subcloning the appropriate heavy chain and light chain domains into pCEP4 from a human spleen cDNA library.
- the plasmid containing the light chain variable domain and its constant domain was designated BD12585.
- the plasmid containing the variable domain and the constant domain of heavy chain was designated BD12584.
- the 3358 (or 543) and 227 (or 3359) antibody variable domains were subcloned into pCEP4 using the same protocol as outlined for rPBA-3. Below are the amino-acid sequences of the variable region antibodies. CDR regions are underlined. The "completed” or complete antibodies comprise human sequence constant regions. Antibody 227 for 3359) variable heavy chain (IgGl)
- Antibody 3358 (or 543) variable heavy chain (IgG2a) OVOLOOPGAELVKPGASVRLSCKAGGYTFTSYWLHWVKQRPGOGLEWIGMIHPNSG
- NIVMTOSPKSMSMSVGERVTFNCRASENVGTWFWYQQKPEOSPRLLrYGASNRYTG VPDRFTGSGSATDFTLTISGVOAEDLADYHCGQSYRHLTFGGGTKLEIK SEQ ID NO:4
- DIKMTQSPSSMYTSLGERVTITCKASODINSCLSWFQQKPGKSPKALIF RANILVDGVPS RFSGSGQDYSLTISSLEYEDLG ⁇ YYCLOYDEFPWTFGGGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:27)
- Recombinant Antibody 3358 (or 543) heavy chain (IgG2a) full length sequence QVQLQQPGAELVKPGASVRLSCKAGGYTFTSYWLHWVKQRPGQGLEWIGMIHPNSG SYDYSETFRTKATLTVDKSSDTAYMQLTSLTSEDSAVYYCARGGSNYDIFAYWGQGT TLTVSSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSQALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDICRVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQ
- variable heavy and variable light chain domains of PCG-4 were created synthetically based on the sequences for the light and heavy chains obtained from Genbank (accession numbers X82691 and X82692).
- the variable domain of both the heavy and the light chains were individually synthesized from 12 synthetic oligos by overlap extension PCR (57).
- the full- length product was cut with Sad and Bbsl (sites included in terminal PCR primers; Bbsl site designed to generate CCTC overhangs compatible with vector) and cloned between the AppA leader sequence (for periplasmic export) and domain I of the human IgG heavy chain in vector pKW-1 (pBK-CMV derivative).
- An appA leader sequence was added to the light chain before the insert was cut with
- variable domains were subcloned from the Fab construct into the same pCEP4 vector system described for rPBA-3 for production of a chimeric full-length IgG construct.
- constant region sequences are human sequences known in the art. Any constant region sequence can be used, e.g., any human constant region sequence can be used to design and make an antibody of the invention.
- the invention provides antibodies comprising the heavy chain variable region sequence encoded in SEQ ID NO:1 and the light chain variable region sequence encoded in SEQ ID NO:2 and the remainder of the antibody (e.g., constant region) comprising human sequence, thus making a "humanized” chimeric antibody (similarly; and in alternative aspects the "humanized” chimeric antibody comprises the variable region sequence combinations SEQ ID NO:3 and SEQ ID NO:4, or, SEQ ID NO:5 and SEQ ID NO:6, or, SEQ ID NO:7 and SEQ ID NO: 8). Exemplary methods to make antibodies of the invention are described herein.
- any constant region sequence can be used, e.g., any human constant region sequence can be used to design and make an antibody of the invention, e.g., and antibody having a variable region comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO.3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8, or substantially similar sequences, as set forth herein.
- Human constant region sequences are well known in the art, e.g., see discussion on Kabat sequences and Ab databases, above.
- mice A total of 30 mice (5 BALB/c and 10 Swiss-Webster) were immunized by 4 25 ⁇ g injections every 21 days with either ToxA: 1800-2710 or ToxB:1807-2366 at Strategic BioSolutions (Newark, DE). Removal of the hexhistidine tags was performed by thrombin cleavage and dialysis overnight in MWCO 10000 dialysis tubing prior to injection. After 12 weeks, all mice had developed anti-toxin A and/or anti-toxin B antibody titers. Sera of the third bleed were tested in toxin neutralization cell assays and by surface plasmon resonance to rank the mice.
- Fusions were initiated with spleen cells of mice demonstrating high anti-toxin titer, toxin neutralization in cell assays and cross-reactivity for toxin A and toxin B in toxin neutralization assays and in ELISA.
- the heavy and light chain plasmids of both rPBA-3 and rPCG-4 were transformed into XLl -blue bacteria and stocked in glycerol.
- Large scale plasmid DNA was prepared as described by the manufacturer (Qiagen, endotoxin-firee MAXIPREPTM kit Cat#12362). Plasmids were transfected into the adenovirus-transformed human embryonic kidney cell line 293F using 293fectin and using 293F-FreeStyle Media for culture.
- Light and heavy chain plasmids were both transfected at 0.5 ⁇ g/mL. Generally, the cells were spun down after 3 days and 1/2 the media was replenished with fresh media.
