Nothing Special   »   [go: up one dir, main page]

WO2004022717A2 - Variants tres faiblement immunogenes de l'anticorps humanise col-1 contre l'antigene carcino-embryonnaire - Google Patents

Variants tres faiblement immunogenes de l'anticorps humanise col-1 contre l'antigene carcino-embryonnaire Download PDF

Info

Publication number
WO2004022717A2
WO2004022717A2 PCT/US2003/027976 US0327976W WO2004022717A2 WO 2004022717 A2 WO2004022717 A2 WO 2004022717A2 US 0327976 W US0327976 W US 0327976W WO 2004022717 A2 WO2004022717 A2 WO 2004022717A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
hucol
humanized
col
cea
Prior art date
Application number
PCT/US2003/027976
Other languages
English (en)
Other versions
WO2004022717A3 (fr
Inventor
Syed V.S. Kashmiri
Jeffrey Schlom
Eduardo A. Padlan
Original Assignee
The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services filed Critical The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority to AU2003270369A priority Critical patent/AU2003270369A1/en
Publication of WO2004022717A2 publication Critical patent/WO2004022717A2/fr
Publication of WO2004022717A3 publication Critical patent/WO2004022717A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present disclosure relates to humanized monoclonal antibodies that bind a tumor antigen. More specifically, the present disclosure relates to humanized monoclonal antibodies with amino acid substitutions that have minimal immunogenicity and retain antigen binding affinity for the carcinoembryonic antigen (CEA).
  • CEA carcinoembryonic antigen
  • mAbs Monoclonal antibodies raised against tumor antigens hold promise for diagnosis and therapy of human cancers (reviewed in: Weiner, Semin. Oncol. 26: 43, 1999; Green et al, Cancer Treat. Rev. 26:269, 2000; Carter, Nature Rev. 1:118, 2001).
  • a major impediment to the clinical use of murine mAbs is the human anti-murine antibody (HAMA) response these mAbs elicit in patients (Seccamani et al, Int. J. Rad. Appl. Instrum. B. 16:167, 1989; Reynolds et al, Int. J. Rad. Appl. Instrum. B. 16:121, 1989; Colcher et al, J.
  • humanized Abs have been developed by grafting the complementarity determining regions (CDRs) of the murine Abs onto the frameworks of the variable light (V L ) and variable heavy (V H ) regions of human mAbs (reviewed in Winter and Harris. Immunol. Today 14:243, 1993).
  • CDRs complementarity determining regions
  • Humanization of a xenogenic antibody does not necessarily eliminate the immunogenicity of the molecule since the humanized molecule can evoke anti-N region response (Blanco et al, JClin. Immunol. 17:96, 1997; Schneider et al, J. Immunol 150:3086, 1993; Stephens et al, Immunology 85:668, 1995; Sharkey et al. Cancer Res. 55: 5935s, 1995; Iwahashi et al, Mol. Immunol. 36:1079, 1999; Tamura et al, J. Immunol. 164:1432, 2000).
  • CEA is a member of the immunoglobulin superfamily that includes normal fecal antigen, non-specific cross-reacting antigen, meconium antigen, and biliary glycoprotein. It is composed of seven domains linked to the cell membrane through a glycosylphosphatidylinositol anchor and has a molecular weight of 180 kDa.
  • CEA is normally expressed in a variety of glandular epithelial tissues where it appears to be localized to the apical surface of the cells, although it is expressed in numerous carcinomas including gastrointestinal, colorectal, breast, ovarian and lung carcinomas.
  • CEA is an especially well characterized human tumor antigen and is widely used for the diagnosis of human colon cancer.
  • Monoclonal antibodies have been generated to detect various epitopes on CEA, and in using these antibodies the differential expression of CEA has been determined (Muraro et al, Cancer Res., 45:5769, 1985; Ohuchi et al, Cancer Res. 47:3565, 1987; Wilkinson et al, Proc. Natl. Acad. Sci. 98:10256, 2001).
  • COL-1 is of clinical importance because it has a high affinity for CEA.
  • COL-1 reacts specifically with CEA and not with CEA-related antigens such as normal fecal antigen and non-specific cross-reacting antigen (Kuroki et al, Int. J.
  • the present disclosure relates to humanized COL-1 monoclonal antibodies that are minimally immunogenic and that retain CEA binding affinity.
  • a humanized COL-1 monoclonal antibody in one embodiment, includes a light chain Complementarity Determining Region (LCDR)1, a LCDR2, and a LCDR3; and a heavy chain Complementarity Determining Region (HCDR)1, a HCDR2, and a HCDR3 from monoclonal antibody COL-1, wherein the LCDR1, LCDR2, LCDR3 are in a human light chain framework and wherein the LCDR1, LCDR-2 and LCDR3 are in a human heavy chain framework.
  • the humanized COL-1 monoclonal antibody also includes an amino acid substitution of a non-ligand contact residue in HCDR2, for example at position 61. It is shown herein that the humanized COL-1 retains, or has increased, binding affinity for CEA and has reduced immunogenicity, as compared to a parental antibody.
  • a humanized COL-1 antibody is disclosed that is encoded by the nucleic acid sequence deposited as ATCC Accession number PTA- 4642, ATCC Accession number PTA-4643, or ATCC Accession number PTA-4644 (Deposited September 5, 2002).
  • a humanized COL-1 monoclonal antibody that includes an amino acid substitution at position 24, 25, or 27 in LCDR1 of a murine COL-1 antibody and is minimally immunogenic in a subject.
  • FIG. 1A-B are schematic representations of the nucleotide sequence of the genes encoding the V regions of HuCOL-1 and their leader peptides.
  • Nucleotide sequences of the humanized N L (FIG. 1 A; SEQ ID NO: 11) and V H (FIG. IB; SEQ ID NO: 12) genes were generated and amplified by PCR amplification, using four overlapping synthetic oligonucleotides (indicated by arrows) that together encompass, on alternating strands, the entire sequence of each of the genes and its leader. Sequences on the flanks of the genes encoding the variable region domains and their leader peptides are shown by lower case letters.
  • the N region (FIG.
  • N H region (FIG. IB) starts from position 70 and ends at 441.
  • the restriction enzyme sites incorporated in the oligomers to facilitate cloning are shown in italics.
  • FIG. 2A-B are schematic representations of the nucleotide and amino acid sequences of murine monoclonal antibody COL-1 (mCOL-1). Nucleotide sequences of the NL (FIG. 2A; SEQ ID NO: 13) and N H (FIG. 2B; SEQ ID NO: 14) regions of COL- 1 were determined and the amino acid sequences of the NL (SEQ ID NO: 15) and N H (SEQ ID NO: 16) regions were deduced from the nucleotide sequences.
  • FIG. 3A-C are schematic representations of the humanization protocols for mAb COL-1.
  • FIG. 3 A shows the amino acid sequences of the V L regions of murine COL-1 (SEQ ID NO: 15), human Ab VJFCL (SEQ ID NO: 17), HuCOL-1 derived from mCOL-1 and VJFCL (SEQ ID NO: 18), and the HuCOL-1 variant 24>25>27 L (SEQ ID NO: 19).
  • FIG. 3B shows the amino acid sequences of the V H regions of mCOL-1 (SEQ ID NO: 19), human antibody MO30 (SEQ ID NO: 20), HuCOL-1 derived from mCOL-1 and MO30 (SEQ ID NO: 21), and HuCOL-1 variant 61 H (SEQ ID NO: 22).
  • 3C shows the amino acid sequences of CDR1 in the V L regions of murine COL-1 (SEQ ID NO: 23), human Ab VJFCL (SEQ ID NO: 24), HuCOL- 1 derived from mCOL- 1 and VJFCL (SEQ ID NO: 25), and the HuCOL-1 variant 24 ' 25>27 L (SEQ ID NO: 26) and the amino acid sequences of CDR2 in the V H regions of mCOL-1 (SEQ ID NO: 27), human antibody MO30 (SEQ ID NO: 28), HuCOL-1 derived from mCOL-1 and MO30 (SEQ ID NO: 29), and HuCOL-1 variant H (SEQ ID NO: 30).
  • Dashes indicate residues that are identical between the antibodies. Shaded areas show CDR residues that are substituted in HuCOL- 1 24 ' 25,27 L, HuCOL-l 6 1H, and HuCOL-l 24 ' 25,27 L/ 61 H.
  • FIG. 4A-B are schematic representations of the dual expression constructs derived from the baculovirus vectors.
  • Genes encoding the light (L) and heavy (H) chains of cCOL-1 were cloned in pAcUW51 (FIG. 4A) and those of HuCOL- 1 or HuCOL-1 variants were cloned in pBACx-1 (FIG. 4B) vectors, downstream from the appropriate promoters.
  • PIO and polh represent PIO and polyhedrin promoters; ori and fl are SV40 and fl origin of replication; Amp R represents an ampicillin-resistant gene; arrows show the direction of transcription.
  • FIG. 5A-B are a set of digital images demonstrating SDS-PAGE analysis of the purified monoclonal Abs derived from murine COL-1.
  • Samples of the monoclonal Abs were analyzed in non-reducing (FIG. 5 A) and reducing (FIG. 5B) conditions.
  • FIG. 6A-B are a set of graphs demonstrating the reactivity of various murine COL-1 -derived antibodies (identified by their symbols, below) in a competition RIA assay. Increasing concentrations of mAbs mCOL-1 ( ⁇ ), cCOL-1 ( ⁇ ), HuCOL-1 (A),
  • FIG. 7A-H are a series of graphs demonstrating the flow cytometric analysis of the binding of HuCOL- 1 and its variants to cells expressing cell surface CEA.
  • FIG. 8 is a graph representing the binding of pre-adsorbed patients' sera JS, EM, MB, and serum from a normal donor to immobilized HuCOL-1.
  • the sera were diluted (1:5) and applied, using an external pump, to the surfaces of the flow cells 1 and 2. Shown are response differences in the association phase between flow cell 1 (immobilized HuCOL- 1) and flow cell 2 (immobilized control protein).
  • FIG. 9A-C are a series of graphs that represent sera reactivity, by SPR, of HuCOL- 1 and its variants.
  • Increasing concentrations of HuCOL- 1 (A), 24,25J27 L (O), 61 H (D), and 24>25>27 L/ 61 JJ (•) mAbs were used to compete with the anti-V region Abs to COL-1 present in sera from patients EM (FIG. 9A), JS (FIG. 9B) and MB (FIG. 9C) for binding to HuCOL- 1 immobilized on a sensor chip.
  • Percent binding of the sera to HuCOL- 1 was calculated from the sensograms and plotted as a function of the concentration of the competitor.
  • FIG. 10 is a graph that represents sera reactivity, by SPR, of mCOL-1 and the engineered Abs derived from it.
  • Increasing concentrations of mCOL-1 ( ⁇ ), HuCOL- 1 (A) and 24>25>27 / 61 H (•) were used to compete with the anti-V region Abs to COL-1 present in serum from patient MB for binding to mCOL-1 immobilized on a sensor chip.
  • Percent binding of the sera to mCOL-1 was calculated from the sensograms and plotted as a function of the concentration of the competitor.
  • nucleic and amino acid sequences listed in the sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for a ino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence listing :
  • SEQ ID NO: 1 is the nucleic acid sequence of a murine COL-1 variable heavy chain 5' primer.
  • SEQ ID NO: 2 is the nucleic acid sequence of a murine COL-1 variable heavy chain 3' primer.
  • SEQ ED NO: 3 is the nucleic acid sequence of a murine COL-1 variable light chain 5' primer.
  • SEQ ID NO: 4 is the nucleic acid sequence of a murine COL-1 variable light chain 3' primer.
  • SEQ ID NO: 5 is the nucleic acid sequence of a humanized COL-1 variable heavy chain 5' primer.
  • SEQ ID NO: 6 is the nucleic acid sequence of a humanized COL-1 variable heavy chain 3 ' primer.
  • SEQ ID NO: 7 is the nucleic acid sequence of a humanized COL-1 variable light chain 5' primer.
  • SEQ ID NO: 8 is the nucleic acid sequence of a humanized COL-1 variable light chain 3' primer.
  • SEQ ED NO: 9 is the nucleic acid sequence of a humanized COL-1 variant variable heavy chain 3' primer.
  • SEQ ID NO: 10 is the nucleic acid sequence of a humanized COL-1 variant variable light chain 3' primer.
  • SEQ ED NO: 11 is the nucleotide sequence of the gene encoding the humanized V L region of HuCOL- 1 and its leader peptide.