- Transfections were performed at a cell density of 10 6 cells/mL. Supernatants were collected by centrifugation at 1200 rpm for 8 minutes at 25° C 7 days after transfection. Expression levels varied from ⁇ 0.25-1.5 ⁇ g/mL. Supernatants were stored as described above for hybridoma cultures.
- Native toxin domains were purified on an AKTA FPLC (Amersham Biosciences) using a two-step procedure. The supernatants from sonicated cell pellets were first applied at 3 mL/min onto a Ni 2+ -bound HITRAPTM chelating column (Amersham, Cat#l 7-0409-01). Toxin domains were eluted by applying a 50-300 mM imidazole gradient. HisTagged toxins were eluted between 200 and 250 mM imidazole and collected in a 96-well plate. Toxins A and B have very different pis, therefore the CWB-constructs of each toxin were applied to different ion exchange resins.
- Ni 2+ -purified toxin A domains were dialyzed overnight against a 50 mM MES, 100 mM NaCl, pH 7.0 buffer and applied a HITRAP-CMTM prepacked ion exchange resin (Amersham, Cat#l 7-5056-01).
- the toxin A constructs were eluted with a gradient of 0.1 - 1.0 M NaCl.
- Ni 2+ - purified toxin B domains were dialyzed overnight against a 50 mM Tris, 100 mM NaCl, pH 7.9 buffer and applied to a HITRAP-SPTM prepacked ion exchange resin (Amersham, Cat#l 7-1151- 01).
- the toxin B constructs were eluted with a gradient of 0.1-1.0 M NaCl. All purified toxin domains were dialyzed extensively against a 5 mM phosphate buffer, pH 7.4 for storage at 4 °C and future analysis (Pierce SLIDE-A-LYZERTM Cassette, 3500 MWCO, cat# 66110). Stock concentrations were determined by UV absorbance using the method of Pace and coworkers (2). ToxinA:l 800-1945, ToxinA:2078-2234 and ToxinB:2207-2366 were found primarily
- insoluble ToxA 1800-1945 and ToxA:2078-2234 were solubilized with urea at pH 7.5 and captured with a Ni 2+ -NTA resin (Qiagen).
- the protein material was eluted from the resin with EDTA and dialyzed overnight against PBS. Alternately, the EDTA extractions were injected directly onto a reverse phase C5 JUPITERTM HPLC column (Phenomenex Inc.). H 2 O/acetonitrile gradients with 0.1% trifluoroacetic acid were used to elute the toxin domains from the hydrophobic matrix.
- Denatured toxins eluted between 80% and 90% acetonitrile in a broad peak typical of aggregated/unfolded protein material. No purified toxin domains from the insoluble fractions of the cell pellets ever refolded.
- PBA-3, 3358 (or 543), 227 and 251 mouse monoclonal antibodies and rPBA-3 and rPCG-4 chimeric antibodies were purified by passing culture supernatants over protein G columns (Amersham, cat#17-0405-01) at 4 mL/min. Multiple passage of supernatants over the columns was unnecessary as >95% of all IgG material from each supernatant bound to the column on the first pass.
- Mobile phases consisted of IX PBS-Tween (Sigma Aldrich, Running Buffer, cat# P-3563) and 0.1 M glycine pH 2.7 (Fisher Chemicals, Elution Buffer, cat# G48- 500).
- Antibody collections in 0.1 M glycine were diluted 20% (v/v) with 1 M TrisHCl, pH 8.0, to neutralization the pH.
- IgGl collections were pooled and dialyzed exhaustively against IX PBS (Pierce SLIDE-A-LYZERTM Cassette, 3500 MWCO, cat# 66110). The concentration of each IgGl stock solution was determined by Bradford analysis (Bio-Rad Protein Assay, Hercules, CA cat# 500-0006) using a commercial myeloma IgGl stock solution as a standard and by UV absorbance.
- ToxA 1800-2710 and ToxB : 1808-2366 were biotinylated using the EZ-LINK-Biotin- LC-ASATM kit (PIERCE catalog # 29982). Briefly, EZ-LINK-BIOTIN-LC-ASATM was dissolved in DMSO and added to toxin A or toxin B at a 4:1 molar ratio. Protein/biotin conjugation was induced for 20 minutes under a UV lamp in a PBS buffer. Conjugated toxins were removed from unreacted biotin by application of the reaction mixture to a desalting column (PIERCE D-SaIt Dextran Plastic Desalting Columns, catalog # 43230). 500 ⁇ L fractions from the desalting procedure were tested for protein absorption at 280 nm to detect the presence of biotinylated toxins.
- Microtiter Streptavidin plates (Sigma Chemical, St. Louis, MO, catalog #M5432) were coated with 200 ng per well of biotinylated ToxA: 1800-2710 or ToxB: 1808-2366 diluted into PBS buffer and incubated at 4 0 C overnight. The plates were then washed 3 times with TBST buffer. All samples were diluted in Tris buffer, pH 8.0 TBST buffer (Sigma, cat#T9039). Aliquots of 100 ⁇ L of each diluted serum sample or fusion supernatant were transferred to the toxin-coated plates and incubated for 1-2 hours at room temperature.