  • SEQ ID NO: 12 is the nucleotide sequence of the gene encoding the humanized V H region of HuCOL- 1 and its leader peptide.
  • SEQ ID NO: 13 is the nucleotide sequence of the mCOL-1 V L region.
  • SEQ ID NO: 14 is the nucleotide sequence of the mCOL-1 V H region.
  • SEQ ID NO: 15 is amino acid sequence of the mCOL-1 V L region.
  • SEQ ID NO: 16 is amino acid sequence of the mCOL-1 V region.
  • SEQ ID NO: 17 is amino acid sequence of the V L region of the human antibody VJFCL (GenBank Accession Number Z00022).
  • SEQ ED NO: 18 is the amino acid sequence of the V L region of HuCOL- 1 derived from mCOL-1 and VJFCL.
  • SEQ ID NO: 19 is the amino acid sequence of the V L region of HuCOL- 1 variant 24>25 ' 27 L (GenBank Accession Number PTA-4643).
  • SEQ ID NO: 20 is amino acid sequence of the V H region of the human antibody MO30 (GenBank Accession Number A32483).
  • SEQ ID NO: 21 is the amino acid sequence of the V H region of HuCOL- 1 derived from mCOL-1 and MO30 (GenBank Accession Number PTA-4661).
  • SEQ ID NO: 22 is the amino acid sequence of the V H region of HuCOL- 1 variant 61 H (GenBank Accession Number PTA-4642).
  • SEQ ID NO: 23 is the amino acid sequence of CDRl in the V L region of murine COL-1.
  • SEQ ID NO: 24 is the amino acid sequence of CDRl in the V L region of human Ab VJFCL.
  • SEQ ED NO: 25 is the amino acid sequence of CDRl in the V region of HuCOL-1 derived from mCOL-1 and VJFCL.
  • SEQ ED NO: 26 is the amino acid sequence of CDRl in the V L region of the
  • SEQ ED NO: 27 is the amino acid sequence of CDR2 in the V H region of mCOL-1.
  • SEQ ID NO: 28 is the amino acid sequence of CDR2 in the V H region of human antibody MO30.
  • SEQ D NO: 29 is the amino acid sequence of CDR2 in the V H region of HuCOL-1 derived from mCOL-1 and MO30.
  • SEQ ED NO: 30 is the amino acid sequence of CDR2 in the V H region of
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non- human mammals.
  • subject includes both human and veterinary subjects.
  • the antigen is CEA.
  • Monoclonal and humanized immunoglobulins are encompassed by the disclosure.
  • a murine monoclonal antibody that recognizes CEA is COL-1.
  • a humanized COL-1 antibody is HuCOL- 1 (ATCC Accession Number PTA-4661).
  • variant HuCOL-1 antibodies are HuCOL- 1 61 H (ATCC Accession Number PTA-4642), HuCOL- 1 24 ' 25 ' 27 L (ATCC Accession Number PTA-4643), or HuCOL- 1 2 ,25,27 L/ 61 H (ATCC Accession Number PTA-4644).
  • the disclosure also includes synthetic and genetically engineered variants of these immunoglobulins.
  • a naturally occurring antibody e.g., IgG
  • IgG includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody.
  • antigen-binding fragments are also intended to be designated by the term "antibody.”
  • binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the V L , V H , C L and rl domains; (ii) an Fd fragment consisting of the V H and C H I domains; (iii) an Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544, 1989) which consists of a V H domain; and (v) an F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al. Science 242:423, 1988; and Huston et al. Proc. Natl. Acad. Sci. 85:5879, 1988) by recombinant methods.
  • single chain Fv single chain Fv
  • dsFv disulfide stabilized Fv
  • dimeric Fvs diabodies
  • antibody fragments for use in this disclosure are those which are capable of cross-linking their target antigen, e.g., bivalent fragments such as F(ab') 2 fragments.
  • an antibody fragment which does not itself cross-link its target antigen e.g., a Fab fragment
  • a secondary antibody which serves to cross-link the antibody fragment, thereby cross-linking the target antigen.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described for whole antibodies.
  • An antibody is further intended to include humanized monoclonal molecules that specifically bind the target antigen.
  • Specifically binds refers to the ability of individual antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an antibody molecule and the antigen.
  • the antigen is CEA.
  • Binding specificity is typically deterrnined from the reference point of the ability of the antibody to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • An antibody that specifically binds to a particular epitope is referred to as a "specific antibody”.
  • Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as 32 P, fluorophores, chemiluminescent agents, and enzymes.
  • the chemical or biochemical modifications that incorporate toxins in the antibody are also encompassed in the disclosure.
  • the toxin is chemically conjugated to the antibody.
  • a fusion protein is genetically engineered to include the antibody and the toxin.
  • toxins are radioactive isotopes, chemotherapeutic agents, bacterial toxins, viral toxins, or venom proteins.
  • the disclosure also includes chemical or genetically engineered modifications that link a cytokine to an antibody (such as by a covalent linkage).
  • cytokines are interleukin (IL)-2, IL-4, IL-10, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma.
  • Antigen Any molecule that can bind specifically with an antibody.
  • An antigen is also a substance that antagonizes or stimulates the immune system to produce antibodies. Antigens are often foreign substances such as allergens, bacteria or viruses that invade the body.
  • An antigen is CEA.
  • Carcinoembryonic antigen (CEA) A member of the immunoglobulin superfamily that includes normal fecal antigen, non-specific cross-reacting antigen, meconium antigen, and biliary glycoprotein.
  • CEA is composed of seven domains linked to the cell membrane through a glycosylphosphatidylinositol anchor and has a molecular weight of 180 kDa (GenBank Accession Number A36319, herein incorporated by reference).
  • CEA is normally expressed in a variety of glandular epithelial tissues, where it appears to be localized to the apical surface of the cells, although it is also expressed in numerous carcinomas including gastrointestinal, colorectal, breast, ovarian and lung carcinomas (Robbins et al, Int'lJ. Cancer, 53:892- 897, 1993; Greiner et al, J. Clin. Oncol, 10:735-746, 1992; Ohuchi et al, Cancer Res.
  • CEA is an especially well characterized human tumor antigen and is widely used for the diagnosis of human colon cancer.
  • Monoclonal antibodies, designated COL-1 through COL-15, have been generated to detect various epitopes on CEA (Muraro et al. , Cancer Res. , 45:5769-5780, 1985, herein incorporated by reference), and in using these antibodies the differential expression of CEA has been determined (Muraro et al, Cancer Res., 45:5769-5780, 1985; Ohuchi et al, Cancer Res. 47:3565-3571, 1987; Wilkinson et al, Proc. Natl.
  • COL-1 is of clinical importance because it has a high affinity for CEA.
  • cDNA complementary DNA
  • Chimeric antibody An antibody which includes sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies include human and murine antibody domains, generally human constant and murine variable regions.
  • Complementarity Determining Region Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site.
  • the light and heavy chains of an Ig each have three CDRs, designated L- CDR1, L-CDR2, L-CDR3 and H-CDRl, H-CDR2, H-CDR3, respectively.
  • the CDRs of the light chain are bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded by the residues at positions 31 and 35b (H-CDRl ), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineated by Kabat et al, (1991) Sequences of Proteins of Immunological Interest, 5 t Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda (NIH Publication No. 91-3242). Constant Region: The portion of the antibody molecule which confers effector functions.
  • the variant antibodies include constant regions derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of various subclasses (such as the IgG subclass of heavy chains) are responsible for different effector functions. Thus, by choosing the desired heavy chain constant region, humanized antibodies with the desired effector function can be produced.
  • the light chain constant region can be of the kappa or lambda type.
  • Cytotoxin An agent that is toxic for cells.
  • cytotoxins include radioactive isotopes, chemotherapeutic drugs, bacterial toxins, viral toxins, and proteins contained in venom (e.g. insect, spider, reptile, or amphibian venom).
  • a cytokine such as interleukin-2 or interferon, can also be a cytotoxin.
  • DNA Deoxyribonucleic acid.
  • DNA is a long chain polymer which constitutes the genetic material of most living organisms (some viruses have genes composed of ribonucleic acid (RNA)).
  • the repeating units in DNA polymers are four different nucleotides, each of which contains one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide.
  • codon is also used for the corresponding (and complementary) sequence of three nucleotides in the mRNA that is transcribed from the DNA.
  • Encode A polynucleotide is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • Epitope A site on an antigen recognized by an antibody, as determined by the specificity of the antibody amino acid sequence. Epitopes are also called antigenic determinants.
  • Framework Region Amino acid sequences interposed between CDRs include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.
  • HAMA Human anti-murine antibody response: An immune response in a human subject to the variable and constant regions of a murine antibody that has been administered to the patient. Repeated antibody administration may lead to an increased rate of clearance of the antibody from the patient's serum and may also elicit allergic reactions in the patient.
  • Humanized antibody A human antibody genetically engineered to include mouse hypervariable regions.
  • the DNA encoding hypervariable loops of mouse monoclonal antibodies or variable regions selected in phage display libraries is inserted into the framework regions of human Ig genes.
  • Antibodies can be "customized” to have a desired binding affinity or to be minimally immunogenic in the humans treated with them.
  • Humanized COL-1 antibodies COL-1 antibodies humanized by grafting mCOL-1 (murine COL-1) CDRs onto the frameworks of the relevant human antibodies.
  • the murine CDRs in the resultant humanized COL-1 (HuCOL- 1) could evoke an anti- idiotypic response when administered in human subjects.
  • COL-1 can be humanized by grafting only a subset of the COL-1 CDR residues, for example those that are important for antigen binding, onto the variable light and variable heavy framework regions of, for example, VJFCL and MO30 human antibodies.
  • an HuCOL-1 antibody COL-1 CDR residues that are not involved in antigen binding (non-ligand contact residues) are substituted with the corresponding residues of a human antibody such as those from, for example, VJFCL or MO30.
  • a humanized COL-1 monoclonal antibody is HuCOL- 1 (ATCC Accession Number PTA-4661).
  • a humanized COL-1 antibody is HuCOL- 1 24 ' 25 ' 27 L (ATCC Accession Number PTA-4643).
  • a humamzed COL-1 antibody is HuCOL-l 61 H (ATCC Accession Number PTA-4642) and HuCOL- 1 24 ' 25 ' 27 L/ 61 H (ATCC Accession Number PTA-4644).
  • HuCOL- 1 is referred to herein as the parent HuCOL- 1 antibody and both mCOL-1 and HuCOL-1 are referred to herein as parental antibodies.
  • HuCOL-l 6 1H, HuCOL-l 24,25,27 L, and HuCOL-l 24,25 ' 27 L/ 61 H are referred to herein as variant HuCOL- 1 antibodies.
  • IC 5 o value The concentration of a competitor antibody (for example, concentration of a variant HuCOL-1) required for half-maximal (50%) inhibition of binding of sera to another antibody (for example, mCOL-1 or the parent HuCOL- 1).
  • concentration of a competitor antibody for example, concentration of a variant HuCOL-1
  • a higher IC 50 value for a particular antibody indicates a decreased reactivity of that antibody to the serum, suggesting that the antibody with the higher IC 50 value has reduced immunogenicity in a subject.
  • a variant HuCOL-1 antibody has an IC 50 value that is greater than that of the parent HuCOL- 1 antibody, suggesting that the variant HuCOL- 1 antibody has a decreased immunogenicity in a subject compared to the parent HuCOL- 1 antibody.
  • Idiotype the property of a group of antibodies or T cell receptors defined by their sharing a particular idiotope (an antigenic determinant on the variable region); t.e., antibodies that share a particular idiotope belong to the same idiotype. "Idiotype” may be used to describe the collection of idiotopes expressed by an Ig molecule. An "anti- idiotype” antibody may be prepared to a monoclonal antibody by methods known to those of skill in the art and may be used to prepare pharmaceutical compositions.
  • Immune cell Any cell involved in a host defense mechanism.
  • Immune response A response of a cell of the immune system, such as a neutrophil, a B cell, or a T cell, to a stimulus.
  • the response is specific for a particular antigen (an "antigen-specific response").
  • the response is against an antibody, such as HAMA response, including an anti-variable region response.
  • Immunoconjugate A covalent linkage of an effector molecule to an antibody.
  • the effector molecule can be a toxin or a detectable label.
  • toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (such as PE35, PE37, PE38, and PE40), diphtheria toxin, anthrax toxin, botulinum toxin, or modified toxins thereof.
  • Pseudomonas exotoxin and diphtheria toxin are highly toxic compounds that typically bring about death through liver toxicity.
  • Pseudomonas exotoxin and diphtheria toxin can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (e.g., domain la of Pseudomonas exotoxin and the B chain of diphtheria toxin) and replacing it with a different targeting moiety, such as an antibody.
  • native targeting component of the toxin e.g., domain la of Pseudomonas exotoxin and the B chain of diphtheria toxin
  • chemotherapeutic drugs include cytokines, for example interleukin-2 or interferon, radioactive isotopes, viral toxins, or proteins contained within, for example, insect, spider, reptile, or amphibian venom.
  • detectable labels include, but are not limited to, radioactive isotopes, enzyme substrates, co- factors, ligands, chemiluminescent agents, fluorescent agents, haptens, or enzymes.
  • a "chimeric molecule” is a targeting moiety, such as a ligand or an antibody, conjugated (attached or coupled) to an effector molecule.
  • conjugated refers to making two polypeptides into one contiguous polypeptide molecule.
  • an antibody is joined to an effector molecule.
  • an antibody joined to an effector molecule is further joined to a lipid or other molecule to a protein or peptide to increase its half- life in the antibody.
  • Therapeutic, diagnostic or detection moieties can be linked to an antibody using any number of means known to those of skill in the art. Both covalent and noncovalent linkage means may be used.
  • the procedure for linking an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; e.g., carboxyl (COOH), amino (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the linkage of the effector molecule.
  • the antibody is derivatized to expose or link additional reactive functional groups.
  • the derivatization may involve linkage of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the tumor site e.g. when exposed to tumor-associated enzymes or acidic pH
  • Immunogenicity A measure of the ability of a targeting protein or therapeutic moiety to elicit an immune response (humoral or cellular) when administered to a subject.
  • Immunoreactivity A measure of the ability of an Ig to recognize and bind to a specific antigen.
  • Isolated An biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and exfrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been "isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant DNA expression in a host cell as well as chemically synthesized nucleic acids.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, chemiluminescent tags, haptens, enzymatic linkages, and radioactive isotopes.
  • Ligand contact residue A residue within a CDR that is involved in contact with a ligand or antigen.
  • a ligand contact residue is also known as a specificity determining residue (SDR).
  • a non-ligand contact residue is a residue in a CDR that does not contact a ligand.
  • a non-ligand contact residue can also be a framework residue.
  • Lymphocytes A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B-cells and T-cells. Mammal: This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Minimally immunogenic An antibody that generates a reduced, for example low, immune response when administered to a subject, such as a human subject.
  • a minimally immunogenic antibody is an antibody that is administered to a subject without eliciting a HAMA response.
  • immunogenicity is measured in a competitive binding assay. En one specific, non-limiting example, immunogenicity is the ability of a variant HuCOL- 1 antibody to prevent a mCOL-1 or parental HuCOL- 1 antibody from binding to COL-1 anti-idiotypic antibodies in a patient's serum. If a variant HuCOL-1 antibody competes with an equal molar amount of the parental HuCOL-1 antibody (i.e.
  • the variant HuCOL-1 antibody is immunogenic. If a variant HuCOL-1 antibody competes poorly with an equal molar or less amount of the parent HuCOL- 1 antibody (t.e. elicits about 50% or less inhibition of parental HuCOL- 1 binding to anti-idiotypic antibodies in a patient's serum) then the variant HuCOL-1 antibody is minimally immunogenic.
  • IC 50 is the concentration of the competitor antibody (for example, concentration of a variant HuCOL-1) required for half-maximal (50%) inhibition of binding of sera to mCOL- 1 , or the parent HuCOL-1.
  • Monoclonal antibody An antibody produced by a single clone of B- lymphocytes. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Oligonucleotide A linear single-stranded polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polymer of deoxyribonucleotides or ribonucleotides which is at least 6 nucleotides, for example at least 15, 50, 100 or even 200 nucleotides long.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Phage display A technique wherein DNA sequences are amplified and cloned into filamentous phage vector to create a library of fusion phages ("phage library") in which the phages display on their surface the proteins encoded by the foreign DNA.
  • phage library a phage library is produced that expresses HuCOL-1 variant immunoglobulins. From the rescued phages, the individual phage clones are selected through interaction of the displayed protein with a ligand, and the specific phage is amplified by infection of bacteria. Antigen specific immunoglobulins can then be expressed and characterized for their antigen binding and sera reactivity (potential immunogenicity) .
  • Pharmaceutical agent A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.
  • “Incubating” includes a sufficient amount of time for a drug to interact with a cell.
  • “Contacting” includes incubating a drug in solid or in liquid form with a cell.
  • a “therapeutically effective amount” is a quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor or to decrease a sign or symptom of the tumor in the subject. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W.
  • compositions and formulations suitable for pharmaceutical delivery of humanized COL-1 monoclonal antibodies disclosed herein are suitable for pharmaceutical delivery of humanized COL-1 monoclonal antibodies disclosed herein.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide A single-stranded linear nucleotide sequence, including sequences of greater than 100 nucleotide bases in length.
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred in nature.
  • polypeptide or protein as used herein encompasses any amino acid sequence and includes, but may not be limited to, modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantiy or synthetically produced.
  • Substantially purified polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • a non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain.
  • substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl.
  • a hydrophilic residue is substituted for (or by) a hydrophobic residue
  • a proline is substituted for (or by) any other residue
  • a residue having a bulky side chain e.g., phenylalanine
  • an electropositive side chain e
  • Variant amino acid sequences may, for example, be 80, 90 or even 95 or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI web site.
  • Preventing or treating a disease refers to inhibiting completely or in part the development or progression of a disease, for example in a person who is known to have a predisposition to a disease.
  • An example of a person with a known predisposition is someone with a history of cancer in the family, or who has been exposed to factors that predispose the subject to the development of a tumor.
  • Treating a disease refers to a therapeutic intervention that inhibits, or suppressed the growth of a tumor, eliminates a tumor, ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.
  • Protein A biological molecule encoded by a gene and comprised of amino acids.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or was made artificially. Artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Similarly, a recombinant protein is one encoded by a recombinant nucleic acid molecule.
  • Relative binding affinity constant Affinity of an antibody for an antigen can be expressed relative to the binding affinity of another antibody to the same antigen.
  • the relative affinity constant of a variant HuCOL- 1 antibody is less than, similar to, or greater than, that of a murine COL-1 or a parent HuCOL- 1 antibody.
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al. Mol. Immunol. , 16:101, 1979.
  • One of skill in the art can readily identify a statistical test that determines a statistically significant result for example, the Student's t-test, the Wilcoxon two sample test, or the Median test.
  • a variant HuCOL- 1 antibody retains CEA binding affinity if the variant HuCOL- 1 antibody binds CEA and has a relative binding affinity constant at least about 1.0 x 10 "8 M. In other embodiments, a variant HuCOL- 1 antibody retains CEA binding affinity if the relative binding affinity constant is at least about 1.2 x 10 "8 , about 1.5 x 10 "8 , about 2.0 x 10 "8 , about 2.5 x 10 "8 , about 2.8 x 10 "8 , about 3.0 x 10 "8 , about 3.2 x 10 " 8 , about 3.5 x 10 -8 , about 4.0 x 10 "8 , about 4.5 x 10 "8 , or about 5.0 x 10 -8 M.
  • a binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a binding affinity is measured by a competition radioimmunoassay. In a further embodiment, a binding affinity is measured by flow cytometry as the number of gated cells labeled with HuCOL- 1 antibody.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • Therapeutically effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.
  • Treatment Refers to both prophylactic inhibition of initial infection or disease, and therapeutic interventions to alter the natural course of an untreated infection or disease process, such as a tumor growth.