- alkaline phosphatase-conjugated rabbit anti-mouse IgG(HH-L) (Zymed, cat#61-6522) was added to each well at a 1 : 1000 dilution.
- the reaction was carried out for 1 hr at room temperature, plates were washed 3 times with TBST and 100 ⁇ L of ⁇ -nitrophenylphosphate substrate was added (Sigma, Catalog # A3469). The absorption was determined at 405 nm using a Molecular Devices v max kinetic microplate reader.
- CD spectra were taken on an Aviv model 215TM spectrophotometer equipped with a thermoelectric cuvette holder. All final spectra were the average of at least three scans utilizing a signal averaging time of 3s/ ⁇ and a 1 nm bandwidth. Temperatures were held constant using a Peltier heating/cooling device coupled to a circulating water bath maintained at 20°C. All scans were performed in a 1 mm cuvette at 100 ⁇ g/mL toxin concentrations and using a 5 mM phosphate, 10 mM NaCl buffer, pH 7.4. Near-UV spectra were performed in a 1 cm cuvette and were the average of 5 scans with a 3s/ ⁇ signal averaging time and a 2 nm bandwidth.
- Temperature dependent far-UV CD spectra were collected at 5 C° intervals from 25 to 75°C on all three samples using a 1 mm cuvette.
- Near-UV CD scans of ToxA:2459-2710 were performed as described above using the same temperature range.
- Precise thermal denaturations of ToxA:2459-2710 were performed by monitoring the far-UV CD signal at 230 nm between 10 and 90 °C using a 1 cm cuvette with constant stirring and 1°C temperature intervals. The temperature equilibration period was 3 minutes/deg and the UV-averaging time was set to 30s/°C.
- Far-UV CD curves of ToxA:2459-2710 were fit to a two-state unfolding model
- Typical antibody concentration series were 0.5, 2, 6, 20 and 100 nM injections. Chip surfaces regenerated reliably with a 10 ⁇ L injection of 0.1 M glycine, pH 1.5 followed by a 10 ⁇ L injection of 50 mM NaOH. The flow rate was 30 ⁇ L/min. For all runs, there was a flow cell dependent baseline drift between 0.0002 and 0.001 RU/sec, which could be accounted for in the 1:1 fitting model used to analyze the kinetics.
- Monoclonal antibodies 3358 (or 543), 3350 (or 227) and rPCG4 were immobilized to 3 separate surfaces on a research grade CM5 Chip.
- 30 nM ToxinA:1800-1945 and 15 nM ToxinA: 1800-2710 were separately injected over the flow cells at 10 ⁇ L/min.
- the toxins Prior to injection, the toxins were incubated with 0, 1, 3, 5, 8, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 105 and 120 nM concentrations of 3358 (or 543), 227, rPCG4 (full-length antibody) and rPCG4 (Fab format).
- the flow rate was increased to 30 mL/min and the antibody surfaces were regenerated with 10 ⁇ L of 0.1 M glycine, pH 2.0. Regeneration did not affect the 3358 (or 543) and 227 surfaces, but resulted in approximately 0.5% signal loss per injection for the rPCG-4 antibody. Every sixth injection was performed with 100% free toxin, to continually monitor the free toxin signal.
- CHO-Kl cells (ATCC CCL-61) were maintained in Dulbecco's modified Eagle's medium (DMEM - Gibco, cat# 12430-054) supplemented with 10% fetal bovine serum at 37 °C in a CO 2 incubator. Prior to the experiment, cells were split into T75 flasks at 1.5 x 10 6 cells/flask. Cells reached confluency within 2 days. CHO cells were seeded from these stocks into 96-well cell culture plates (Costar, cat#3598) and incubated for 4-6 hours. Toxin A and toxin B were purchased from List Biological Laboratories.
- Toxin and toxin/antibody mixtures were incubated at 37 0 C in DMEM with 10% FBS (Gibco, cat#l 0082-147) for 1 hour and added to the 96-well plates. Cell rounding was monitored over a 48 hour period. At 48 hours, 10 ⁇ L WST-I (Roche, cat#1644807) was added to each well and the plates were incubated for 1 hour at 37 °C. Absorbance at 450 nm was determined using a Molecular Devices SPECTRAMAX PLUS 384TM 96-well plate reader.
- Kinetic measurement of toxin A mediated cell rounding at various calcium concentrations was performed by culturing the CHO cells in calcium depleted DMEM (Sigma, cat#M8167) doped with L-glutamine and 5% FBS. 96-well plates were seeded with CHO cells as described above. CaCl 2 was doped into the solution to create a calcium gradient consisting of 100 ⁇ M, 300 ⁇ M, 700 ⁇ M, 1 niM, 2 roM, 5 mM and 10 mM Ca 2+ . Data from the 10 mM cell killing assay was discarded due to significant levels of precipitated CaCl 2 .
- Time points were taken every hour after the addition of toxin A (up to 6 hrs) and after overnight incubation.