  • Tumor A neoplasm that may be either malignant or non-malignant.
  • Tumors of the same tissue type are primary tumors originating in a particular organ (such as breast, gastrointestinal, prostate, ovarian, bladder or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).
  • Breast cancers can be divided histologically into scirrhous, infiltrative, papillary, ductal, medullary and lobular.
  • cells in a tumor express CEA.
  • Variable region also variable domain or V domain: The regions of both the light-chain and the heavy-chain on an Ig that contain antigen-binding sites.
  • the regions are composed of polypeptide chains containing four relatively invariant "framework regions” (FRs) and three highly variant “hypervariable regions” (HVs). Because the HVs constitute the binding site for antigen(s) and determine specificity by forming a surface complementarity to the antigen, they are more commonly termed the “complementarity-determining regions,” or CDRs, and are denoted CDRl, CDR2, and CDR3. Because both of the CDRs from the heavy- and light-chain domains contribute to the antigen-binding site, it is the three-dimensional combination of the heavy and the light chain that determines the final antigen specificity.
  • the framework regions surround the CDRs. Proceeding from the N-ter inus of a heavy or light chain, the order of regions is: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the term "variable region” is intended to encompass a complete set of four framework regions and three complementarity-determining regions. Thus, a sequence encoding a "variable region” would provide the sequence of a complete set of four framework regions and three complementarity-determining regions.
  • Variant HuCOL-1 A humanized COL-1 antibody that has at least one amino acid substitution in a murine CDR region.
  • HuCOL- 1 monoclonal antibodies that have an amino acid substitution in the heavy chain complementarity determining region (HCDR) 2 of the COL-1 antibody.
  • HuCOL- 1 antibodies with an amino acid substitution are also referred to herein as variant HuCOL-1 antibodies.
  • the variant HuCOL- 1 antibody has an amino acid substitution of a non- ligand contact residue in HCDR2.
  • the variant HuCOL- 1 antibody has an amino acid substitution of a non-ligand contact residue at position 59, 60, 61, 62, 63, 64, or 65 of HCDR2 (FIG. 3).
  • the variant HuCOL-1 antibody has an amino acid substitution at position 61 of HCDR2, such as a proline to glutamine substitution (see HuCOL-l 61 H in Table 1).
  • the variant HuCOL- 1 antibody has no more than one amino acid substitution in HCDR2.
  • the variant HuCOL-1 antibody can include no more than two, no more than three, no more than four, or no more than seven amino acid substitutions in HCDR2.
  • the variant HuCOL- 1 antibody has an amino acid substitution at position 61 of HCDR2 and an additional amino acid substitution at 59, 60, 62, 63, 64, or 65 of HCDR2.
  • the variant HuCOL- 1 antibody has an amino acid substitution at position 61 of HCDR2 and an additional amino acid substitution of a ligand contact residue in HCDR2.
  • the variant HuCOL- 1 antibody can have an amino acid substitution in the light chain complementarity determining region (LCDR) 1 of the COL-1 antibody.
  • the variant HuCOL- 1 antibody has an amino acid substitution of a non- ligand contact residue in LCDRl.
  • the HuCOL- 1 antibody has an amino acid substitution of a non-ligand contact residue at position 24, 25, 26, 27, 27a, 27b, or 27c of LCDRl (FIG. 3).
  • the variant HuCOL-1 antibody has no more than one amino acid substitution in LCDRl .
  • the variant HuCOL- 1 antibody includes no more than two, no more than three, no more than four, or no more than seven amino acid substitutions in LCDRl.
  • the variant HuCOL-1 antibody has an amino acid substitution at position 24, 25 and 27 of LCDRl. In one specific, non-limiting example, the variant HuCOL-1 antibody has an arginine to lysine substitution at position 24 of LCDRl . In another specific, non-limiting example, the variant HuCOL- 1 antibody has an alanine to serine substitution at position 25 of LCDRl . In yet another specific, non-limiting example, the variant HuCOL- 1 antibody has a lysine to glutamine substitution at position 27 of LCDRl .
  • the variant HuCOL- 1 antibody has an arginine to lysine substitution at position 24, an alanine to serine substitution at position 25 of LCDRl, and a lysine to glutamine substitution at position 27 of LCDRl (see HuCOL- 1 4 ' 5 ' 27 L in Table 1).
  • the variant HuCOL-1 antibody has an amino acid substitution at position 24, 25, and 27 of LCDRl and an additional amino acid substitution of a ligand contact residue in LCDRl .
  • a variant HuCOL-1 monoclonal antibody includes an amino acid substitution in HCDR2 and an additional amino acid substitution.
  • an additional substitution is an amino acid substitution in another HCDR or in an LCDR.
  • a variant HuCOL- 1 monoclonal antibody that includes amino acid substitutions in more than one CDR is a HuCOL- 1 monoclonal antibody with an amino acid substitution of a non-ligand contact residue in HCDR2 and an amino acid substitution of a non-ligand contact residue in LCDRl .
  • the HuCOL- 1 antibody has an amino acid substitution at position 61 of HCDR2 and at positions 24, 25, and 27 of LCDRl (see HuCOL-l 24 ' 25 ' 27 L/ 61 H in Tablel).
  • the variant HuCOL- 1 with an amino acid substitution in HCDR2 has an additional amino acid substitution of a ligand contact residue in another HCDR or in an LCDR.
  • the variant HuCOL- 1 antibody has an additional amino acid substitution in a framework residue.
  • Immunogenicity of variant HuCOL- 1 antibodies can be measured in a competitive binding assay as the ability of a variant HuCOL- 1 antibody to prevent a murine COL-1 or the parent HuCOL- 1 antibody from binding to anti-idiotypic antibodies in a human subject's serum.
  • a variant HuCOL-1 antibody with an amino acid substitution in HCDR2 such as HuCOL-l 61 H, HuCOL- 1 24 ' 25 ' 27 L, or HuCOL-l 24,25 ' 27 L/ 61 H, are minimally immunogenic in a subject.
  • At least about a two-fold higher molar concentration of the variant HuCOL- 1 antibody, compared to the molar concentration of mCOL-1 or the parent HuCOL-1 antibody, is required to elicit 50% inhibition of mCOL-1 or parent HuCOL- 1 antibody binding, respectively, to its cognate anti-idiotypic antibodies in a subject's sera.
  • At least about a three-fold, at least about a fivefold, at least about a ten-fold, at least about fifteen-fold, at least about a twenty fivefold, at least about a fifty-fold, at least about a seventy-fold, or at least about a one hundred-fold higher molar concentration of the variant HuCOL- 1 antibody, compared to the molar concentration of the mCOL-1 or the parent HuCOL- 1 antibody, is required to elicit 50% inhibition of mCOL-1 or parent HuCOL- 1 antibody binding, respectively, to its cognate anti-idiotypic antibodies in a subject's serum.
  • HuCOL- 1 has minimal immunogenicity, compared to the mCOL-1 antibody, since at least a 3-fold higher molar concentration of HuCOL- 1 was required to attain 50% inhibition of mCOL-1 binding to patient serum. In another embodiment, HuCOL- 1 suffered a partial loss in antigen-binding affinity (2.82 x 10 "8 M) compared to the mCOL-1 antibody (5.17 x 10 "8 M).
  • HuCOL- 1 ' ' LI H has minimal immunogenicity, compared to the mCOL-1 and HuCOL-1 antibodies, since at least 17-fold and 5.5-fold higher molar concentrations of HuCOL-l 24 ' 25,27 L/ 61 H were required to attain 50% inhibition of mCOL-1 and HuCOL-1 binding, respectively, to patient serum.
  • HuCOL- 1 24 ' 25 ' 27 L/ 61 H suffered a partial loss in antigen binding affinity (1.03 x 10 " M) compared to that of HuCOL-l (2.64 x lO "8 M).
  • the variant HuCOL- 1 antibodies disclosed herein retain CEA relative binding affinity.
  • a variant HuCOL- 1 antibody retains CEA binding affinity if the relative binding affinity constant is at least about 1.0 x 10 "8 M.
  • a variant HuCOL- 1 antibody retains CEA binding affinity if the relative binding affinity constant is at least about 1.2 x 10 "8 , about 1.5 x 10 "8 , about 2.0 x 10 "8 , about 2.5 x 10 "8 , about 2.8 x 10 "8 , about 3.0 x 10 "8 , about 3.2 x 10 “8 , about 3.5 x 10 "8 , about 4.0 x 10 "8 , about 4.5 x 10 "8 , or about 5.0 x 10 "8 M.
  • HuCOL- 1 24 ' 25 ' 27 L, HuCOL-l 61 H, and HuCOL-l 24,25,27 L/ 61 H have relative binding affinity constants for CEA that are about 1.20 x 10 "8 M, about 2.64 x 10 "8 M, and about 1.03 x 10 "8 M, respectively.
  • the variant HuCOL- 1 antibody retains CEA binding affinity if the variant HuCOL- 1 antibody can inhibit the binding of mCOL-1 or HuCOL- 1 to CEA.
  • the variant HuCOL- 1 antibody retains CEA binding affinity, compared to the mCOL-1 or the parent HuCOL- 1 antibody, if the variant has a similar antigen/antibody dissociation rate as that of the mCOL-1 or the parent HuCOL- 1 antibody.
  • the variant HuCOL-1 antibody retains CEA binding affinity if a similar amount of the variant antibody is required for a 50% inhibition of the binding of 125 I-labeled HuCOL-1 to CEA, compared to the murine COL-1 antibody or the parent HuCOL-1 antibody.
  • the variant HuCOL-1 antibody retains CEA binding affinity when cells expressing CEA are labeled with the variant HuCOL- 1 antibody.
  • the number of CEA-expressing cells labeled with the variant HuCOl-1 antibody can be measured by flow cytometry.
  • the variant HuCOL- 1 antibody retains CEA binding affinity when the number of CEA-expressing cells labeled by a variant HuCOL- 1 antibody is similar to the number of CEA-expressing cells labeled by the murine COL-1 antibody or the parent HuCOL- 1 antibody.
  • HuCOL- 1 has a relative binding affinity constant for CEA (2.82 x 10 "8 M) that is 1.8-fold less than that of mCOL-1 (5.17 x 10 "8 M)
  • HuCOL- 1 24,25 ' 27 L, HuCOL-l 61 H, and HuCOL-l 24 ' 25,27 L/ 6 1H have relative binding affinity constants for CEA (1.20 x 10 "8 M, 2.64 x 10 "8 M, and 1.03 x 10 "8 M, respectively) that are 4.3-, 1.9-, and 5.0-fold less than that of mCOL-1.
  • HuCOL- 1 24 ' 25 ' 27 L and HuCOL-l 24,25 ' 27 L/ 61 H have relative binding affinity constants for CEA (1.20 x 10 "8 M and 1.03 x 10 "8 M, respectively) that are 2-fold less than that of HuCOL- 1.
  • HuCOL- l 6l H has a relative binding affinity constant (2.64 x 10 "8 M ) that is similar to that of HuCOL- 1 (2.82 x 10 "8 M).
  • the variant HuCOL- 1 antibody has an increase in CEA binding affinity, compared to the murine COL-1 antibody, if the variant has a lower antigen/antibody dissociation rate compared to that of the murine COL-1 or the parent HuCOL- 1 antibody.
  • the variant HuCOL- 1 antibody has an increase in CEA binding affinity if less of the variant antibody is required for a 50% inhibition of the binding of I-labeled HuCOL- 1 to CEA, compared to the murine COL-1 antibody or the parent HuCOL- 1 antibody.
  • the variant HuCOL- 1 antibody has an increase in CEA binding affinity when the number of CEA- expressing cells labeled with variant HuCOL- 1 antibody is significantly greater than the number of CEA-expressing cells labeled by the murine COL-1 antibody or the parent HuCOL-1 antibody.
  • the variant HuCOL- 1 antibody has a CH2 domain deletion (Slavin-Chiorini et al, Int. J. Cancer, 53:97-103, 1993; Slavm-CMorini et al, Cancer Research, 55:5957s-5967s, 1995; Slavin-Chiorini et al, Cancer Biother. Radiopharm., 12:305-316, 1997, incorporated herein by reference).
  • the generation and characterization of CH2 domain deleted antibodies is described in Mueller et al, Proc. Natl. Acad. Sci. USA., 87:5702-5705, 1990.
  • a variant HuCOL- 1 antibody with a CH2 domain deletion is cleared more quickly from the plasma compared to the murine COL-1 monoclonal antibody or the parent HuCOL- 1 antibody.
  • a variant HuCOL- 1 antibody with a CH2 domain deletion has reduced immunogenicity compared to the murine COL-1 antibody or the parent HuCOL- 1 antibody.
  • a variant HuCOL- 1 antibody with a CH2 domain deletion has reduced immunogenicity compared to the murine COL-1 antibody or the parent HuCOL- 1 antibody, and retains CEA binding affinity.
  • Effector molecules can be linked to a variant HuCOL- 1 antibody that specifically binds CEA, using any number of means known to those of skill in the art.
  • a variant HuCOL- 1 antibody with an amino acid substitution can have any one of a number of different types of effector molecules linked to it.
  • the variant HuCOL- 1 antibody is linked to a detectable label.
  • the variant HuCOL- 1 antibody is linked to a radioactive isotope, an enzyme substrate, a co-factor, a ligand, a chemiluminescent agent, a fluorescent agent, a hapten, or an enzyme.
  • the variant HuCOL- 1 antibody is linked to a cytotoxin.
  • the variant HuCOL- 1 antibody is linked to a chemotherapeutic drug, a radioactive isotope, a bacterially- expressed toxin, a virally-expressed toxin, or a venom protein.
  • the variant HuCOL- 1 antibody is linked to a cytokine.
  • Specific, non- limiting examples ofcytoMnes are IL-2, IL-4, IL- 10, TNF-alpha and IFN-gamma.
  • the variant HuCOL- 1 antibody is linked to an effector molecule by a covalent or non-covalent means.
  • compositions are disclosed herein that include a variant
  • HuCOL-1 monoclonal antibody such as HuCOL-l 61 H, HuCOL- 1 24 ' 25 ' 27 L, or HuCOL- 1 24,25,27 L/ 61 H, and can be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen.
  • a variant HuCOL- 1 monoclonal antibody linked to an effector molecule i.e., toxin, chemotherapeutic drug, or detectable label
  • an effector molecule i.e., toxin, chemotherapeutic drug, or detectable label
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • the pharmaceutical composition to be administered can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Topical preparations can include eye drops, ointments, sprays and the like.
  • Enhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like.
  • Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules).
  • Suppository preparations can also be solid, gel, or in a suspension form.
  • conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • compositions that include a variant HuCOL- 1 monoclonal antibody can be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • the pharmaceutical compositions may be administered as an immunoprophylactic in a single dose schedule or as an immunotherapy in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions.
  • Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
  • a unit dosage can be about 0.1 to about 10 mg per patient per day. Dosages from about 0.1 up to about 100 mg per patient per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity, into a lumen of an organ, or directly into a tumor. In one embodiment, about 10 mCi of a radiolabeled variant HuCOL- 1 monoclonal antibody is administered to a subject.
  • a radiolabeled variant HuCOL- 1 monoclonal antibody is administered to a subject.
  • the amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.
  • the compounds of this disclosure can be administered to humans on whose tissues they are effective in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository.
  • the particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. the subject, the disease, the disease state involved, and whether the treatment is prophylactic).
  • a therapeutically effective amount of a variant HuCOL- 1 antibody is the amount of variant HuCOL- 1 antibody necessary to inhibit further growth of a CEA- expressing tumor or suppress the growth of a CEA-expressing tumor, without eliciting a HAMA response in the patient receiving the treatment.
  • a therapeutically effective amount of variant HuCOL- 1 antibody is the amount of variant HuCOL- 1 antibody necessary to eliminate or reduce the size of a CEA-expressing tumor, without eliciting a HAMA response.
  • a therapeutically effective amount of variant HuCOL- 1 antibody is an amount of variant HuCOL- 1 antibody that is effective at reducing a sign or a symptom of the tumor and induces a minimal immune response.
  • a therapeutically effective amount of a variant HuCOL-1 monoclonal antibody such as HuCOL-l 61 H, HuCOL- 1 24 ' 25 ' 27 L, or HuCOL-l 24 ' 25,27 L/ 61 H, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. In one embodiment, treatment continues until a therapeutic result is achieved. However, the effective amount of variant HuCOL- 1 antibody will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration of the therapeutic(s).
  • Controlled release parenteral formulations of a variant HuCOL-1 monoclonal antibody can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres the therapeutic is dispersed throughout the particle.
  • Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly (see ICreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992).
  • Polymers can be used for ion-controlled release.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, R., Accounts Chem. Res. 26:537, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al, Pharm. Res. 9:425, 1992; and Pec et al, J. Parent. Sci. Tech. 44:58, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al, Int. J. Pharm. 112:215, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri, et al, Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Pat. No.
  • Site-specific administration of the disclosed compounds can be used, for instance by applying the variant HuCOL- 1 antibody to a pre-cancerous region, a region of tissue from which a tumor has been removed, or a region suspected of being prone to tumor development.
  • sustained intra-tumoral (or near-tumoral) release of the pharmaceutical preparation that includes a therapeutically effective amount of variant HuCOL-1 antibody may be beneficial.
  • the present disclosure also includes therapeutic uses of variant HuCOL- 1 monoclonal antibodies, such as HuCOL-l 61 H, HuCOL- 1 2 ' 25 ' 27 L, or HuCOL- 1 24 ' 25,27 L/ 61 H, that are non-covalently or covalently linked to effector molecules.
  • the variant HuCOL- 1 monoclonal antibody is covalently linked to an effector molecule that is toxic to a tumor or cell expressing CEA.
  • the effector molecule is a cytotoxin.
  • the effector molecule is a radioactive isotope, a chemotherapeutic drug, a bacterially-expressed toxin, a virally-expressed toxin, a venom protein, or a cytokine.
  • Variant HuCOL- 1 monoclonal antibodies covalently linked to an effector molecule have a variety of uses.
  • a variant HuCOL- 1 antibody linked to a radioactive isotope is of use in immunotherapy.
  • a variant HuCOL-1 antibody covalently linked to a radioactive isotope is of use to localize a tumor in radioimmunoguided surgery, such that the tumor can be removed.
  • the present disclosure also includes combinations of a variant HuCOL- 1 monoclonal antibody, such as HuCOL-l 61 H, HuCOL- 1 24 ' 25 ' 27 L, or HuCOL- 1 ' ' L/ H, with one or more other agents useful in the treatment of tumors.
  • a therapeutically effective amount of the compounds of this disclosure can be administered in combination with effective doses of immunostimulants, anti-cancer agents, anti-inflammatory agents, anti-infectives, and/or vaccines.
  • administration in combination” or “co-administration” refers to both concurrent and sequential administration of the active agents.
  • a subject that has a tumor, or is predisposed to the development of a tumor, will be a candidate for treatment using the therapeutic methods disclosed herein.
  • a method is provided herein for the in vivo or in vitro detection of CEA- expressing tumors or cells.
  • An in vivo detection method can localize any tumor or cell that expresses CEA in a subject.
  • a variant HuCOL- 1 antibody such as HuCOL-l 61 H, HuCOL-l 24 ' 25,27 L, or HuCOL-l 24 ' 25,27 L/ 61 H, is administered to the subject for a sufficient amount of time for the antibody to localize to the tumor or cell in the subject and to form an immune complex with CEA.
  • the immune complex is detected.
  • detection of an immune complex is performed by immunoscintography.
  • immune complex detection include radiolocalization, radioimaging, or fluorescence imaging.
  • the antibody is linked to an effector molecule.
  • the effector molecule is a detectable label.
  • detectable labels include a radioactive isotope, an enzyme substrate, a co-factor, a ligand, a chemiluminescent agent, a fluorescent agent, a hapten, or an enzyme.
  • a method of detecting tumors in a subject includes the administration of a variant HuCOL- 1 antibody complexed to an effector molecule, such as a radioactive isotope.
  • a variant HuCOL- 1 antibody complexed to an effector molecule such as a radioactive isotope
  • an effector molecule such as a radioactive isotope
  • a variant HuCOL- 1 antibody complexed to an effector molecule such as a radioactive isotope
  • the detection step is performed prior to surgery.
  • the detection step is performed during surgery, for example to detect the location of the tumor prior to removing it, as in radioimmunoguided surgery.
  • the detection step is performed after surgery to ensure the complete removal of the tumor, or to detect a recurrence of the tumor.
  • a radiolabeled immune complex is detected using a hand-held gamma detection probe.
  • Primary tumors, metastasized tumors, or cells expressing CEA can be detected.
  • a variant HuCOL- 1 antibody and a secondary antibody are administered to the subject for a sufficient amount of time for the variant HuCOL- 1 antibody to form an immune complex with CEA on a tumor or cell, and for the secondary antibody to form an immune complex with the variant HuCOL- 1 antibody.
  • the variant HuCOL-1 antibody is complexed with the secondary antibody prior to their administration to the subject.
  • the secondary antibody is linked to a detectable label.
  • the immune complex which includes CEA, the variant HuCOL- 1 antibody, and the secondary antibody linked to a detectable label, is detected as described above.
  • the in vitro detection method can screen any biological sample containing any tumor or cell that expresses CEA.
  • samples include, but are not limited to, tissue from biopsies, autopsies, and pathology specimens.
  • Biological samples also include sections of tissues, such as frozen sections taken for histological purposes.
  • Biological samples further include body fluids, such as blood, serum, saliva, or urine.
  • a biological sample is typically obtained from a mammal, such as a human.
  • the subject has a colorectal tumor.
  • the subject has a gastrointestinal tumor, a breast tumor, a lung tumor, or an ovarian tumor.
  • Other biological samples that can be detected by the in vitro detection method include samples of cultured cells that express CEA.
  • kits for detecting a CEA-expressing tumor or cell will typically comprise a variant HuCOL- 1 antibody that specifically binds CEA.
  • an antibody fragment such as an Fv fragment is included in the kit.
  • the antibody is an immunoconjugate.
  • the antibody is conjugated to a detectable label (for example, radioactive isotope, enzyme substrate, co-factor, ligand, fluorescent agent, hapten, enzyme, or chemiluminescent agent).