- the percentage of cell killing was determined by counting the number of flat versus round cells. At least 150 cells were counted and at least three separate spots on each well were used to account for any variability in killing throughout each well.
- CHO cells were cultivated at a ratio of 1 : 10 and grown in 10 cm dishes, washed two times with PBS, scraped, and pelleted at 1100 rpm at 4°C for 5 minutes. Cell staining was performed with 5 x 10 5 cells/tube in wash buffer (PBS supplemented with 2.5 mM Hepes, 0.1% sodium azide, and 2% FBS). ToxA:1800-1945, ToxA:2078-2234, ToxA:2459-2710 and ToxA: 1800-2710 were all tested for CHO-cell binding at various protein and Ca 2+ concentrations.
- ToxA:2459-2170 (50 ⁇ g/mL) was chosen for the flow cytometry assay based on its native folding properties and cell binding characteristics.
- 250 or 500 ⁇ g/mL antibody was combined with ToxA:2459-2710 to a total volume of 100 ⁇ L and incubated with the cells for 20 minutes. Cells were washed two times with 2 mL wash buffer. Adhered toxins were detected via their histidine tags by incubation with the Alexafluor 488 conjugated PENTA-HIS monoclonal antibody (Qiagen, cat#35310) for 20 minutes. Cells were washed twice with 2 mL wash buffer and resuspended in 500 ⁇ L wash buffer.
- the CaCl 2 concentration was held strictly to 1 mM where the addition of ToxA:2459- 2710 consistently led to a 35-50% population of fluorescently labeled cells.
- Ca 2+ contributed by the 2% FBS was less than 100 ⁇ M, Invitrogen/Gibco.
- StreptmutansGtfC GTVTFNGQRIJYFKPNGVQAK (SEQ ID NO:15)
- StreptmutansGtfB GARTINGQLLYFRANGVQVK (SEQ ID NO:16)
- PhageCP-1 GWVKIGDGWYYFDNSGAMAT (SEQ ID NO:18)
- CDiffToxA GWQTINGKKYYFNTNTAAAA SEQ ID NO:20
- CDiffToxB GLVXIDDKKYYFDDDGIMQX SEQ ID NO:21
- FIG. 8B illustrates Far-UV CD spectra of Toxin Domains.
- a positive peak at 230 run is only present for the full-length CWB domains of toxins A and B and the truncated domain, ToxA:2459-2710.
- the spectra of ToxA: 1800-1945 and ToxA:2078-2234 does not demonstrate a thermal unfolding transition as is observed for ToxA:2459-2710.
- the denatured spectrum of ToxA:2459-2710 at 75°C is similar to the spectra of ToxA:1800-1945 and ToxA:2078-2234 at both 25 and 75°C.
- LytA forms a unique ⁇ -solenoid structure using a minimum of six repeats (5).
- the CWB-domains of toxin A and B had the added complexity of a second-type of repeat sequence occurring after approximately every six classical repeats.
- Three constructs of Toxin A and B (ToxA: 1800-1945, ToxA:2078-2234 and ToxB:2208-
- ToxA:1800-1935 and ToxA:2078-2234 isolated from inclusion bodies using Ni 2+ capture in urea and/or reverse phase HPLC did not refold to the native secondary structure of the limited soluble protein fraction and had far-UV CD spectra indicative of random coil.
- ToxA:2459-2710 unfolded in a two-state fashion.
- the spectrum of thermally unfolded ToxA:2459-2710 resembled the spectra of the smaller, 6-7 repeat indicating the presence of ⁇ -solenoid-like secondary structure even in the denatured state of ToxA:2459-2710, see Figure 8C.
- This unfolding temperature agreed with early literature demonstrating that toxins A and B are no longer active at 56° C (7).
- Figure 9 illustrates photographs of adherent CHO cells cultured in the absence (media only) and presence of 20 ng (100 ⁇ L total volume) toxin A with and without anti-toxin A antibodies present. Antibody concentrations are provided on the images. Antibodies provided significant protection from toxin A at concentrations of 2 ⁇ g. Interestingly, antibody combinations, i.e., 543 and 227, 227 and rPCG-4 at l ⁇ g are each more neutralizing that the single antibodies alone.
- Figures 1 OA to E illustrate the antibody competition for toxin binding sites using static concentrations of toxin and titrating the amount of antibody in solution.
- the amount of toxin with available binding sites was determined by capture of toxin with available binding sites to antibodies immobilized to CM5 chip surfaces. Titration of ToxA: 1800-1945 with rPCG-4 Fab (A). Titration of ToxA: 1800-2710 with rPCG-4 Fab (B), full-length rPCG-4 antibody (C), antibody 227 (D) and antibody 543 (F).
- Figures 1 IA to F illustrate: Fig. 11 (A) the thermal denaturation of ToxA:2459-2710 in the absence and presence of CWB-binding ligands monitored by the CD signal of the protein at 230 nm.
- Fig. 1 IB-E Calcium dependent binding of ToxA:2459-2710 to CHO cell surfaces determined by flow cytometry. 100 ⁇ L of 50 ⁇ g/mL ToxA:2459-2710 was incubated with 0.5 x 10 6 CHO cells for 20 minutes on ice. The cells were washed twice with 2 mL buffer and added to 100 mL of an anti-His tag ALEXAFLUORTM-conjugated antibody (1:500 dilution) for 20 minutes.
- Fig. H(F) The ratio of rounded CHO cells after incubation for 5 hours with 80 ng toxin A was determined in the presence of 100 ⁇ M, 300 ⁇ M, 700 ⁇ M, 1 mM, 2 mM and 5 mM CaCl 2 concentrations.
- ToxA:2459-2710 bound choline (similar to pneumococcal LytA), but did not bind
- Gal ⁇ l-3Gal ⁇ l-4GlcNac as has been reported for full-length toxin A. Binding was assessed by addition of various concentrations of choline or Galocl-3Gal ⁇ l-4GlcNac and testing for an increase in the T M of the protein upon binding (Fig. 1 IA).
- the CWB-domain bound choline with an apparent dissociation constant of 13 ⁇ 5 mM assuming a 1:1 interaction.
- the domains almost certainly bound numerous choline moieties considering what was known for LytA (5).
- Binding of Gal ⁇ l-3Gal ⁇ l-4GlcNac was very weak with an estimated K d > 100 mM in the absence or presence of additional Ca 2+ .
- Recombinant forms are produced from the publicly available antibodies PCG-4 (see, e.g., Lyerly (1986) Infect Immun. 54:70-76; Frey (1992) Infect. Immun. 60: 2488-2492) and PBA-3, were produced.
- Antibodies were raised against ToxA: 1800-2710 and ToxB:1807-2366 in Swiss-Webster mice.
- One anti-toxin B monoclonal was found to effectively neutralize toxin B.
- All anti-toxin A antibodies recognized multiple CWB-mini-domains as determined by surface plasmon resonance (Table 6). Each monoclonal antibody (and rPCG-4 Fab) was tested for binding to all four recombinant toxin A domains. Although the monoclonal antibodies were functionally bivalent and ToxA: 1800-2710 certainly had multiple antibody binding sites, the data sets fit well to a 1 :1 model. The apparent kinetic/equilibrium constants were used to evaluate relative strength of antibody binding.
- Table 6 shows the apparent antibody affinities for toxin A CWB as determined by surface plasmon resonance. The potential number of toxin A-binding sites was estimated based on concentration dependent antibody competition studies. Toxin neutralization was determined using CHO cells with a concentration of toxin A killing 100% of the cells. The anti-toxin polyclonal antibody was used as a positive control with approximately 100% staying alive in presence of the mixture toxin A and antibody.
- the two neutralizing monoclonal antibodies 3359 (or 227) and 3358 (or 543) performed differently than rPCG-4 in the competition assay.
- the 3359 (or 227) antibody did not saturate the toxin even at an 8 molar excess of antibody (Fig.lOD).
- the 3359 (or 227) antibody also did not compete with 3358 (or 543), but instead synergistically enhanced 3358 (or 543)'s binding to ToxA: 1800-2710.
- the 3359 (or 227) antibody only weakly competed with rPCG-4.
- the 3358 (or 543) antibody saturated its own binding sites at a 1:7 toxin: antibody ratio suggesting that 3358 (or 543) had a maximum of approximately fourteen high affinity binding sites (Fig.lOE).
- 3358 (or 543) competed for rPCG-4 binding sites exactly as it competed against itself; an indication that the two antibodies had strongly overlapping binding sites. 3358 (or 543) did not displace 3359 (or 227) at concentrations below 40 nM and only partially displaced 3359 (or 227) at higher concentrations suggesting the two antibodies had at least one or two fully independent binding sites.
- the anti-toxin A and anti-toxin B antibodies were tested for their ability to neutralize full-length, active toxin A and B in a cell-killing assay.
- 4 0.8 and 0.2 ⁇ g/mL toxin A and 60 and 20 ng/mL toxin B were used in the assays. All three toxin A concentrations and 60 ng/mL toxin B were 100% killing in the cell assay while 20 ng/mL toxin B was generally partially killing when incubated with CHO cells.
- the antibodies were introduced with toxin at 20, 10 and 5 ⁇ g/mL concentrations.
- the rPCG-4, 3358 (also designated 543) and 3359 (also designated 227) antibodies are all capable of partially neutralizing 0.2 ⁇ g/mL concentrations of toxin A (Table 6).
- rPBA-3 only weakly neutralized at a concentration of 8 ⁇ g/mL. While some variability was observed in the cell neutralization assay depending upon the specific batch of toxin A and the age and/or healthiness of the CHO cells used in the assay, the indicated trends were observed over multiple (3 or more) separate cell assays.
- the 3358 (or 543) and 3359 (or 227) B-cell lines were selected originally from murine B-cell supernatants based on a synergistic neutralizing affect discovered when testing the two supernatants in combination.
- the clonally selected, purified antibodies demonstrated a similar synergistic ability to neutralize toxin at antibody concentrations lower than the concentrations necessary to observe partial neutralization by each antibody alone (Table 6, Fig. 9).
- the 3359 (or 227) antibody also combined favorably with rPCG-4 towards toxin neutralization.
- rPCG-4 and 3358 were weakly synergistic in their ability to neutralize toxin A, however the neutralization was generally much weaker than what is observed for the 3359 (or 227)/3358 (or 543) and 3359 (or 227)/rPCG-4 pairs. Photographs of cell cultures grown in the presence and absence of toxin and antibodies are shown in Fig.9. The presence of unique epitopes recognized by the 3359 (or 227) antibody can explain how it preferentially coupled with 3358 (or 543) and rPCG-4 for enhanced toxin neutralization. The fact that 3358 (or 543) and rPCG-4 competed for overlapping binding sites also provides an explanation as to why these antibodies did not display as strong a synergistic effect.
- Table 7 shows the apparent affinities and toxin neutralization ability of anti-toxin B antibodies. Toxin neutralization was determined using CHO cells with a concentration of toxin B killing 100% of the cells. The Techlab polyclonal antibody was used as a positive control with approximately 100% staying alive in presence of the mixture toxin A and antibody. Table 7
- Ca 2+ Regulates the ability of the CWB-Domains to Bind Cell Surfaces and Accelerates Toxin Mediated Cell Rounding.
- the CWB-domains of toxin A bound CHO cell surfaces in a calcium-dependent fashion as determined using flow cytometry.
- the full length CWB-domain of toxin A, ToxA: 1800-2710, and the folded 11 repeat domain of toxin A, ToxA:2459-2710 did not bind CHO cells (Fig. 11 B,C).
- Addition of up to 10 mM Ca 2+ induced a strong interaction between ToxA:2459-2710 and CHO-cell surfaces (Fig.l IB-E). Calcium-dependent binding of ToxA: 1800-2710 to cell surfaces was also detectable, but the binding was limited by the comparatively low molar amount of protein in the assay.
- ToxA: 1800-1945 and ToxA:2078- 2234 bind weakly to cell surfaces in the absence of Ca 2+ . This may be a result of their partially folded nature, however, and not necessary an intrinsic function of these domains (12,13).
- the addition of calcium moderately increased the binding of ToxA:1800-1945 and ToxA:2078-2234 to cell surfaces, but not to the same extent observed for the folded ToxA:2459-2710 domain.
- the control His-tagged protein, bovine IgGl C H 3 (10) was not found associated with CHO cell surfaces at protein concentrations as high as 200 ⁇ g/mL and calcium concentrations as high as 1O mM.
- toxin A mediated CHO cell killing assays.
- CHO cells were treated with 0.4 and 0.08 ⁇ g of toxin A and incubated anaerobically at 37°C. Both toxin concentrations were 100% lethal after overnight incubation.
- Ca + had a repeatable and definitive affect on the kinetics of cell rounding. The largest disparity in cell rounding between cells incubated in Ca 2+ -depleted versus calcium rich media appeared at the 5 hour time point (Fig.1 IF).
- Toxin A mediated cell rounding was more rapid at calcium concentrations above 1 mM.
- the level of calcium necessary for accelerating cytotoxicity correlates well with the apparent affinity of calcium for the CWB-domain of toxin A.
- Low levels of calcium (100 ⁇ M and below) also led to toxin susceptibility, potentially due to cellular responses to low calcium rather than direct calcium induced structural or physical changes within the CWB-domain itself.
- At 0.4 ⁇ g toxin A cells rounded rapidly independent of the calcium concentration probably due to the saturating level of toxin.
- the neutralizing anti-toxin A antibodies demonstrated lead to various changes in the ability of ToxA:2459-2710 to bind CHO cell surfaces (Fig.12). Both the 3358 (or 543) and rPCG-4 antibodies significantly increased the amount of CWB-domain detected at the cell surface. These two antibodies also demonstrated overlapping binding epitopes, suggesting a similar mechanism for neutralization. Alternatively, the 3359 (or 227) antibody inhibited cell surface association of ToxA:2459-2710. Its epitope was different according to the competition and mini-domain binding studies.
- toxins A and B are unknown at this time. Contrary to what is observed for LytA (5), 6-7 contiguous toxin A or B repeats did not contain all the necessary elements for forming the native tertiary structure of the full-length toxin CWB-domains, even though they form non-random, probably ⁇ -solenoid-like secondary structure. Toxins A and B had a unique 30 residue peptide stretch after approximately every sixth 20 residue P-motif. This unique motif can add complexity to the tertiary fold of the CWB-domains of toxins A and B differentiating these domains from LytA which is capable of forming a tertiary fold using only 6 repeats.
- the toxin A CWB-domains alone were capable of binding CHO cell surfaces. While cell surface association of toxin A to the CWB-domains has not been definitively linked, a recent study by Pfeifer et al. (32) demonstrated the localization of residues 547-2366 of toxin B in membrane fractions of Vero cells using radioactively labeled-protein. A recent study by Pfeifer et al. (32) demonstrated the localization of residues 547-2366 of toxin B in membrane fractions of Vero cells using radioactively labeled-protein.
- toxin B lacks the cytotoxic domain of the molecule but includes the putative transmembrane region, the 700 residues domain of unknown function as well as the CWB-domain. This large fragment of toxin B has been reported to form pores within membranes by a pH inducible mechanism (55).
- Aktories and coworkers demonstrated that a construct very similar to ToxA:2459-2710, designated REP231, could bind F9-cell surfaces, but only at relatively high concentrations, 200 ⁇ g/mL, and with pretreatment with 4% paraformaldehyde (34). No binding was ever detected in their assay with CHO cells.
- REP231 weakly inhibited the association of toxin A with F9-cells potentially demonstrating CWB-domain importance for cell surface binding.
- Other studies report the effect of deleting the CWB-domains from the toxins in an attempt to more fully characterize their function. Deletion of the entire CWB-domain of toxin B only attenuates its toxicity 10- fold (45), while removal of even half the CWB-domain of toxin A appears to completely neutralize the enterotoxin (25). Similar to what has been observed for toxin B, removal of one or two 20-residue repeats from the LytC choline binding domain only attenuates its function, but does not delete it.
- the CWB-domain of toxin A does bind choline as has been described for LytA (51).
- the fact that 2% choline ( ⁇ 300 mM) is necessary to inhibit the cell wall binding of LytA to pneumococci (52) indicates that its relative affinity for choline may be low, similar to the millimolar affinity determined for the CWB-domain of C. difficile toxin A.
- Dimerization/oligomerization of the choline binding domain of LytA is functionally important for positioning its amidase domain into the peptidoglycan layer (52, 54).
- ToxA 1800-2710
- ToxB 1807-2366
- ToxA :2459-2710 are all monomelic both in the presence of 10 mM Ca 2+ and in the presence of 10 mM choline suggesting that calcium or choline induced oligomerization may not be a function of the CWB-domains.
- Choline binding does suggest that toxin A (and potentially toxin B) is linked to the lipoteichoic acid layer on the surface of C. difficile before secretion or at an early stage of delivery to targeted mammalian cells.
- Monoclonal antibodies directed at the CWB-domains of toxin A and B do not appear to be as neutralizing as polyclonal antibody mixtures in toxin neutralization cell assays. Poor kinetic association/dissociation profiles, as observed for rPBA-3, were not necessarily predictive of an antibody's ability to neutralize. Instead, neutralization appeared to depend upon the number and exact location of individual epitopes recognized by an antibody.
- One toxin A CWB-binding antibody, 251 demonstrated promising kinetic properties ⁇ i.e., rapid association and very slow dissociation); however, this antibody behaved similarly to rPBA-3 in the cell assays. Binding of 251 was limited to only one of the three purified mini-domains.
- rPCG-4 The two most effective antibodies, 3358 (or 543) and rPCG-4, recognized numerous epitopes of the toxin A CWB-domain with high affinity. The exact surface recognized by these two antibodies was shown to be overlapping. rPCG-4 is known to bind two epitopes in particular (6). These experiments demonstrate that rPCG-4 binds between 2 and 4 additional epitopes with high affinity, one of which is located within residues 1800-1945. rPCG-4 also has a relatively weak (-30 nM) affinity for ToxA:2459-2710.
- the 3358 (or 543) and 3359 (or 227) antibodies were shown to bind separate epitopes in competition experiments. Interestingly, combination of these two antibodies results in neutralization similar to what can be achieved with the standard TechLab polyclonal antibody.
- Combination of 3359 (or 227) with rPCG-4 was also highly effective.
- Combination of 3358 (or 543) with rPCG-4 was effective as well, but to a lesser extent than the 3358 (or 543)/3359 (or 227) or rPCG-4/3359 (or 227) mixtures, perhaps because they compete with one another for binding to toxin A. Similar synergies were uncovered for toxin B binding antibodies (Table 7).
- 3358 (or 543)/3359 (or 227) combinations and rPCG-4/3359 (or 227) combinations can be the most effective combinations because they incorporate both neutralization mechanisms and recognize non-overlapping toxin epitopes. With multiple antibodies, these studies demonstrate the ability to provide more superior toxin/epitope coverage and incorporate multiple mechanisms of toxin neutralization for additional synergy.
- Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities. J. Biol. Chem. 273, 16021-16026.
- Example 3 Engineering antibodies for resistance in simulated intestinal fluid
- Simulated intestinal fluid was prepared fresh daily as described in the United States Pharmacopoeia.
- IX SIF buffer consisted of 10 mg/mL pancreatin, (Sigma Chemical Co., St. Louis, MO), and 6.8 mg/ml KH 2 PO 4 .
- a master tube was prepared in a 1.5 mL microcentrifuge tube containing 18.6 uL of sample, 70 uL of 1 OX SIF (1OX SIF was centrifuged before use) in a final volume of 770 uL. The reaction was incubated at 37 0 C.
- the plates were then washed 3 times with Tris buffered saline, pH 8.0 with Tween-20 (TBST - Sigma, cat#T9039). Aliquots of 100 ⁇ L of each antibody sample (diluted into TBST) were transferred to the protein G-coated plates and incubated for 1-2 hours at room temperature. Following 3 washes with TBST, alkaline phosphatase-conjugated goat anti-human Fab (Pierce, 31312) was added to each well at a 1:1000 dilution. The reaction was carried out for 1 hr at room temperature, the plate(s) was washed 3 times with TBST and 100 ⁇ L ofp-nitrophenylphosphate substrate was added (Sigma, Catalog # A3469). The absorption was determined at 405 nm using a Molecular Devices v max kinetic microplate reader.
- pancreatin Various antibody classes were tested in simulated intestinal fluids. All antibody classes, IgGl, IgG2, IgG3 and IgG4 were proteolyzed by pancreatin (see also, Figure 1). Interestingly, the pattern of degradation appeared similar at 0 and 30 min. Determination of pancreatin cleavage sites to mutate: Trypsin and chymotrypsin are the most abundant enzymes present in pancreatin. Potential pancreatin cleavage motifs in the sequence of human IgG were determined based on known trypsin and chymotrypsin cleavage rales (Table 8). Trypsin specifically recognizes Arg and Lys residues at the site where it cleaves peptide bonds.
- Arg and Lys residues with greater than 40% solvent exposure were identified as potential candidates for directed mutagenesis in the heavy chain and light chain constant domains.
- Chymotrypsin specifically recognizes Phe, Tyr or Trp. Therefore, Phe, Tyr and Trp residues with greater than 25% solvent exposure were identified as potentially candidates for directed mutagenesis in the heavy chain and light chain constant domains.
- the selection of residues to replace the potential cleavage sites was based on information from an "unbiased" database of IgG Fc sequences. Mutations were made to the next most frequently observed residue within the dataset of IgG sequences.
- Table 8 shows a list of chymotrypsin and trypsin putative cleavage sites in the constant domain region of the light and heavy chains.
- CT chymotrypsin
- T trypsin
- MFR most frequent residue
- SMFR second most frequent residue
- Antibody region CL: light chain constant domain
- CH heavy chain constant domain
- X ray # residue position based on crystal structure
- Replacement residue selected to replace the cleavage site.
- thermotolerance screening was performed for every member of the library to determine whether mutation at each chymotrypsin and trypsin-labile position was tolerated.
- supernatants with recombinant antibody were heat-challenged for 10 minutes at 70, 75 and 80 0 C.
- Antibodies can denature irreversibly with heat.
- the amount of antibody remaining in the supernatant subsequent to thermal challenge was detected by ELISA and compared to ELISA data obtained with the wildtype protein.
- Most mutants demonstrate comparable thermotolerance and/or expression compared to the wildtype antibody. Interestingly, even single mutations can confer some degree of resistance to pancreatin digestion.
- Table 9 shows ELISA results after pancreatin digestion of the wildtype and the mutated antibody molecules.
- the parent antibody molecule (2934) as well as the mutants were expressed in mammalian cells, purified, and dialyzed. Antibody mutants were digested with pancreatin at 37 0 C for the time indicated. ELISA assays were performed to measure the amount of remaining antibody. Mutations are listed below. A score was given to each variant to describe its expression (Ex): +: Expression was greater than wildtype; : Equivalent expression compared to wildtype; -: Less material was expressed than the wildtype; -: No expression.
- thermotolerance score (T) according to the following criteria: +: A greater percentage of folded protein remaining at 75°C and/or 80°C compared to wildtype; : Equivalent percentage of folded protein remaining at each temperature point compared to wildtype; -: A lesser percentage of folded protein remaining at 75°C than wildtype; -: Thermal unfolding observed at 7O 0 C.
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AU2005321974A AU2005321974B2 (en) | 2004-12-27 | 2005-12-22 | Orally deliverable and anti-toxin antibodies and methods for making and using them |
US11/794,491 US20090087478A1 (en) | 2004-12-27 | 2005-12-22 | Orally Deliverable and Anti-Toxin Antibodies and Methods for Making and Using Them |
CA002592015A CA2592015A1 (en) | 2004-12-27 | 2005-12-22 | Orally deliverable and anti-toxin antibodies and methods for making and using them |
EP05857223A EP1833510A4 (en) | 2004-12-27 | 2005-12-22 | Orally deliverable and anti-toxin antibodies and methods for making and using them |
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- 2005-12-22 AU AU2005321974A patent/AU2005321974B2/en not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
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EP1833510A2 (en) | 2007-09-19 |
US20090087478A1 (en) | 2009-04-02 |
WO2006071877A3 (en) | 2007-04-05 |
AU2005321974B2 (en) | 2011-11-17 |
EP1833510A4 (en) | 2010-02-10 |
CA2592015A1 (en) | 2006-07-06 |
AU2005321974A1 (en) | 2006-07-06 |
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