  • kits includes instructional materials disclosing means of use of an antibody that specifically binds CEA or fragment thereof (for example, for detection of CEA-expressing cells in a sample).
  • the instructional materials may be written, in an electronic form (e.g. computer diskette or compact disk) or may be visual (for example, video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (for example, enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kit contains a secondary antibody that is conjugated to a detectable label.
  • the kits may additionally include buffers and other reagents, such as an antigen (for example, purified CEA) routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting CEA or fragment thereof in a biological sample generally comprises the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to CEA.
  • the antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
  • the invention is illustrated by the following non-limiting Examples.
  • the long overlapping oligomers and oligonucleotide primers used for DNA amplifications were supplied by Lofstrand Labs. (Gaithersburg, MD) and Midland Certified Reagent Company (Midland, TX).
  • the sequences of the four primers that were used to generate DNA fragments encoding the V H and V L domains of the murine COL-1 monoclonal antibody were as follows:
  • Each of the above 5' primers (SEQ ID NO:l and SEQ ID NO:3) carries a Hind III site followed by a sequence encoding a signal peptide.
  • the 3' V H primer (SEQ ID NO:2) carries an Apa I, while the 3' V L primer (SEQ ID NO:4) has a Sac II site.
  • the four 119- to 133- base pair (bp)-long oligonucleotides that were used to generate each of the V H and V L genes of HuCOL- 1 are shown by long arrows in FIG. 1.
  • the sequence of the 20- to 21- bp-long end primers used for DNA amplification were as follows: 5' V H (SEQ ID NO:5):
  • the 5' primers (SEQ ID NO:5 and SEQ ID NO:7) carry a. Hind III site. While a site for Ap ⁇ I has been incorporated in the 3' V H primer (SEQ ID NO:6), the sequence for the Sac II site has been included in the 3' V L primer (SEQ ID NO:8).
  • the sequences of two additional mutagenic primers (supplied by Milligen/Bioresearch, Burlington, VT) that were used for the generation and amplification of the genes encoding the V domains of HuCOL- 1 variants are as follows:
  • PCRs were carried out in a final volume of 100 ⁇ l of PCR buffer containing 200 ⁇ M of dNTPs, 3 units of Taq polymerase (Boehringer Mannheim, Indianapolis, IN), 0.2 ⁇ M each of the end primers and 100 ng of DNA template.
  • Initial denaturation at 94°C for 2 minutes was followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute. It was followed by a final primer extension step at 72°C for 10 minutes.
  • V L and V H genes of the HuCOL- 1 were synthesized by the overlap extension PCR (FIG. 1) that has previously been described (Kashmiri et al. Hybridoma 14:461, 1995). Primer induced mutagenesis was carried out by a dual step PCR as described by Landt et al. (Landt et al Gene 96: 125, 1990). The first PCR was carried out in a final volume of 100 ⁇ l containing 10 ng of template, 20 pmol of each primer, 100 ⁇ M of dNTPs and 5 units of Taq Polymerase (Gibco BRL, Gaithersburg, MD).
  • the genes encoding the L chain and the Fd region of the H chain of mAb COL-1 were generated by repertoire cloning methodology using the suggested sets of the 5' and 3' primers for the PCR amplification of the murine K L chain and the murine H chain Fd. (Kang, et al, Methods: A Companion to Methods in Enzymology. 2:111,
  • cDNA synthesized from total RNA extracted from COL-1 hybridoma (Muraro et al, Cancer Res. 45:5769, 1985) was used as a template. Since mAb COL-1 is a murine IgG2a, the 3' primer used to amplify the Fd region of the H chain was specific to the 3'-end of the IgG2a C H I region. The cloned genes were sequenced (FIG. 2) before the phagemids were used as templates for the subsequent PCR amplification.
  • Murine COL-1 was humamzed by grafting the CDRs of the L and H chains onto the V L and V H frameworks of the VJFCL and MO30 human Abs, respectively, while retaining those framework residues that were deemed essential for preserving the structural integrity of the combining site (Jones et al, Nature 321:522, 1986; Riechmann et al, Nature 332:323, 1988; Verhoeyen and Riechmann Bioessays 8:74, 1988).
  • the Ig CDRs have been defined as comprising residues 31-35b, 50-65, and 95- 102 in the H chain and residues 24-34, 50-56, and 89-97 in the L chain (Kabat et al, Sequence of proteins of immunological interests, 5 th Ed. U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD (NEH Publication no. 91- 3242) 1991).
  • the framework residues that were deemed critical were identified on the basis of the atomic coordinates of the Abs of known structures available in the database (see, for example, Padlan, Mol. Immunol. 31:169, 1994).
  • VJFCL The human Ab sequences that are most similar to mCOL-1 are VJFCL (Klobeck et al, Nucleic Acids Res. 13:6515, 1985.) (GenBank Accession Number Z00022) for V and MO30 (Larrick et al, Biochem. Biophys. Res. Commun. 160:1250, 1989) (GenBank Accession Number
  • V H V H .
  • the alignments of the V L sequences of mCOL-1 and VJFCL, and the V H sequences of mCOL-1 and MO30 are shown in FIG. 3. Also indicated in FIG. 3 are the locations of the framework residues that are critical for Ag binding.
  • the humanization protocols for the V L and V H genes, shown in FIG. 3, are based on putting the CDR sequences of mAb COL- 1 together with the frameworks of the human V L and V H templates while replacing some of the human framework residues with those murine framework residues that may be critical for Ag binding.
  • a nucleotide sequence was then deduced from the amino acid sequence of each of the designed humanized V L and V H domains.
  • the nucleotide sequences were refined to provide high frequency usage of codons and by eliminating, with the help of programs FOLD and MAPSORT (Devereux et al, Nucleic Acids Res. 12:387, 1984), any self-annealing regions and any sites for restriction endonucleases that might complicate cloning of the designed genes in the desired vectors.
  • FOLD and MAPSORT Disevereux et al, Nucleic Acids Res. 12:387, 1984
  • DNA fragments encoding the humanized V L or V H regions were generated and amplified by overlap extension PCR technique (Kashmiri et al, Hybridoma 14:461, 1995).
  • the humanized V L or V H regions thus generated were extended to the Apa I and Sac II sites located 10 bp and 17 bp downstream from the 5' end of the C regions of the H and L chains, respectively.
  • the PCR products were cloned in pBSc to generate PBSCHUCOL-IV L and pBScHuCOL- 1 V H constructs. The inserts were sequenced to check the fidelity of the PCR products.
  • HuCOL- 1 H were generated by primer-induced mutagenesis, using PBSCHUCOL-IV L and pBScHuCOL-lVn constructs, respectively, as templates.
  • Variant HuCOL- 1 61 H was generated by replacing residue 61 of mCOL-1 HCDR2 (numbering convention of Kabat et al. (Sequence of proteins ofimmunological interests, Ed. U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD (NEH Publication no. 91-3242) 1991)) with the corresponding residue of the human mAb MO30 HCDR2 (FIG. 3, Table 1).
  • the resulting PCR product was gel purified and used as a 5' primer for the subsequent step of the PCR in which a 21-mer oligonucleotide was utilized as a 3' primer.
  • the PCR products of the variants were cloned in pBSC vector and sequenced.
  • PAcUW51 vector contains the plO and polyhedrin (polh) promoters placed in opposite orientation.
  • One of the target genes can be cloned at the BamH I site located downstream oiihepolh promoter, while the other gene can be driven by the plO promoter by inserting the gene either at Bgl II or EcoR I site located 3 ' to the promoter.
  • the vector carries the fl origin of replication. Co-transfection of insect cells with pAcUW51 and the baculovirus BaculoGold DNA (Pharmingen) rescues the lethal deletion in this DNA and results in the production of 99% of viable recombinant virions expressing the target gene.
  • the pBAC4x-l baculovirus transfer plasmid is designed for cloning and co-expression of up to four target genes.
  • the plasmid contains two of each o t epolh andplO promoters, with a unique cloning site placed downstream of each promoter. The homologous promoters are placed in opposite orientation to minimize recombination.
  • the plasmid is compatible with baculovirus DNA, BacVector2000 (Novagen).
  • the V region sequences of the H and L chain genes were PCR amplified using the phagemid constructs of the cDNAs encoding the Fd and the L chain of mCOL-1 as templates.
  • Primers with nucleic acid sequences set forth as SEQ ID NO:3 and SEQ ID NO:4 were used as forward and reverse primers, respectively, to amplify a 420 bp sequence encoding the N L domain along with the signal peptide located upstream.
  • the 3' primer was designed to extend the 3 '-end of the amplified sequence to a unique Sac II site located 10 bp downstream from the start of the human K C region.
  • a D ⁇ A fragment encoding the human K C region was excised from a pre-existing construct pL ⁇ CXHuCC49HuK (Kashmiri et al. Hybridoma 14:461 , 1995) by Sac ⁇ VCla I treatment.
  • the construct carried an EcoR I site immediately upstream of Cla I site.
  • the V and the C regions of the L chain were joined to the Hind IWCla I linearized pBluescript II S/K + (pBSc) plasmid (Stratagene, La Jolla, CA) by three-way ligation.
  • a 460 bp sequence encoding the N H domain and its leader peptide was PCR amplified using primers, with nucleic acid sequences as set forth as SEQ ID NO: 1 and SEQ ID NO:2, as the 5' and 3' primers, respectively.
  • the design of the 3' primer facilitated amplification of the V H sequence to extend to the Apa I site located 17 bp downstream from the start of the C H I domain.
  • an Apa VCla I DNA fragment carrying the human ⁇ l C region was excised from a pre-existing construct pLgpCXHuCC49HuGl (Kashmiri et al. Hybridoma 14:461 , 1995).
  • the Apa VCla I fragment along with the 460 bp PCR product were inserted into the Hind IWCla I linearized pBSc.
  • the DNA encoding the entire H chain was released by Hind IWCla I treatment of the pBSc construct.
  • the termini of the target DNA were filled in using Klenow fragment of the DNA polymerase, and the DNA fragment was subcloned in the L-chain construct of pAcUW51 at the blunt-ended BamH I site located downstream of the polh promoter (FIG. 4A).
  • the entire H chain of the HuCOL- 1 or its variant was excised from its pBSc construct by Hind WXho I treatment and it was cloned unidirectionally in the L-chain construct of pBAC4x-l at the Hind IWXho I site, downstream of polh promoter (FIG. 4B).
  • Three expression constructs were generated; one containing the variant L chain and the parental humanized H chain (HuCOL- 1 ' ' L), the other containing the variant H chain and the parental humanized L chain (HuCOL-l 61 H), while the third carried variants of both the L and H chains (HuCOL-l 24 ' 25 ' 27 L/ 61 H) (Table I).
  • Serum-free-adapted Sf9 insect cells (Gibco BRL) were cultured at 27°C in Sf900-II medium (Gibco BRL) with 50 ⁇ g/ml of gentamicin.
  • Sf900-II medium (Gibco BRL)
  • gentamicin 50 ⁇ g/ml of gentamicin.
  • To develop transfectomas secreting cCOL-1 insect cells were co-transfected with the pAcUW51 derived expression construct and the linearized BaculoGold Baculovirus DNA.
  • Transfectomas producing HuCOL- 1 and its variants were generated by transfecting insect cells with one of the pBAC4x-l derived expression constructs and the linearized BacVector2000 Baculovirus DNA.
  • a cationic liposome-mediated system DOTAP (Boehringer Mannheim) was used for all transfections.
  • the supernatants containing the recombinant virus were harvested and screened for Eg expression and Ag binding by ELISA.
  • Viral plaques were generated by infecting Sf9 cells (2 x 10 6 cells/60-mm dish) with recombinant virus followed by overlaying the infected cells with 0.5% baculovirus agarose (Invitrogen, Carlsbad, CA). Individual viral plaques were isolated and expanded for three rounds by infecting increasingly larger number of freshly seeded monolayers of Sf9 cells, using the highest producing clone as a source of inoculum each time. Supernatants harvested from each round were assayed for Ag binding by ELISA (Bei et al. J. Immunol.
  • the titer of recombinant virus was determined by plaque assay. To produce the recombinant protein, 6 x 10 8 Sf9 cells were infected with the supernatant at a multiplicity of infection of 5.
  • the supernatants were collected and centrifuged at 2,000 x g for 10 minutes to remove cellular debris. After adding Tris-HCI pH 7.5 to a final concentration of 20 mM and incubating at 4°C for 2 hours, a second centrifugation was performed at 10,000 x g to remove any contaminating proteins. The supernatant was then loaded on a protein G agarose column (Gibco BRL). The bound protein was eluted from the column with 0.1 M glycine hydrochloride, pH 2.5 and the pH of the eluted material was immediately adjusted to 7.4 with 1.0 M Tris pH 8.0.
  • the protein was concentrated using Centricon 30 (Amicon, Beverly, MA) and dialyzed in PBS buffer using Slide- A-Lyzer cassette (Pierce, Rockford, EL).
  • the protein concentration was determined by the method of Lowry et al. (Lowry, et al, J. Biol. Chem. 193:265, 1951) and the purity of the eluted proteins was evaluated by SDS-PAGE, under reducing and non-reducing conditions, using pre-cast 4-20% Tris-glycine gel (Novex, San Diego, CA).
  • the protein bands were visualized by Coomassie blue staining (Novex).
  • ELISA assays were carried out by coating 96-well polyvinyl microtiter plates with CEA (100 ng/well) (Research Diagnostic Inc., Flanders, NJ) or with Fc ⁇ -fragment- specific goat anti-human IgG (100 ng/well) (Jackson Immunoresearch Lab, West Grove, PA). Anti-human IgG or the CEA-coated plates were used to test for the production of Ig by the insect cells or to assess its Ag reactivity, respectively. The plates were blocked with 5% BSA in PBS for 1 hour at 37°C and then washed with 1% BSA in PBS. Fifty microliters of culture supematants were loaded in duplicate wells.
  • the concentration of the secreted Abs was comparable (2 to 3 ⁇ g/ml) in culture supernatants of all five transfectants.
  • ELISA assays were carried out using serial dilutions of the purified recombinant Abs. Results of the assays suggested that mAbs HuCOL-l 24 ' 25 ' 27 L and HuCOL-l 24 ' 25 ' 27 L/ 61 H had lower CEA-binding reactivity than those of cCOL-1, HuCOL-1 and the variant HuCOL-l 61 H mAbs.
  • the relative Ag binding of the mCOL-1 and the recombinant antibodies derived from it were determined using competition radioimmunoassay (RIA). Twenty-five ⁇ l of serial dilutions of the Abs to be tested as well as the mCOL-1, prepared in 1% BSA in PBS, were added to microtiter plates containing 200 ng of CEA saturated with 5% BSA in PBS. 125 I-labeled mCOL-1 or 125 I-labeled HuCOL-1 (100,000 cpm in 25 ⁇ l) was then added to each well. After an overnight incubation at 4°C, the plates were washed and counted in a ⁇ -scintillation counter. The relative affinity constants were calculated by a modification of the Scatchard method (Frankel and Gerhard, Mol. Immunol. 16:101, 1979).
  • the cell suspension was stained with FITC-conjugated mouse anti-human Ab (Pharmingen) for 30 minutes on ice.
  • a second washing cycle was performed and then the samples were analyzed with a FACScan (Becton Dickinson, Mountain View, CA) using CellQuest for Macintosh. Data from analysis of 10,000 cells were obtained.
  • the circulating CEA and anti-murine Fc Abs were removed by sequential pre-adsorption of the sera with a purified mCOL-4 mAb that reacts with epitopes of CEA different from the one recognized by mCOL-1.
  • Murine COL-6 has the same isotype as that of mCOL-1.
  • serum samples were added to mCOL-6 coupled to Reacti-gel according to the method of Hearn et al. (Hearn et al, J. Chromatogr. 185:463, 1979). The mixtures were incubated overnight at 4°C with end- to-end rotation and centrifuged at 1000 x g for 5 min.
  • Pre-adsorption was repeated until the supernatants displayed no detectable anti-murine Fc activity.
  • SPR Surface Plasmon Resonance
  • the pre-adsorbed serum was used as a mobile reactant.
  • Proteins were immobilized on carboxymethylated dextran CM5 chips (Biacore, Piscataway, NJ) by amine coupling using standard procedure (Johnsson et al, Anal. Biochem. 198:268, 1991; Schuck et al., Measuring protein interactions by optical biosensors. En Current Protocols in Protein Science, Vol. 2. J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W. Speicher, and P. T.
  • HuCOL-1 was immobilized on the surface of flow cell 1, while the surface of flow cell 2 was coated with an unrelated protein, rabbit gamma globulin (Biorad, Hercules, CA).
  • COL-1 variants The reactivity of COL-1 variants to anti-V region antibodies was determined using a recently developed SPR-based competition assay (De Pascalis et al, J. Immunol. In Press). Competition experiments were performed at 25 °C using a CM5 sensor chip containing either mCOL-1 or HuCOL- 1 in flow cell 1 and rabbit gamma globulin (Biorad), as a reference, in flow cell 2. Typically, mCOL-1, HuCOL- 1 or its variants, were used at different concentrations, to compete with the Ab immobilized on the sensor chip for binding to serum anti-V region Abs.
  • IC 50 for each antibody the concentration required for 50% inhibition of the binding of the serum to either mCOL-1 or HuCOL- 1, was calculated.
  • the Abs were characterized for their reactivity to sera from gastrointestinal carcinoma patients who were administered 131 I-mCOL-l in a phase I clinical trial (Yu et al, J. Clin. Oncol. 14:1798, 1996).
  • any circulating CEA and anti-murine Fc Abs were removed from the sera by immunoadsorption with mCOL-4, a murine anti-CEA Ab of IgG 2a isotype that reacts with a CEA epitope different from the one recognized by mAb COL-1.
  • Pre-adsorbed sera were tested for the presence of anti-V region Abs to mAb COL-1.
  • FIG. 9 shows the competition profiles generated by HuCOL-1 and its variants when they were used to compete with the HuCOL-1 immobilized on the sensor chip for binding to the anti-V region Abs to COL-1 present in the sera of patients EM (FIG. 9A), JS (FIG. 9B), and MB (FIG. 9C).
  • the competition profiles were used to calculate IC 50 values that are presented in Table 2.
  • the ICs 0 values of all three variants are 2- to 3-fold higher than that of HuCOL- 1.
  • Studies with the serum from patient EM show that the variants HuCOl-l 24,25,27 L and HuCOL- 1 24)25,27 L/ 61 H have 50% higher IC 50 values, while the variant HuCOL-l 61 H has significantly lower IC 50 value, than that of HuCOL-1.
  • the IC 50 of the variant HuCOL-1 • ' LI H is twice as much as that of HuCOL-1, while the IC 50 values of the variant HuCOL-l 61 H and HuCOL- 1 24 ' 25 ' 27 L are comparable to that of parental HuCOL- 1.
  • Patients with recurrent colorectal cancer are assessed to determine the maximum tolerated dose of intravenously administered 177 Lutetium radiolabeled HuCOL- 1 6 H, 177 Lutetium radiolabeled HuCOL-l 24 ' 25 ' 27 L, and 177 Lutetium radiolabeled HuCOL- ⁇ 24,2 5 ,27 L/ 6i H (Mulligan, (1995) Clin. Cancer Res. 1:1447-1454).
  • Colorectal cancer patients are given a test dose of 0.1 mg (intravenous bolus) of HuCOL- 1 61 H, HuCOL- 1 24,25,27 L, or HuCOL-l 24,25,27 L/ 61 H and are observed for 30 minutes prior to administration of the 177 Lu-labeled HuCOL-l 61 H, 177 Lu-labeled HuCOL- 1 24 ' 25 ' 27 L, or 177 Lu-labeled HuCOL-l 24,25,27 L/ 61 H.
  • the radiolabeled antibodies are given as an intravenous infusion over the course of a one hour time interval. Blood samples are collected prior to and at the end of the infusion, as well as 0.5, 1 and 2 hours following the completion of the infusion. In addition, blood samples are collected daily over the subsequent 7 days. Patients return for a follow-up examination at 3, 6 or 8 weeks. Blood samples are again collected during these visits. Sera are separated and stored at - 20°C.
  • the sera from the patients are evaluated for the presence of human anti-murine antibodies (HAMA) in response to radiolabeled HuCOL-l 61 H, HuCOL- 1 24 ' 25 ' 27 L, or HuCOL-l 24,25,27 L/ 61 H using the SPR-based assay described in Example 8, above.
  • the sera are pre-absorbed with an mCOL-4 monoclonal antibody that recognizes an epitope of CEA which is different from the epitope recognized by the humanized COL-1 monoclonal antibody.
  • Pre-absorption using the COL-4 antibody removes circulating CEA and anti-murine Fc antibodies from the sera.
  • HuCOL- 1 61 H, HuCOL- 1 24,25 ' 27 L, or HuCOL-1 ' ' IJ H is coated on the surface of flow cell 1 and a reference protein (HuIgG2a, bovine serum albumin, or rabbit gamma globulin) is immobilized on the surface of flow cell 2.
  • a reference protein HuIgG2a, bovine serum albumin, or rabbit gamma globulin
  • Sensograms to flow cell l and flow cell 2 are generated and the response difference between the two cells is plotted for each serum sample, thus providing a measure of the anti- variable region response against HuCOL-l 61 H, HuCOL-l 24 ' 25 ' 27 L, or HuCOL- 1 24 ' 25,27 L/ 61 H in each particular serum sample.
  • Results indicate that the patients' sera have a minimal anti- variable region response against the HuCOL- 1 61 H, HuCOL-l 24 ' 25,27 L, or HuCOL- l2 4,2 s ,27 L/ 6i H antibodies.
  • This disclosure provides humanized COL-1 monoclonal antibodies.
  • the disclosure further provides methods of diagnosing and treating tumors using these humanized COL-1 antibodies. It will be apparent that the precise details of the methods described may be varied or modified without departing from the spirit of the described disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des anticorps monoclonaux humanisés COL-1 qui sont très faiblement immunogènes et qui retiennent l'affinité de la liaison à l'antigène carcino-embryonnaire. Dans un mode de réalisation, un anticorps humanisé COL-1 comprend une substitution d'acide aminé en position 61 dans une région 2 déterminant la complémentarité de la chaîne lourde de l'anticorps murin COL-1. Dans un autre mode de réalisation, un anticorps humanisé COL-1 comprend une substitution d'acide aminé en position 24, 25 et 27 dans une région 1 déterminant la complémentarité de la chaîne légère de l'anticorps murin COL-1. Plusieurs modes de réalisation concernent des procédés d'utilisation d'un anticorps humanisé COL-1 pour la détection ou le traitement d'une tumeur chez un patient. L'invention concerne également une trousse comprenant l'anticorps humanisé COL-1 décrit ci-dessus.
PCT/US2003/027976 2002-09-05 2003-09-05 Variants tres faiblement immunogenes de l'anticorps humanise col-1 contre l'antigene carcino-embryonnaire WO2004022717A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003270369A AU2003270369A1 (en) 2002-09-05 2003-09-05 Minimally immunogenic variants of humanized col-1 antibody against carcinoembryonic antigen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US40870302P 2002-09-05 2002-09-05
US60/408,703 2002-09-05

Publications (2)

Publication Number Publication Date
WO2004022717A2 true WO2004022717A2 (fr) 2004-03-18
WO2004022717A3 WO2004022717A3 (fr) 2004-09-02

Family

ID=31978660

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/027976 WO2004022717A2 (fr) 2002-09-05 2003-09-05 Variants tres faiblement immunogenes de l'anticorps humanise col-1 contre l'antigene carcino-embryonnaire

Country Status (2)

Country Link
AU (1) AU2003270369A1 (fr)
WO (1) WO2004022717A2 (fr)

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005112564A2 (fr) * 2004-04-15 2005-12-01 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Régions variables d'anticorps humanisés et lignée germinale et méthodes de fabrication et d'utilisation
WO2006074071A1 (fr) * 2004-12-30 2006-07-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Anticorps col-1 humanises a residus charpente substitues et utilisation
WO2007090596A1 (fr) * 2006-02-06 2007-08-16 Medizinische Universität Wien Vaccin et mimotopes d'antigènes dirigés contre les maladies cancéreuses associées à l'antigène
US7662925B2 (en) 2002-03-01 2010-02-16 Xencor, Inc. Optimized Fc variants and methods for their generation
US7973136B2 (en) 2005-10-06 2011-07-05 Xencor, Inc. Optimized anti-CD30 antibodies
US8039592B2 (en) 2002-09-27 2011-10-18 Xencor, Inc. Optimized Fc variants and methods for their generation
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
US8124731B2 (en) 2002-03-01 2012-02-28 Xencor, Inc. Optimized Fc variants and methods for their generation
US8188231B2 (en) 2002-09-27 2012-05-29 Xencor, Inc. Optimized FC variants
US8318907B2 (en) 2004-11-12 2012-11-27 Xencor, Inc. Fc variants with altered binding to FcRn
US8388955B2 (en) 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US8394374B2 (en) 2006-09-18 2013-03-12 Xencor, Inc. Optimized antibodies that target HM1.24
US8524867B2 (en) 2006-08-14 2013-09-03 Xencor, Inc. Optimized antibodies that target CD19
US8546543B2 (en) 2004-11-12 2013-10-01 Xencor, Inc. Fc variants that extend antibody half-life
US8802820B2 (en) 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
US9040041B2 (en) 2005-10-03 2015-05-26 Xencor, Inc. Modified FC molecules
US9051373B2 (en) 2003-05-02 2015-06-09 Xencor, Inc. Optimized Fc variants
US9200079B2 (en) 2004-11-12 2015-12-01 Xencor, Inc. Fc variants with altered binding to FcRn
US9657106B2 (en) 2003-03-03 2017-05-23 Xencor, Inc. Optimized Fc variants
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
US11401348B2 (en) 2009-09-02 2022-08-02 Xencor, Inc. Heterodimeric Fc variants
US11820830B2 (en) 2004-07-20 2023-11-21 Xencor, Inc. Optimized Fc variants
US11932685B2 (en) 2007-10-31 2024-03-19 Xencor, Inc. Fc variants with altered binding to FcRn

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8362210B2 (en) 2010-01-19 2013-01-29 Xencor, Inc. Antibody variants with enhanced complement activity

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6333405B1 (en) * 1996-10-31 2001-12-25 The Dow Chemical Company High affinity humanized anti-CEA monoclonal antibodies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6333405B1 (en) * 1996-10-31 2001-12-25 The Dow Chemical Company High affinity humanized anti-CEA monoclonal antibodies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KASS E.S. ET AL: 'Carcinoembryonic antigen as a target for specific antitumor immunotherapy of head and neck cancer' CANCER RESEARCH vol. 62, 01 September 2002, pages 5049 - 5057, XP002978237 *
RABEN D. ET AL: 'Enhancement of radiolabeled antibody binding and tumor localization through adenoviral transduction of the human carcinoembryonic antigen gene' GENE THERAPY vol. 3, 1996, pages 567 - 580, XP002061094 *
YU B.B. ET AL: 'Phase I trial of iodine 131-labeled COL-1 in patients with gastrointestinal malignancies: Influence of serum carcinoembryonic antigen and tumor bulk on pharmacokinetics' JOURNAL OF CLINICAL ONCOLOGY vol. 14, no. 6, June 1996, pages 1798 - 1809, XP002978417 *

Cited By (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7662925B2 (en) 2002-03-01 2010-02-16 Xencor, Inc. Optimized Fc variants and methods for their generation
US8124731B2 (en) 2002-03-01 2012-02-28 Xencor, Inc. Optimized Fc variants and methods for their generation
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
US8093357B2 (en) 2002-03-01 2012-01-10 Xencor, Inc. Optimized Fc variants and methods for their generation
US9353187B2 (en) 2002-09-27 2016-05-31 Xencor, Inc. Optimized FC variants and methods for their generation
US9193798B2 (en) 2002-09-27 2015-11-24 Xencor, Inc. Optimized Fc variants and methods for their generation
US8858937B2 (en) 2002-09-27 2014-10-14 Xencor, Inc. Optimized Fc variants and methods for their generation
US8039592B2 (en) 2002-09-27 2011-10-18 Xencor, Inc. Optimized Fc variants and methods for their generation
US8809503B2 (en) 2002-09-27 2014-08-19 Xencor, Inc. Optimized Fc variants and methods for their generation
US8383109B2 (en) 2002-09-27 2013-02-26 Xencor, Inc. Optimized Fc variants and methods for their generation
US8093359B2 (en) 2002-09-27 2012-01-10 Xencor, Inc. Optimized Fc variants and methods for their generation
US10184000B2 (en) 2002-09-27 2019-01-22 Xencor, Inc. Optimized Fc variants and methods for their generation
US10183999B2 (en) 2002-09-27 2019-01-22 Xencor, Inc. Optimized Fc variants and methods for their generation
US8188231B2 (en) 2002-09-27 2012-05-29 Xencor, Inc. Optimized FC variants
US9663582B2 (en) 2003-03-03 2017-05-30 Xencor, Inc. Optimized Fc variants
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US10584176B2 (en) 2003-03-03 2020-03-10 Xencor, Inc. Fc variants with increased affinity for FcγRIIc
US8388955B2 (en) 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US10113001B2 (en) 2003-03-03 2018-10-30 Xencor, Inc. Fc variants with increased affinity for FcyRIIc
US8735545B2 (en) 2003-03-03 2014-05-27 Xencor, Inc. Fc variants having increased affinity for fcyrllc
US9657106B2 (en) 2003-03-03 2017-05-23 Xencor, Inc. Optimized Fc variants
US9051373B2 (en) 2003-05-02 2015-06-09 Xencor, Inc. Optimized Fc variants
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
WO2005112564A3 (fr) * 2004-04-15 2007-01-18 Us Gov Health & Human Serv Régions variables d'anticorps humanisés et lignée germinale et méthodes de fabrication et d'utilisation
WO2005112564A2 (fr) * 2004-04-15 2005-12-01 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Régions variables d'anticorps humanisés et lignée germinale et méthodes de fabrication et d'utilisation
US11820830B2 (en) 2004-07-20 2023-11-21 Xencor, Inc. Optimized Fc variants
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
US8546543B2 (en) 2004-11-12 2013-10-01 Xencor, Inc. Fc variants that extend antibody half-life
US9803023B2 (en) 2004-11-12 2017-10-31 Xencor, Inc. Fc variants with altered binding to FcRn
US8338574B2 (en) 2004-11-12 2012-12-25 Xencor, Inc. FC variants with altered binding to FCRN
US8883973B2 (en) 2004-11-12 2014-11-11 Xencor, Inc. Fc variants with altered binding to FcRn
US11198739B2 (en) 2004-11-12 2021-12-14 Xencor, Inc. Fc variants with altered binding to FcRn
US8367805B2 (en) 2004-11-12 2013-02-05 Xencor, Inc. Fc variants with altered binding to FcRn
US8802820B2 (en) 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
US8852586B2 (en) 2004-11-12 2014-10-07 Xencor, Inc. Fc variants with altered binding to FcRn
US9200079B2 (en) 2004-11-12 2015-12-01 Xencor, Inc. Fc variants with altered binding to FcRn
US10336818B2 (en) 2004-11-12 2019-07-02 Xencor, Inc. Fc variants with altered binding to FcRn
US8318907B2 (en) 2004-11-12 2012-11-27 Xencor, Inc. Fc variants with altered binding to FcRn
US8324351B2 (en) 2004-11-12 2012-12-04 Xencor, Inc. Fc variants with altered binding to FcRn
US7855276B2 (en) 2004-12-30 2010-12-21 The United States Of America As Represented By The Department Of Health And Human Services Framework residue substituted humanized COL-1 antibodies and their use
WO2006074071A1 (fr) * 2004-12-30 2006-07-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Anticorps col-1 humanises a residus charpente substitues et utilisation
US8828717B2 (en) 2004-12-30 2014-09-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Framework residue substituted humanized COL-1 antibodies and their use
US9040041B2 (en) 2005-10-03 2015-05-26 Xencor, Inc. Modified FC molecules
US7973136B2 (en) 2005-10-06 2011-07-05 Xencor, Inc. Optimized anti-CD30 antibodies
US9574006B2 (en) 2005-10-06 2017-02-21 Xencor, Inc. Optimized anti-CD30 antibodies
WO2007090596A1 (fr) * 2006-02-06 2007-08-16 Medizinische Universität Wien Vaccin et mimotopes d'antigènes dirigés contre les maladies cancéreuses associées à l'antigène
US9803020B2 (en) 2006-08-14 2017-10-31 Xencor, Inc. Optimized antibodies that target CD19
US11618788B2 (en) 2006-08-14 2023-04-04 Xencor, Inc. Optimized antibodies that target CD19
US10626182B2 (en) 2006-08-14 2020-04-21 Xencor, Inc. Optimized antibodies that target CD19
US8524867B2 (en) 2006-08-14 2013-09-03 Xencor, Inc. Optimized antibodies that target CD19
US9040042B2 (en) 2006-09-18 2015-05-26 Xencor, Inc. Optimized antibodies that target HM1.24
US8394374B2 (en) 2006-09-18 2013-03-12 Xencor, Inc. Optimized antibodies that target HM1.24
US11932685B2 (en) 2007-10-31 2024-03-19 Xencor, Inc. Fc variants with altered binding to FcRn
US11401348B2 (en) 2009-09-02 2022-08-02 Xencor, Inc. Heterodimeric Fc variants

Also Published As

Publication number Publication date
WO2004022717A3 (fr) 2004-09-02
AU2003270369A1 (en) 2004-03-29
AU2003270369A8 (en) 2004-03-29

Similar Documents

Publication Publication Date Title
WO2004022717A2 (fr) Variants tres faiblement immunogenes de l'anticorps humanise col-1 contre l'antigene carcino-embryonnaire
US7919607B2 (en) Nucleic acids encoding humanized anti-tag 72 CC49 antbodies
EP2215121B1 (fr) Anticorps anti-mésothéline et leurs utilisations
US8828717B2 (en) Framework residue substituted humanized COL-1 antibodies and their use
EP2612869B1 (fr) Antigène ED-A du fibrinogène associé aux lymphomes
WO2005112564A2 (fr) Régions variables d'anticorps humanisés et lignée germinale et méthodes de fabrication et d'utilisation
US7915396B2 (en) Nucleic acid molecules encoding minimally immunogenic variants of SDR-grafted humanized antibody CC49
US10202442B2 (en) Antigen associated with lung cancers and lymphomas

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP