US20070154477A1 - Humanized anti-CD3 specific antibodies - Google Patents

Humanized anti-CD3 specific antibodies Download PDF

Info

Publication number
US20070154477A1
US20070154477A1 US11/704,940 US70494007A US2007154477A1 US 20070154477 A1 US20070154477 A1 US 20070154477A1 US 70494007 A US70494007 A US 70494007A US 2007154477 A1 US2007154477 A1 US 2007154477A1
Authority
US
United States
Prior art keywords
ser
gly
thr
val
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/704,940
Inventor
Sarah Bolt
Michael Clark
Scott Gorman
Edward Routledge
Herman Waldman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BTG International Ltd
Original Assignee
BTG International Ltd
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 BTG International Ltd filed Critical BTG International Ltd
Priority to US11/704,940 priority Critical patent/US20070154477A1/en
Publication of US20070154477A1 publication Critical patent/US20070154477A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/06Immunosuppressants, e.g. drugs for graft rejection
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/40Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41Glycosylation, sialylation, or fucosylation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74Inducing cell proliferation

Definitions

  • This invention relates to novel antibodies, in particular to antibodies directed against the CD3 antigen complex.
  • Antibodies, or immunoglobulins comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds in a “Y” shaped configuration.
  • the two “arms” of the antibody are responsible for antigen binding, and include regions where the polypeptide structure varies, these “arms” being termed Fab′ fragments (fragment-antigen-binding) or F(ab′) 2 which represents two Fab′ arms linked together by disulphide bonds.
  • the “tail” or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment (fragment-crystalline).
  • Each heavy chain has at one end a variable domain followed by a number of constant domains.
  • Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CH1).
  • the constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
  • the light chain constant domain and the CH1 domain of the heavy chain account for 50% of each Fab′ fragment.
  • variable domains of each pair of light and heavy chains form the antigen binding site.
  • the domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three complementarity-determining regions (CDRS) (Kabat et al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1987)).
  • CDRS complementarity-determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
  • the human CD3 antigen consists of a minimum of four invariant polypeptide chains, which are non-covalently associated with the T-cell receptors on the surface of T-cells, and is generally now referred to as the CD3 antigen complex. It is intimately involved in the process of T-cell activation in response to antigen recognition by the T-cell receptors.
  • CD3 monoclonal antibodies can be used to sensitise T-cells to secondary proliferative stimuli such as IL1 (interleukin 1) and IL2 (interleukin 2).
  • certain CD3 monoclonal antibodies are themselves mitogenic for T-cells. This property is isotype dependent and results from the interaction of the CD3 antibody Fc domain with Fc receptors on the surface of accessory cells.
  • Rodent CD3 antibodies have been used to influence immunological status by suppressing, enhancing or re-directing T-cell responses to antigens. They therefore have considerable therapeutic potential in the human for use as an immunosuppressive agent, for example for the treatment of rejection episodes following the transplantation of renal, hepatic and cardiac allografts. However their value is compromised by two main factors. The first is the antiglobulin response evoked due to the xenogeneic nature of the antibody. The second is the “first dose” syndrome experienced by patients following the initial administration of the antibody. The symptoms, which range in severity from fever and chills to pulmonary edema, and which in rare cases can cause death, are caused by the elevated levels of circulating cytokines associated with CD3-antibody induced T-cell activation. This phenomenon requires the cross-linking of the CD3 antigen on the surface of T-cells to accessory cells through Fc receptors; such proliferation does not occur with F(ab′) 2 fragments of CD3 antibodies.
  • the first problem can be addressed by re-shaping or “humanising” the variable region genes of antibodies and expressing them in association with relevant human constant domain genes. This reduces the non-human content of the monoclonal antibody to such a low level that an antiglobulin response is unlikely.
  • a reshaped antibody with a binding affinity for the CD3 antigen complex is described in UK Patent Application No. 9121126.8 (published as GB 2249310A) and its equivalents (European Patent Application No. 91917169.4, Japanese Patent Application No. 516117/91 and U.S. Patent Application No. 07/862543).
  • the invention provides an aglycosylated IgG antibody having a binding affinity for the CD3 antigen complex.
  • aglycosylated is employed in its normal usage to indicate that the antibodies according to the invention are not glycosylated.
  • the present invention can be applied to antibodies having a binding affinity for a non-human CD3 antigen complex, for example various other mammalian CD3 antigens for veterinary use, the primary value of the invention lies in aglycosylated antibodies having an affinity for the human CD3 antigen complex for use in the human and the following discussion is particularly directed to that context.
  • CD3 antigens are to be found in the report of the First International Workshop and Conference on Human Leukocyte Differentiation Antigens and description of various glycosylated antibodies directed against the CD3 antigen is also to be found in the reports of this series of Workshops and Conferences, particularly the Third and Fourth, published by Oxford University Press.
  • Specific examples of such antibodies include those described by Van Ller et al., Euro. J. Immunol., 1987, 17, 1599-1604, Alegre et al., J. Immunol., 1991, 140, 1184, and by Smith et al., ibid, 1986, 16, 478, the last publication relating to the IgG1 antibody UCHT1 and variants thereof.
  • the antibody OKT3 is discussed in publications such as Chatenaud et al., Transplantation, 1991, 51, 334 and the New England Journal of Medicine paper, 1985, 313, 339, and also in European Patent No. 0 018 795 and U.S. Pat. No. 4,361,539.
  • the antibody YTH 12.5.14.2 (hereinafter referred to as YTH 12.5) is discussed in publications such as Clark et al., European J. Immunol., 1989, 19, 381-388 and reshaped YTH 12.5 antibodies are the subject of UK Patent Application No. 9121126.8 and its equivalents, this application describing in detail the CDRs present in this antibody.
  • the antibodies of the invention preferably have at least one CDR selected from the amino acid sequences:
  • the CDRs are situated within framework regions of the heavy chain (for (a), (b) and (c)) and light chain (for (d), (e) and (f)) variable domains.
  • the antibody also comprises a constant domain.
  • the aglycosylated antibody has three CDRs corresponding to the amino acid sequences (a), (b) and (c) above or conservatively modified variants thereof and/or three CDRs corresponding to amino acid sequences (d), (e) and (f) or conservatively modified variants thereof, the heavy chain CDRs (a), (b) and (c) being of most importance.
  • a preferred aglycosylated antibody with a binding affinity for the CD3 antigen thus has a heavy chain with at least one CDR and particularly three CDRs selected from the amino acid sequences:
  • an aglycosylated antibody according to the invention contains preferred CDRs as described hereinbefore it conveniently contains both one or more of the specified heavy chain CDRs and one or more of the specified light chain CDRs.
  • the CDRs (a), (b) and (c) are arranged in the heavy chain in the sequence: framework region 1/(a)/framework region 2/(b)/framework region 3/(c)/framework region 4 in a leader ⁇ constant domain (n-terminal to C-terminal) direction and the CDRs (d), (e) and (f) are arranged in the light chain in the sequence: framework region 1/(d)/framework region 2/(e)/framework region 3/(f)/framework region 4 in a leader ⁇ constant domain direction.
  • the heavy chain CDRs are arranged in the sequence (a), (b), (c) in a leader ⁇ constant domain direction and the light chain CDRs are arranged in the sequence (d), (e), (f) in a leader ⁇ constant domain direction.
  • aglycosylated antibodies according to the invention may contain quite different CDRs from those described hereinbefore and that, even when this is not the case, it may be possible to have heavy chains and particularly light chains containing only one or two of the CDRs (a), (b) and (c) and (d), (e) and (f), respectively.
  • CDRs aglycosylated antibodies according to the present invention
  • all six CDRs will most usually be present in the most preferred antibodies.
  • a particularly preferred aglycosylated antibody therefore has a heavy chain with three CDRs comprising the amino acid sequences (a), (b) and (c) or conservatively modified variants thereof and a light chain with three CDRs comprising the amino acid sequences (d), (e) and (f) or conservatively modified variants thereof in which the heavy chain CDRs are arranged in the order (a), (b), (c) in the leader constant region direction and the light chain CDRs are arranged in the order (d), (e), (f) in the leader constant region direction.
  • the CDRs may be of different origin to the variable framework region and/or to the constant region and, since the CDRs will usually be of rat or mouse origin, this is advantageous to avoid an antiglobulin response in the human, although the invention does extend to antibodies with such regions of rat or mouse origin.
  • the CDRs are either of the same origin as the variable framework region but of a different origin from the constant region, for example in a part human chimaeric antibody, or, more commonly, the CDRs are of different origin from the variable framework region.
  • variable domain framework region can take various forms, it is conveniently of or derived from those of a rodent, for example a rat or mouse, and more preferably of or derived from those of human origin.
  • the antibody is conveniently of or derived from those of a rodent, for example a rat or mouse, and more preferably of or derived from those of human origin.
  • the antibody is preferably in the humanised form as regards both the variable domain framework region and as discussed further hereinafter, the constant region.
  • the invention further comprises an aglycosylated antibody which has a binding affinity for the human CD3 antigen and in which the variable domain framework regions and/or the constant region are of or are derived from those of human origin.
  • variable domain framework sequences will be preferable for the grafting of the preferred CDR sequences, since the 3-dimensional conformation of the CDRs will be better maintained in such sequences and the antibody will retain a high level of binding affinity for the antigen. Desirable characteristics in such variable domain frameworks are the presence of key amino acids which maintain the structure of the CDR loops in order to ensure the affinity and specificity of the antibody for the CD3 antigen, the lambda type being preferred for the light chain.
  • V region frameworks which are particularly suitable for use in conjunction with the above CORs have been previously identified in UK Patent Application No. 9121126.8.
  • the heavy chain variable (V) region frameworks are those coded for by the human VH type III gene VH26.D.J. which is from the B cell hybridoma cell line 18/2 (Genbank Code: Huminghat, Dersimonian et al., Journal of Immunology, 139, 2496-2501).
  • the light chain variable region frameworks are those of the human V L ⁇ type VI gene SUT (Swissprot code; LV6CSHum, Solomon et al. In Glenner et al (Eds), Amyloidosis, Plenum Press N.Y., 1986, p.449.
  • the one or more preferred CDRs of the heavy chain of the rat anti-CD3 antibody are therefore preferably present in a human variable domain framework which has the following amino acid sequence reading in the leader ⁇ constant region direction, CDR indicating a CDR (a), (b) or (c) as defined hereinbefore, a conservatively modified variant thereof or an alternative CDR:-Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly-Sly- Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Ser-/CDR/-Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/- Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-
  • the heavy chain variable region comprises the following sequence:— (SEQUENCE ID NO. 11) Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val- Gln-Pro-GLy-GLy-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala- Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Mer-Ala-Trp- Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val- Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr- Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg- Asp-Asn-Ser-L
  • the one or more preferred CDRs of the light chain of the rat CD3 antibody are therefore preferably present in a human variable domain framework which has the following amino acid sequence reading in the leader ⁇ constant region direction, CDR indicating a CDR (d), (e) and (f) as defined hereinbefore, a conservatively modified variant thereof or an alternative CDR:-Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys- Thr-Val-Ile-Ile-Ser-Cys-/CDR/-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-Ile-Phe-/CDR/-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser- Ile-Asp-Arg-Ser-Ser-Asn-Ser-Ala-S
  • the light chain variable region comprises the following sequence:— (SEQUENCE ID NO. 16) Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu- Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu- Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp- Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val- Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro- Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn- Ser-Ala-Ser-Leu
  • variable domains for example comprising one or more preferred CDRs as described above, preferably in the humanised form having human antibody-derived variable framework regions, are attached to appropriate constant domains.
  • the heavy and light chain constant regions can be based on antibodies of different types as desired subject to the antibody being an IgG antibody, but although they may be of or derived from those of rat or mouse origin they are preferably of or are derived from those of human origin.
  • the constant region is preferably of the lambda type and for the heavy chain it is preferably of an IgG isotype, especially IgG1, modified to effect aglycosylation as appropriate.
  • All human constant regions of the IgG isotype are known to be glycosylated at the asparagine residue at position 297, which makes up part of the N-glycosylation motif Asparagine 297 -X 298 -Serine 299 or Threonine 299 , where X is the residue of any amino acid except proline.
  • the antibody of the invention may thus be aglycosylated by the replacement of Asparagine 297 in such a constant region with another amino acid which cannot be glycosylated. Any other amino acid residue can potentially be used, but alanine is the most preferred.
  • glycosylation at Asparagine 297 can be prevented by altering one of the other residues of the motif, e.g.
  • the replacement of one amino acid in a CDR with another amino acid having similar properties may not substantially alter the properties or structure of the peptide or protein in which the substitution or substitutions were made.
  • the aglycosylated antibodies of the present invention include those antibodies containing the preferred CDRs but with a specified amino acid sequence in which such a substitution or substitutions have occurred without substantially altering the binding affinity and specificity of the CDRs.
  • deletions may be made in the amino acid residue sequence of the CDRs or the sequences may be extended at one or both of the N- and C-termini whilst still retaining activity.
  • Preferred aglycosylated antibodies according to the present invention are such that the affinity constant for the antigen is 10 5 mole ⁇ 1 or more, for example up to 10 12 mole ⁇ 1 .
  • Ligands of different affinities may be suitable for different uses so that, for example, an affinity of 10 6 , 10 7 or 10 8 mole ⁇ 1 or more may be appropriate in some cases. However antibodies with an affinity in the range of 10 6 to 10 8 mole ⁇ 1 will often be suitable. Conveniently the antibodies also do not exhibit any substantial binding affinity for other antigens. Binding affinities of the antibody and antibody specificity may be tested by assay procedures such as those described in the Examples section hereinafter, (Effector Cell Retargetting Assay), or by techniques such as ELISA and other immunoassays.
  • Antibodies according to the invention are aglycosylated IgG CD3 antibodies having a “Y” shaped configuration which may have two identical light and two identical heavy chains and are thus bivalent with each antigen binding site having an affinity for the CD3 antigen.
  • the invention is also applicable to antibodies in which only one of the arms of the antibody has a binding affinity for the CD3 antigen.
  • Such antibodies may take various forms.
  • the other arm of the antibody may have a binding affinity for an antigen other than CD3 so that the antibody is a bispecific antibody, for example as described in U.S. Pat. No. 4,474,893 and European Patent Applications Nos. 87907123.1 and 87907124.9.
  • the antibody may have only one arm which exhibits a binding affinity, such,an antibody being termed “monovalent”.
  • Monovalent antibodies may be prepared in a number of ways. Glennie and Stevenson (Nature, 295, 712-713, (1982)) describe a method of preparing monovalent antibodies by enzymic digestion. Stevenson et al. describe a second approach to monovalent antibody preparation in which-enzymatically produced Fab′ and Fc fragments are chemically cross-linked (Anticancer Drug Design, 3, 219-230 (1989)). In these methods the resulting monovalent antibodies have lost one of their Fab′ arms. A third method of preparing monovalent antibodies is described in European Patent No. 131424. In this approach the “Y” shape of the antibody is maintained, but only one of the two Fab′ domains will bind to the antigen. This is achieved by introducing into the hybridoma a gene coding for an irrelevant light chain which will combine with the heavy chain of the antibody to produce a mixture of products in which the monovalent antibody is the one of interest.
  • the monovalent aglycosylated CD3 antibodies of the invention are prepared by the following method. This involves the introduction into a suitable expression system, for example a cell system as described hereinafter, together with genes coding for the heavy and light chains, of a gene coding for a truncated heavy chain in which the variable region domain and first constant region domain of the heavy chain are absent, the gene lacking the exon for each of these domains.
  • a suitable expression system for example a cell system as described hereinafter
  • genes coding for the heavy and light chains of a gene coding for a truncated heavy chain in which the variable region domain and first constant region domain of the heavy chain are absent, the gene lacking the exon for each of these domains.
  • Such an antibody fragment (c) is monovalent since it has any only one Fab′ arm. Production of a monovalent antibody in the form of such a fragment by this method is preferred for a number of reasons. Thus, the resulting antibody fragment is easy to purify from a mixture of antibodies produced by the cell system since, for example, it may be separable simply on the basis of its molecular weight. This is not possible in the method of European Patent No. 131424 where the monovalent antibody produced has similar characteristics to a bivalent antibody in its size and outward appearance.
  • the production of a monovalent antibody fragment by the new method uses conditions which can more easily be controlled and is thus not as haphazard as an enzyme digestion/chemical coupling procedure which requires the separation of a complex reaction product, with the additional advantage that the cell line used will continue to produce monovalent antibody fragments, without the need for continuous synthesis procedures as required in the enzyme digestion/chemical coupling procedure.
  • aglycosylated antibodies according to the invention do not occur in nature and these aglycosylated antibodies may in general be produced synthetically in a number of ways. Most conveniently, however, appropriate gene constructs for the constant and variable regions of the heavy and light chains which are present in the antibody are separately obtained and then inserted in a suitable expression system.
  • Genes encoding the variable domains of a ligand of the desired structure may be produced and conveniently attached to genes encoding the constant domains of an antibody which have undergone site directed mutagenesis. These constant genes may be obtained from hybridoma cDNA or from the chromosomal DNA and have undergone mutagenesis (site directed) to produce the aglycosylated constant regions. Genes encoding the variable regions may also be derived by gene synthesis techniques used in the identification of the CDRs contained herein. Suitable cloning vehicles for the DNA may be of various types.
  • Expression of these genes through culture of a cell system to produce a functional CD3 ligand is most conveniently effected by transforming a suitable prokaryotic or particularly eukaryotic cell system, particularly an immortalised mammalian cell line such as a myeloma cell line, for example the YB2/3.01/Ag20 (hereinafter referred to as YO) rat myeloma cell, or Chinese hamster ovary cells (although the use of plant cells is also of interest), with expression vectors which include DNA coding for the various antibody regions, and then culturing the transformed cell system to produce the desired antibody.
  • a suitable prokaryotic or particularly eukaryotic cell system particularly an immortalised mammalian cell line such as a myeloma cell line, for example the YB2/3.01/Ag20 (hereinafter referred to as YO) rat myeloma cell, or Chinese hamster ovary cells (although the use of plant cells is also of interest),
  • the present invention thus includes a process for the preparation of an aglycosylated IgG antibody having a binding affinity for the CD3 antigen which comprises culturing cells capable of expressing the antibody in order to effect expression thereof.
  • the invention also includes a cell line which expresses an aglycosylated antibody according to the invention.
  • Preferred among such cell lines are those which comprise DNA sequences encoding the preferred CDRs described hereinbefore.
  • a group of nucleotide sequences coding for the CDRs (a) to (f) described hereinbefore is as indicated under (a) to (f) below, respectively, but it will be appreciated that the degeneracy of the genetic code permits variations to be made in these sequences whilst still encoding for the CDRs' amino acid sequences.
  • Such cell lines will particularly contain larger DNA sequences which comprise (1) DNA expressing human heavy chain variable framework regions and one or more of (a), (b) and (c), and (2) DNA expressing human light chain variable framework regions and one or more of (d), (e) and (f).
  • a specific example of such DNA is that sequence (1) indicated below which codes for the CDRs (a), (b) and (c) arranged in the heavy chain framework coded for by the human VH type III gene VH26.D.J. as discussed hereinbefore and that sequence (2) indicated below which codes for the CDRs (d), (e) and (f) arranged in the light chain framework coded for by the human V L ⁇ type VI gene SUT.
  • the cell lines will of course also particularly contain DNA sequences expressing the heavy and light chain constant regions.
  • the humanised aglycosylated antibodies in accordance with the invention have therapeutic value.
  • such aglycosylated antibodies especially a humanised aglycosylated antibody with a specificity for the human CD3 antigen, has valuable applications in immunosuppression, particularly in the control of graft rejection, where it is especially desirable that immunosuppression is temporary rather than total, and thus that T-cells are not completely destroyed, but instead rendered non-functional by antibody blockade of the CD3 antigen-TCR complex.
  • the aglycosylated CD3 antibodies may have potential in other areas such as in the treatment of cancer, specifically in the construction of bispecific antibodies (for effector cell retargetting) or antibody-toxin conjugates, where the efficacy of the therapeutic agent would be compromised by Fc-mediated killing of the effector cells or non-specific killing of Fc receptor bearing cells respectively.
  • the invention thus includes a method of treating patients with cancer, particularly a lymphoma, or for immunosuppression purposes, for instance in a case where graft rejection may occur, comprising administering a therapeutically effective amount of an aglycosylated antibody in accordance with the invention.
  • FIGS. 1 - 8 show the results of proliferation assays of peripheral blood lymphocytes to CD3 antibodies.
  • Four different healthy volunteers were used.
  • the humanised anti-lymphocyte antibody CDw52 was included as a negative control.
  • FIGS. 9 - 12 show the comparison of aglycosylated CD3 antibody and glycosylated CD3 antibody in a mixed lymphocyte reaction. Aglycosylated antibody specific for the mouse CD8 antigen was included as a negative control.
  • FIGS. 13 & 14 show the results of an Effector Cell Retargetting Assay comparing glycosylated and aglycosylated IgG-type CD3 antibodies.
  • the CDw52 antibody was used as a negative control.
  • YTH 12.5 is a rat hybridoma cell line secreting an IgG2b monoclonal antibody specific for the CD3 antigen complex.
  • the methodology was based on that of Orlandi et al., 1989, PNAS USA, 86, 3833, using the polymerase chain reaction (PCR).
  • the V H gene (heavy chain variable region gene) was cloned using oligonucleotide primers VH1FOR and VH1BACK.
  • the PCR products were ligated into the vector M13-VHPCR1 in which site directed mutagenesis was performed using 6 oligonucleotide primers.
  • the V L gene (light chain variable region gene) was cloned using primers designed based on the published V L ⁇ sequences.
  • the gene was cloned into the vector M13-VKPCR, together with the human lambda light chain constant region. In this vector mutagenesis of the V L framework was performed using 5 oligonucleotides.
  • the humanised V L gene was then inserted into the expression vector pH ⁇ Apr-1.
  • a vector was generated (p316) in which the reshaped CD3 VH gene could be expressed in conjunction with different immunoglobulin H chain constant region genes, this vector being based on the pH ⁇ Apr-gpt vector (Gunning et al., 1987, P.N.A.S. USA, 85, 7719-7723).
  • a 1.65 Kb fragment of DNA carrying the dihydrofolate reductase (dhft) gene and SV 40 expression signals was inserted into the unique EcoRI site of pH ⁇ Apr-gpt.
  • a 700 bp HindIII-BamHI DNA fragment encoding the reshaped CD3-VH gene was then cloned into the vector's multiple cloning site, downstream and under the control of the ⁇ actin promoter.
  • the desired H chain constant region gene (in genomic configuration) could then be inserted into the unique BamHl restriction enzyme site downstream of the CD3-VH gene.
  • the aglycosyl human IgG1 constant region was derived from the wild type Glm (1, 17) gene described by Takahashi et al., (1982, Cell, 29, 671-679) as follows. The gene was cloned into the vector M13 tg131 where site-directed mutagenesis was performed (Amersham International PLC) to mutate the amino acid residue at position 297 from an asparagine to an alanine residue.
  • Oligosaccharide at Asn-297 is a characteristic feature of all normal human IgG antibodies (Kabat et al., 1987, Sequence of Proteins of Immunological Interest, US Department of Health Human Services Publication), each of the two heavy chains in the IgG molecules having a single branched chain carbohydrate group which is linked to the amide group of the asparagine residue (Rademacher and Dwek, 1984, Prog. Immunol., 5, 95-112). Substitution of asparagine with alanine prevents the glycosylation of the antibody.
  • the 2.3 Kb aglycosyl IgG1 constant region was excised from M13 by double digestion using BamHI and BgIII and ligated into the BamHI site of vector p316 to produce clone p323.
  • Heavy and light chain transfectants were selected for in xanthine/hypoxanthine free IMOM containing 5% (v/v) dialysed foetal calf serum.
  • H-chain expression vectors carrying the non-mutant human IgG2 (Flanagan & Rabbitts, 1982, Nature 300, 709-713), IgG3 (Huck et al., 1986, Nuc. Acid. Res., 14, 1779-1789), IgG4 (Flanagan & Rabbitts, 1982, Nature 300, 709-713), Epsilon (Flanagan & Rabbitts, 1982, EMBO.
  • vectors p317, p318, p320, p321 and p325, respectively were derived from the vector p316.
  • Cells expressing CD3 antibodies were subjected to two rounds of cloning in soft agar, and then expanded into roller bottle cultures. The immunoglobulin from approximately 4 litres of tissue culture supernatant from each cell line was concentrated by ammonium sulphate precipitation, dialysed extensively against PBS and then quantified as follows:
  • a competition assay was designed to specifically quantitate the concentration of antibody with CD3 antigen binding capacity.
  • Human T-cell blasts were incubated with FITC labelled UCHT-1, an antibody which binds to the same epitope of the CD3 antigen as the chimaeric panel.
  • the concentration of FITC reagent used had previously been determined to be half saturating.
  • Unlabelled YTH 12.5 (HPLC purified) was titrated from a known starting concentration and added to wells containing T-cells and UCHT-1 FITC.
  • the unlabelled antibody serves as a competitor for the antigen binding site. This is detected as decrease in the mean fluorescence seen when the cells are studied using FACS analysis.
  • titration of the chimaeric antibodies from unknown starting concentrations yields a series of sigmoidal curves when mean fluorescence is plotted against antibody dilution. These can be directly compared with the standard YTH 12.5 curve.
  • the capacity of a CD3 antibody to support T-cell proliferation in solution is related to the interaction of the Fc region of the antibody with Fc receptors on accessory cells.
  • the aglycosylated chimaeric CD3 antibody prepared as described in Example 1 was compared with a panel of other chimaeric antibodies which shared the same variable region architecture but different H chain constant regions (see Example 1) for the ability to induce proliferation of human peripheral blood lymphocytes.
  • Lymphocytes isolated from healthy donors' blood were separated on a lymphopaque gradient, washed and resuspended in IMDM containing 5% (v/v) heat-inactivated human AB serum and plated at 5 ⁇ 10 4 to 1 ⁇ 10 5 cells per well in plates containing CD3 antibodies in solution.
  • the aglycosyl derivative of the ⁇ 1 monoclonal antibody was the only CD3 antibody which consistently failed to induce T-cell proliferation in any of the donors tested, giving responses equivalent to those of the non-activating control monoclonal antibody Campath-1H.
  • endotoxin contamination as the cause of the proliferation seen with the ⁇ 2 and ⁇ preparations, it was confirmed that proliferation could be blocked by the addition of an excess of the aglycosyl CD3 mAb thus implicating the CD3 antigen in the activation process.
  • Peripheral blood lymphocytes were-isolated from two blood donors.
  • the stimulator cell population was caesium irradiated.
  • the responder population was incubated with titrated antibody for 30 minutes before the irradiated stimulator cells were added ( FIGS. 9, 10 and 11 ).
  • Control wells of responder cells were also incubated with irradiated responder cells at each antibody condition, to determine the specific effect of the antibody on the responder cells ( FIGS. 9, 10 and 12 ).
  • the aglycosylated antibody does block the mixed lymphocyte reaction; as the antibody is titrated out the blockade effect is less and the proliferation increases.
  • the ‘wild type’ IgG1 actually has a mitogenic effect on the T-cells and so any blockade of the MLR is not seen through this response.
  • mice which were transgenic for the human CD3 epsilon subunit including the aglycosylated antibody of Example 1 (IgG1Ag).
  • IgG1Ag aglycosylated antibody of Example 1
  • TNF detected in serum of hCD3 mice following injection with chimaeric CD3 antibodies Prebleed 90 minute 4 hour
  • Sample TNF level TNF level TNF level TNF level Saline 0 units/ml 0 units/ml 0 units/ml IgG1 0 units/ml >400 units/ml 0 units/ml IgG2 0 units/ml >400 units/ml 0 units/ml IgG1Ag 0 units/ml 50 units/ml 0 units/ml IgE 0 units/ml 50 units/ml 25 units/ml YTH 12.5 0 units/ml 50 units/ml 0 units/ml
  • the aglycosylated form is associated with at least an eight-fold less release of TNF than the wild type IgG1 or with the IgG2 antibody.
  • Examples 2-4 show that the aglycosylated CD3 antibody was not mitogenic to T-cells in solution indicating that the antibody had a reduced capacity to interact with Fc receptors on accessory cells.
  • the antibody retained the immunosuppressive properties that are characteristic of CD3 antibodies.
  • the aglycosylated antibody led to a significantly lower release of tumour necrosis factor in human CD3 transgenic mice than the parental IgG1 antibody.
  • this agent may be an ‘improved’ CD3 antibody for the purposes of immunosuppression if the decreased TNF release seen in mice is mirrored in humans.
  • the U937 monocytic cell line expresses human Fc receptors and can be lysed by activated human T-cell blasts in the presence of CD3 monoclonal antibodies capable of cross-linking the two cell types.
  • the results show that when aglycosylated, the human IgG1 antibody of Example 1 is still able to cross-link T-cells to the U937 cells, albeit at a reduced level, and thus redirect T-cell cytotoxicity. This was a surprising finding since, given the published data, the effective killing mediated by aglycosyl ⁇ 1 monoclonal antibody was unexpected.
  • the ECR results indicate that the hierarchy of binding of the IgG chimaeric antibodies is ⁇ 2 ⁇ 3 ⁇ Ag ⁇ 1 ⁇ 4 ⁇ 1. If the assumption is made that the mitogenic activity of an antibody is predicted by its Fc receptor binding ability, then one would expect the above hierarchy to be displayed in the T cell proliferation assays. However, this was not the case; the order of activities in T cell proliferation experiments (1 to 3) was Ag ⁇ 1 ⁇ 2 ⁇ 4 ⁇ 3 ⁇ 1. This demonstrates that the mitogenicity of an antibody cannot be predicted in a straightforward fashion from the results of assays which measure Fc-Fc receptor interactions. This view is supported by the behaviour of the epsilon chimaeric antibody which performed poorly in the ECR assay and yet consistently had the highest mitogenic activity. This suggests that antibodies can activate T cells by binding to something other than Fcy receptors (as displayed on U937 cells) on accessory cells, i.e. an inability to bind to Fcy receptors is no guarantee that an antibody will not be mitogenic.

Abstract

Novel aglycosylated antibodies having a binding affinity for the CD3 antigen complex are of value for use in therapy, particularly in immunosuppression.

Description

  • This invention relates to novel antibodies, in particular to antibodies directed against the CD3 antigen complex.
  • Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds in a “Y” shaped configuration. The two “arms” of the antibody are responsible for antigen binding, and include regions where the polypeptide structure varies, these “arms” being termed Fab′ fragments (fragment-antigen-binding) or F(ab′)2 which represents two Fab′ arms linked together by disulphide bonds. The “tail” or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment (fragment-crystalline). The production of monoclonal antibodies was first disclosed by Kohler and Milstein (Kohler & Milstein, Nature, 256, 495-497 (1975)), Such monoclonal antibodies have found widespread use as diagnostic agents and also in therapy.
  • Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CH1). The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen. The light chain constant domain and the CH1 domain of the heavy chain account for 50% of each Fab′ fragment.
  • The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three complementarity-determining regions (CDRS) (Kabat et al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1987)). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
  • The human CD3 antigen consists of a minimum of four invariant polypeptide chains, which are non-covalently associated with the T-cell receptors on the surface of T-cells, and is generally now referred to as the CD3 antigen complex. It is intimately involved in the process of T-cell activation in response to antigen recognition by the T-cell receptors.
  • All CD3 monoclonal antibodies can be used to sensitise T-cells to secondary proliferative stimuli such as IL1 (interleukin 1) and IL2 (interleukin 2). In addition, certain CD3 monoclonal antibodies are themselves mitogenic for T-cells. This property is isotype dependent and results from the interaction of the CD3 antibody Fc domain with Fc receptors on the surface of accessory cells.
  • Rodent CD3 antibodies have been used to influence immunological status by suppressing, enhancing or re-directing T-cell responses to antigens. They therefore have considerable therapeutic potential in the human for use as an immunosuppressive agent, for example for the treatment of rejection episodes following the transplantation of renal, hepatic and cardiac allografts. However their value is compromised by two main factors. The first is the antiglobulin response evoked due to the xenogeneic nature of the antibody. The second is the “first dose” syndrome experienced by patients following the initial administration of the antibody. The symptoms, which range in severity from fever and chills to pulmonary edema, and which in rare cases can cause death, are caused by the elevated levels of circulating cytokines associated with CD3-antibody induced T-cell activation. This phenomenon requires the cross-linking of the CD3 antigen on the surface of T-cells to accessory cells through Fc receptors; such proliferation does not occur with F(ab′)2 fragments of CD3 antibodies.
  • The first problem can be addressed by re-shaping or “humanising” the variable region genes of antibodies and expressing them in association with relevant human constant domain genes. This reduces the non-human content of the monoclonal antibody to such a low level that an antiglobulin response is unlikely. Such a reshaped antibody with a binding affinity for the CD3 antigen complex is described in UK Patent Application No. 9121126.8 (published as GB 2249310A) and its equivalents (European Patent Application No. 91917169.4, Japanese Patent Application No. 516117/91 and U.S. Patent Application No. 07/862543).
  • There remains however the problem of the first dose response when these antibodies are used in therapy. Aglycosylation of antibodies has been described to reduce their ability to bind to Fc receptors in vitro in some cases. However, it is not predictable that this will be true of all antibodies, particularly in vivo, and aglycosylation may result in the introduction into the antibody of novel and unpredictable properties including novel Fc binding characteristics causing other undesirable effects. It is also possible that other undesirable properties not associated with Fc binding may be introduced to the antibody.
  • Moreover, it is of course of vital importance that aglycosylation is not accompanied by the loss of certain desirable features of Fc binding in addition to the loss of the undesirable features such as those attributable to the first dose response.
  • It has now been found, however, that it is possible to produce aglycosylated CD3 antibodies of the IgG subclass which surprisingly retain their antigen binding specificity and immunosuppressive properties and yet do not induce T cell mitogenesis in vitro and induce a reduced level of cytokine release in vivo, whilst still maintaining some Fc binding ability.
  • Accordingly, the invention provides an aglycosylated IgG antibody having a binding affinity for the CD3 antigen complex.
  • The term aglycosylated is employed in its normal usage to indicate that the antibodies according to the invention are not glycosylated. Although the present invention can be applied to antibodies having a binding affinity for a non-human CD3 antigen complex, for example various other mammalian CD3 antigens for veterinary use, the primary value of the invention lies in aglycosylated antibodies having an affinity for the human CD3 antigen complex for use in the human and the following discussion is particularly directed to that context.
  • Further discussion of CD3 antigens is to be found in the report of the First International Workshop and Conference on Human Leukocyte Differentiation Antigens and description of various glycosylated antibodies directed against the CD3 antigen is also to be found in the reports of this series of Workshops and Conferences, particularly the Third and Fourth, published by Oxford University Press. Specific examples of such antibodies include those described by Van Ller et al., Euro. J. Immunol., 1987, 17, 1599-1604, Alegre et al., J. Immunol., 1991, 140, 1184, and by Smith et al., ibid, 1986, 16, 478, the last publication relating to the IgG1 antibody UCHT1 and variants thereof. However, of particular interest as the basis for aglycosylated antibodies according to the present invention are the CORs contained in the antibodies OKT3 and YTH 12.5.14.2. The antibody OKT3 is discussed in publications such as Chatenaud et al., Transplantation, 1991, 51, 334 and the New England Journal of Medicine paper, 1985, 313, 339, and also in European Patent No. 0 018 795 and U.S. Pat. No. 4,361,539. The antibody YTH 12.5.14.2 (hereinafter referred to as YTH 12.5) is discussed in publications such as Clark et al., European J. Immunol., 1989, 19, 381-388 and reshaped YTH 12.5 antibodies are the subject of UK Patent Application No. 9121126.8 and its equivalents, this application describing in detail the CDRs present in this antibody.
  • Aglycosylated antibodies containing one or more of the CORs described in the above application are of particular interest. Thus the antibodies of the invention preferably have at least one CDR selected from the amino acid sequences:
    • (a) Ser-Phe-Pro-Met-Ala (SEQUENCE ID NO. 1),
    • (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-Arg-Asp-Ser-Val-Lys-Gly (SEQUENCE ID NO. 2),
    • (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr (SEQUENCE ID NO. 3),
    • (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His (SEQUENCE ID NO. 4),
    • (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp (SEQUENCE ID NO. 5),
    • (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val (SEQUENCE ID NO. 6),
    • and conservatively modified variants thereof.
  • The term “conservatively modified variants” is one well known in the art and indicates variants containing changes which are substantially without effect on antibody-antigen affinity.
  • The CDRs are situated within framework regions of the heavy chain (for (a), (b) and (c)) and light chain (for (d), (e) and (f)) variable domains. The antibody also comprises a constant domain.
  • In a preferred embodiment the aglycosylated antibody has three CDRs corresponding to the amino acid sequences (a), (b) and (c) above or conservatively modified variants thereof and/or three CDRs corresponding to amino acid sequences (d), (e) and (f) or conservatively modified variants thereof, the heavy chain CDRs (a), (b) and (c) being of most importance.
  • A preferred aglycosylated antibody with a binding affinity for the CD3 antigen thus has a heavy chain with at least one CDR and particularly three CDRs selected from the amino acid sequences:
    • (a) Ser-Phe-Pro-Met-Ala (SEQUENCE ID NO. 1),
    • (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-Arg-Asp-Ser-Val-Lys-Gly (SEQUENCE ID NO. 2),
    • (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr (SEQUENCE ID NO. 3),
    • and conservatively modified variants thereof, and/or a light chain with at least one CDR and particularly three CDRs selected from the amino acid sequences:
    • (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His (SEQUENCE ID NO. 4),
    • (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp (SEQUENCE ID NO. 5),
    • (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val (SEQUENCE ID NO. 6),
    • and conservatively modified variants thereof.
  • Where an aglycosylated antibody according to the invention contains preferred CDRs as described hereinbefore it conveniently contains both one or more of the specified heavy chain CDRs and one or more of the specified light chain CDRs. The CDRs (a), (b) and (c) are arranged in the heavy chain in the sequence: framework region 1/(a)/framework region 2/(b)/framework region 3/(c)/framework region 4 in a leader→constant domain (n-terminal to C-terminal) direction and the CDRs (d), (e) and (f) are arranged in the light chain in the sequence: framework region 1/(d)/framework region 2/(e)/framework region 3/(f)/framework region 4 in a leader→constant domain direction. It is preferred, therefore, that where all three are present the heavy chain CDRs are arranged in the sequence (a), (b), (c) in a leader→constant domain direction and the light chain CDRs are arranged in the sequence (d), (e), (f) in a leader→constant domain direction.
  • It should be appreciated however, that aglycosylated antibodies according to the invention may contain quite different CDRs from those described hereinbefore and that, even when this is not the case, it may be possible to have heavy chains and particularly light chains containing only one or two of the CDRs (a), (b) and (c) and (d), (e) and (f), respectively. However, although the presence of all six CDRs defined above is therefore not necessarily required in an aglycosylated antibody according to the present invention, all six CDRs will most usually be present in the most preferred antibodies. A particularly preferred aglycosylated antibody therefore has a heavy chain with three CDRs comprising the amino acid sequences (a), (b) and (c) or conservatively modified variants thereof and a light chain with three CDRs comprising the amino acid sequences (d), (e) and (f) or conservatively modified variants thereof in which the heavy chain CDRs are arranged in the order (a), (b), (c) in the leader constant region direction and the light chain CDRs are arranged in the order (d), (e), (f) in the leader constant region direction.
  • The CDRs may be of different origin to the variable framework region and/or to the constant region and, since the CDRs will usually be of rat or mouse origin, this is advantageous to avoid an antiglobulin response in the human, although the invention does extend to antibodies with such regions of rat or mouse origin.
  • More usually the CDRs are either of the same origin as the variable framework region but of a different origin from the constant region, for example in a part human chimaeric antibody, or, more commonly, the CDRs are of different origin from the variable framework region.
  • The preferred CDRs discussed hereinbefore are obtained from a rat CD3 antibody. Accordingly, although the variable domain framework region can take various forms, it is conveniently of or derived from those of a rodent, for example a rat or mouse, and more preferably of or derived from those of human origin. One possibility is for the antibody to have a variable domain framework region corresponding to that in the YTH12.5 hybridoma although the constant region will still preferably be of or derived from those of human origin. However the antibody of the invention is preferably in the humanised form as regards both the variable domain framework region and as discussed further hereinafter, the constant region.
  • Accordingly, the invention further comprises an aglycosylated antibody which has a binding affinity for the human CD3 antigen and in which the variable domain framework regions and/or the constant region are of or are derived from those of human origin.
  • Certain human variable domain framework sequences will be preferable for the grafting of the preferred CDR sequences, since the 3-dimensional conformation of the CDRs will be better maintained in such sequences and the antibody will retain a high level of binding affinity for the antigen. Desirable characteristics in such variable domain frameworks are the presence of key amino acids which maintain the structure of the CDR loops in order to ensure the affinity and specificity of the antibody for the CD3 antigen, the lambda type being preferred for the light chain.
  • Human variable region frameworks which are particularly suitable for use in conjunction with the above CORs have been previously identified in UK Patent Application No. 9121126.8. The heavy chain variable (V) region frameworks are those coded for by the human VH type III gene VH26.D.J. which is from the B cell hybridoma cell line 18/2 (Genbank Code: Huminghat, Dersimonian et al., Journal of Immunology, 139, 2496-2501). The light chain variable region frameworks are those of the human VLλ type VI gene SUT (Swissprot code; LV6CSHum, Solomon et al. In Glenner et al (Eds), Amyloidosis, Plenum Press N.Y., 1986, p.449.
  • The one or more preferred CDRs of the heavy chain of the rat anti-CD3 antibody are therefore preferably present in a human variable domain framework which has the following amino acid sequence reading in the leader→constant region direction, CDR indicating a CDR (a), (b) or (c) as defined hereinbefore, a conservatively modified variant thereof or an alternative CDR:-Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly- Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Ser-/CDR/-Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/- Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-Lys- /CDR/-Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser (SEQUENCE ID NO. 7/CDR/SEQUENCE ID NO. 8/CDR/SEQUENCE ID NO. 9/CDR/SEQUENCE ID NO. 10).
  • In an aglycosylated antibody containing all three preferred CDRs, the heavy chain variable region comprises the following sequence:—
    (SEQUENCE ID NO. 11)
    Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-
    Gln-Pro-GLy-GLy-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-
    Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Mer-Ala-Trp-
    Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-
    Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
    Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-
    Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-
    Ser-Leu-Arg-ALa-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-
    Ala-Lys-Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr-
    Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser.
  • Similarly, the one or more preferred CDRs of the light chain of the rat CD3 antibody are therefore preferably present in a human variable domain framework which has the following amino acid sequence reading in the leader→constant region direction, CDR indicating a CDR (d), (e) and (f) as defined hereinbefore, a conservatively modified variant thereof or an alternative CDR:-Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys- Thr-Val-Ile-Ile-Ser-Cys-/CDR/-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-Ile-Phe-/CDR/-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser- Ile-Asp-Arg-Ser-Ser-Asn-Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-/CDR/-Phe-Gly-Gly-Gly-Thr-Lys- Leu-Thr-Val-Leu (SEQUENCE ID NO. 12/CDR/SEQUENCE ID NO. 13/CDR/SEQUENCE ID NO. 14/CDR/SEQUENCE ID NO. 15).
  • In an aglycosylated antibody containing all three preferred CDRs.the light chain variable region comprises the following sequence:—
    (SEQUENCE ID NO. 16)
    Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-
    Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu-
    Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp-
    Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-
    Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro-
    Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn-
    Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-
    Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-His-Ser-Tyr-Val-Ser-
    Ser-Phe-Asn-Val-Phe-GLy-GLy-Gly-Thr-Lys-Leu-Thr-
    Val-Leu
  • The variable domains, for example comprising one or more preferred CDRs as described above, preferably in the humanised form having human antibody-derived variable framework regions, are attached to appropriate constant domains.
  • The heavy and light chain constant regions can be based on antibodies of different types as desired subject to the antibody being an IgG antibody, but although they may be of or derived from those of rat or mouse origin they are preferably of or are derived from those of human origin. For the light chain the constant region is preferably of the lambda type and for the heavy chain it is preferably of an IgG isotype, especially IgG1, modified to effect aglycosylation as appropriate. All human constant regions of the IgG isotype are known to be glycosylated at the asparagine residue at position 297, which makes up part of the N-glycosylation motif Asparagine297-X298-Serine299 or Threonine299, where X is the residue of any amino acid except proline. The antibody of the invention may thus be aglycosylated by the replacement of Asparagine297 in such a constant region with another amino acid which cannot be glycosylated. Any other amino acid residue can potentially be used, but alanine is the most preferred. Alternatively, glycosylation at Asparagine297 can be prevented by altering one of the other residues of the motif, e.g. by replacing residue 298 by proline, or residue 299 by any amino acid other than serine or threonine. Techniques for performing this site directed mutagenesis are well known to those skilled in the art and may for example be performed using a site directed mutagenesis kit such, for example, as that commercially available from Amersham. The procedure is further exemplified hereinafter.
  • It is well recognised in the art that the replacement of one amino acid in a CDR with another amino acid having similar properties, for example the replacement of a glutamic acid residue with an aspartic acid residue, may not substantially alter the properties or structure of the peptide or protein in which the substitution or substitutions were made. Thus, the aglycosylated antibodies of the present invention include those antibodies containing the preferred CDRs but with a specified amino acid sequence in which such a substitution or substitutions have occurred without substantially altering the binding affinity and specificity of the CDRs. Alternatively, deletions may be made in the amino acid residue sequence of the CDRs or the sequences may be extended at one or both of the N- and C-termini whilst still retaining activity.
  • Preferred aglycosylated antibodies according to the present invention are such that the affinity constant for the antigen is 105 mole−1 or more, for example up to 1012 mole−1. Ligands of different affinities may be suitable for different uses so that, for example, an affinity of 106, 107 or 108 mole−1 or more may be appropriate in some cases. However antibodies with an affinity in the range of 106 to 108 mole−1 will often be suitable. Conveniently the antibodies also do not exhibit any substantial binding affinity for other antigens. Binding affinities of the antibody and antibody specificity may be tested by assay procedures such as those described in the Examples section hereinafter, (Effector Cell Retargetting Assay), or by techniques such as ELISA and other immunoassays.
  • Antibodies according to the invention are aglycosylated IgG CD3 antibodies having a “Y” shaped configuration which may have two identical light and two identical heavy chains and are thus bivalent with each antigen binding site having an affinity for the CD3 antigen. Alternatively, the invention is also applicable to antibodies in which only one of the arms of the antibody has a binding affinity for the CD3 antigen. Such antibodies may take various forms. Thus the other arm of the antibody may have a binding affinity for an antigen other than CD3 so that the antibody is a bispecific antibody, for example as described in U.S. Pat. No. 4,474,893 and European Patent Applications Nos. 87907123.1 and 87907124.9. Alternatively, the antibody may have only one arm which exhibits a binding affinity, such,an antibody being termed “monovalent”.
  • Monovalent antibodies (or antibody fragments) may be prepared in a number of ways. Glennie and Stevenson (Nature, 295, 712-713, (1982)) describe a method of preparing monovalent antibodies by enzymic digestion. Stevenson et al. describe a second approach to monovalent antibody preparation in which-enzymatically produced Fab′ and Fc fragments are chemically cross-linked (Anticancer Drug Design, 3, 219-230 (1989)). In these methods the resulting monovalent antibodies have lost one of their Fab′ arms. A third method of preparing monovalent antibodies is described in European Patent No. 131424. In this approach the “Y” shape of the antibody is maintained, but only one of the two Fab′ domains will bind to the antigen. This is achieved by introducing into the hybridoma a gene coding for an irrelevant light chain which will combine with the heavy chain of the antibody to produce a mixture of products in which the monovalent antibody is the one of interest.
  • More preferably, however, the monovalent aglycosylated CD3 antibodies of the invention are prepared by the following method. This involves the introduction into a suitable expression system, for example a cell system as described hereinafter, together with genes coding for the heavy and light chains, of a gene coding for a truncated heavy chain in which the variable region domain and first constant region domain of the heavy chain are absent, the gene lacking the exon for each of these domains. This results in the production by the cell system of a mixture of (a) antibodies which are complete bivalent antibodies, (b) antibody fragments consisting only of two truncated heavy chains (i.e. an Fc fragment) and (c) fragments of antibody which are monovalent for the CD3 antigen, consisting of a truncated heavy chain and a light chain in association with the normal heavy chain. Such an antibody fragment (c) is monovalent since it has any only one Fab′ arm. Production of a monovalent antibody in the form of such a fragment by this method is preferred for a number of reasons. Thus, the resulting antibody fragment is easy to purify from a mixture of antibodies produced by the cell system since, for example, it may be separable simply on the basis of its molecular weight. This is not possible in the method of European Patent No. 131424 where the monovalent antibody produced has similar characteristics to a bivalent antibody in its size and outward appearance. Additionally, the production of a monovalent antibody fragment by the new method uses conditions which can more easily be controlled and is thus not as haphazard as an enzyme digestion/chemical coupling procedure which requires the separation of a complex reaction product, with the additional advantage that the cell line used will continue to produce monovalent antibody fragments, without the need for continuous synthesis procedures as required in the enzyme digestion/chemical coupling procedure.
  • It is believed that aglycosylated antibodies according to the invention do not occur in nature and these aglycosylated antibodies may in general be produced synthetically in a number of ways. Most conveniently, however, appropriate gene constructs for the constant and variable regions of the heavy and light chains which are present in the antibody are separately obtained and then inserted in a suitable expression system.
  • Genes encoding the variable domains of a ligand of the desired structure may be produced and conveniently attached to genes encoding the constant domains of an antibody which have undergone site directed mutagenesis. These constant genes may be obtained from hybridoma cDNA or from the chromosomal DNA and have undergone mutagenesis (site directed) to produce the aglycosylated constant regions. Genes encoding the variable regions may also be derived by gene synthesis techniques used in the identification of the CDRs contained herein. Suitable cloning vehicles for the DNA may be of various types.
  • Expression of these genes through culture of a cell system to produce a functional CD3 ligand is most conveniently effected by transforming a suitable prokaryotic or particularly eukaryotic cell system, particularly an immortalised mammalian cell line such as a myeloma cell line, for example the YB2/3.01/Ag20 (hereinafter referred to as YO) rat myeloma cell, or Chinese hamster ovary cells (although the use of plant cells is also of interest), with expression vectors which include DNA coding for the various antibody regions, and then culturing the transformed cell system to produce the desired antibody. Such general techniques of use for the manufacture of ligands according to the present invention are well known in the very considerable art of genetic engineering and are described in publications such as “Molecular Cloning” by Sambrook, Fritsch and Maniatis, Cold Spring Harbour Laboratory Press, 1989 (2nd edition). The techniques are further illustrated by the Examples contained herein.
  • The present invention thus includes a process for the preparation of an aglycosylated IgG antibody having a binding affinity for the CD3 antigen which comprises culturing cells capable of expressing the antibody in order to effect expression thereof. The invention also includes a cell line which expresses an aglycosylated antibody according to the invention.
  • Preferred among such cell lines are those which comprise DNA sequences encoding the preferred CDRs described hereinbefore. A group of nucleotide sequences coding for the CDRs (a) to (f) described hereinbefore is as indicated under (a) to (f) below, respectively, but it will be appreciated that the degeneracy of the genetic code permits variations to be made in these sequences whilst still encoding for the CDRs' amino acid sequences.
    (SEQUENCE ID NO. 17)
    (a) AGCTTTCCAA TGGCC
    (SEQUENCE ID NO. 18)
    (b) ACCATTAGTA CTAGTGGTGG TAGAACTTAC TATCGAGACT
    CCGTGAAGGG C
    (SEQUENCE ID NO. 19)
    (c) TTTCGGCAGT ACAGTGGTGG CTTTGATTAC
    (SEQUENCE ID NO. 20)
    (d) ACACTCAGCT CTGGTAACAT AGAAAACAAC TATGTGCAC
    (SEQUENCE ID NO. 21)
    (e) GATGATGATA AGAGACCGGA T
    (SEQUENCE ID NO. 22)
    (f) CATTCTTATG TTAGTAGTTT TAATGTT
  • Such cell lines will particularly contain larger DNA sequences which comprise (1) DNA expressing human heavy chain variable framework regions and one or more of (a), (b) and (c), and (2) DNA expressing human light chain variable framework regions and one or more of (d), (e) and (f). A specific example of such DNA is that sequence (1) indicated below which codes for the CDRs (a), (b) and (c) arranged in the heavy chain framework coded for by the human VH type III gene VH26.D.J. as discussed hereinbefore and that sequence (2) indicated below which codes for the CDRs (d), (e) and (f) arranged in the light chain framework coded for by the human VLλ type VI gene SUT. The CDR sequences (a), (b), (c), (d), (e) and (f) have been underlined.
    (SEQUENCE ID NO. 23)
    (1) GAGGTCCAAC TGCTGGAGTC TGGGGGCGGT TTAGTGCAGC
    CTGGAGGGTC CCTGAGACTC TCCTGTGCAG CCTCAGGATT
    CACTTTCAGT AGCTTTCCAA TGGCCTGGGT CCGCCAGGCT
    CCAGGGAAGG GTCTGGAGTG GGTCTCAACC ATTAGTACTA
    GTGGTGGTAG AACTTACTAT CGAGACTCCG TGAAGGGCCG
    ATTCACTATC TCCAGAGATA ATAGCAAAAA TACCCTATAC
    CTGCAAATGA ATAGTCTGAG GGCTGAGGAC ACGGCCGTCT
    ATTACTGTGC AAAATTTCGG CAGTACAGTG GTGGCTTTGA
    TTACTGGGGC CAAGGGACCC TGGTCACCGT CTCCTCA
    (SEQUENCE ID NO. 24)
    (2) GACTTCATGC TGACTCAGCC CCACTCTGTG TCTGAGTCTC
    CCGGAAAGAC AGTCATTATT TCTTGCACAC TCAGCTCTGG
    TAACATAGAA AACAACTATG TGCACTGGTA CCAGCAAAGG
    CCGGGAAGAG CTCCCACCAC TGTGATTTTC GATGATGATA
    AGAGACCGGA TGGTGTCCCT GACAGGTTCT CTGGCTCCAT
    TGACAGGTCT TCCAACTCAG CCTCCCTGAC AATCAGTGGT
    CTGCAAACTG AAGATGAAGC TGACTACTAC TGTCATTCTT
    ATGTTAGTAG TTTTAATGTT TTCGGCGGTG GAACAAAGCT
    CACTGTCCTT
  • The cell lines will of course also particularly contain DNA sequences expressing the heavy and light chain constant regions.
  • The humanised aglycosylated antibodies in accordance with the invention have therapeutic value. In particular, such aglycosylated antibodies, especially a humanised aglycosylated antibody with a specificity for the human CD3 antigen, has valuable applications in immunosuppression, particularly in the control of graft rejection, where it is especially desirable that immunosuppression is temporary rather than total, and thus that T-cells are not completely destroyed, but instead rendered non-functional by antibody blockade of the CD3 antigen-TCR complex. In addition, the aglycosylated CD3 antibodies may have potential in other areas such as in the treatment of cancer, specifically in the construction of bispecific antibodies (for effector cell retargetting) or antibody-toxin conjugates, where the efficacy of the therapeutic agent would be compromised by Fc-mediated killing of the effector cells or non-specific killing of Fc receptor bearing cells respectively.
  • In a further aspect, the invention thus includes a method of treating patients with cancer, particularly a lymphoma, or for immunosuppression purposes, for instance in a case where graft rejection may occur, comprising administering a therapeutically effective amount of an aglycosylated antibody in accordance with the invention.
  • Aglycosylated antibodies in accordance with the invention may be formulated for administration to patients by administering the said antibody together with a physiologically acceptable diluent or carrier. The antibodies are preferably administered in an injectable form together with such a diluent or carrier which is sterile and pyrogen free. By way of guidance it may be stated that a suitable dose of antibody is about 1-10 mg injected daily over a time period of, for example 10 days, although due to the elimination of the first dose response it will be possible if desired to adminster higher amounts of the antibody, for example even up to 100 mg daily, depending on the.individual patient's needs. Veterinary use is on a similar g/kg dosage basis.
  • The invention is illustrated by the following Examples which are illustrated by the drawings listed below:—
  • FIGS. 1-8: These figures show the results of proliferation assays of peripheral blood lymphocytes to CD3 antibodies. Four different healthy volunteers were used. The humanised anti-lymphocyte antibody CDw52 was included as a negative control.
  • FIGS. 9-12: These figures show the comparison of aglycosylated CD3 antibody and glycosylated CD3 antibody in a mixed lymphocyte reaction. Aglycosylated antibody specific for the mouse CD8 antigen was included as a negative control.
  • FIGS. 13 & 14: These figures show the results of an Effector Cell Retargetting Assay comparing glycosylated and aglycosylated IgG-type CD3 antibodies. The CDw52 antibody was used as a negative control.
  • EXAMPLES Example 1 Preparation of an Aglycosylated Antibody Specific for the Human CD3 Antigen Containing CDRs from the YTH 12.5 Rat Antibody in Human Variable Framework Regions
  • The cloning and re-shaping of the V-region gene of the rat antibody YTH 12.5 specific for the human CD3 antigen was performed as described in Routledge et al., 1991, Eur. J. Immunol., 21, 2717 and in UK Patent Application No. 9121126.8 and its equivalents. YTH 12.5 is a rat hybridoma cell line secreting an IgG2b monoclonal antibody specific for the CD3 antigen complex.
  • Briefly, the methodology was based on that of Orlandi et al., 1989, PNAS USA, 86, 3833, using the polymerase chain reaction (PCR). The VH gene (heavy chain variable region gene) was cloned using oligonucleotide primers VH1FOR and VH1BACK. The PCR products were ligated into the vector M13-VHPCR1 in which site directed mutagenesis was performed using 6 oligonucleotide primers. The VL gene (light chain variable region gene) was cloned using primers designed based on the published VLλ sequences. The gene was cloned into the vector M13-VKPCR, together with the human lambda light chain constant region. In this vector mutagenesis of the VL framework was performed using 5 oligonucleotides. The humanised VL gene was then inserted into the expression vector pHβApr-1.
  • A vector was generated (p316) in which the reshaped CD3 VH gene could be expressed in conjunction with different immunoglobulin H chain constant region genes, this vector being based on the pHβApr-gpt vector (Gunning et al., 1987, P.N.A.S. USA, 85, 7719-7723). A 1.65 Kb fragment of DNA carrying the dihydrofolate reductase (dhft) gene and SV 40 expression signals (Page & Sydenham, 1991, Biotechnology, 9, 64) was inserted into the unique EcoRI site of pHβApr-gpt. A 700 bp HindIII-BamHI DNA fragment encoding the reshaped CD3-VH gene was then cloned into the vector's multiple cloning site, downstream and under the control of the β actin promoter. The desired H chain constant region gene (in genomic configuration) could then be inserted into the unique BamHl restriction enzyme site downstream of the CD3-VH gene.
  • The aglycosyl human IgG1 constant region was derived from the wild type Glm (1, 17) gene described by Takahashi et al., (1982, Cell, 29, 671-679) as follows. The gene was cloned into the vector M13 tg131 where site-directed mutagenesis was performed (Amersham International PLC) to mutate the amino acid residue at position 297 from an asparagine to an alanine residue.
  • Oligosaccharide at Asn-297 is a characteristic feature of all normal human IgG antibodies (Kabat et al., 1987, Sequence of Proteins of Immunological Interest, US Department of Health Human Services Publication), each of the two heavy chains in the IgG molecules having a single branched chain carbohydrate group which is linked to the amide group of the asparagine residue (Rademacher and Dwek, 1984, Prog. Immunol., 5, 95-112). Substitution of asparagine with alanine prevents the glycosylation of the antibody.
  • The 2.3 Kb aglycosyl IgG1 constant region was excised from M13 by double digestion using BamHI and BgIII and ligated into the BamHI site of vector p316 to produce clone p323.
  • Subconfluent monolayers of dhfr Chinese Hamster Ovary cells were co-transfected with the vector p323 containing the heavy chain gene and a second vector p274 containing the re-shaped human λ light chain (Routledge et al., 1991, Eur. J. Immunol., 21, 2717-2725). Prior to tranfection both plasmid DNAs were linearised using the restriction endonuclease PvuI. Transfection was carried out using the DOTMA reagent (Boehringer, Germany) following the manufacturer's recommendations.
  • Heavy and light chain transfectants were selected for in xanthine/hypoxanthine free IMOM containing 5% (v/v) dialysed foetal calf serum.
  • The production of the analogous wild type human IgG1-CD3 heavy chain vector p278 has been described elsewhere (Routledge et al., 1991, Eur. J. Immunol., 21, 2717-2725). H-chain expression vectors carrying the non-mutant human IgG2 (Flanagan & Rabbitts, 1982, Nature 300, 709-713), IgG3 (Huck et al., 1986, Nuc. Acid. Res., 14, 1779-1789), IgG4 (Flanagan & Rabbitts, 1982, Nature 300, 709-713), Epsilon (Flanagan & Rabbitts, 1982, EMBO. Journal 1, 655-660) and Alpha-2 (Flanagan & Rabbitts, 1982, Nature 300, 709-713) constant region genes (vectors p317, p318, p320, p321 and p325, respectively) were derived from the vector p316. Introduction of these vectors, in conjunction with the light chain vector p274, into dhfr CHO cells as described earlier, produced cell lines secreting CD3 antibody of the γ1, γ2, γ3, γ4, ε and α-2 isotype respectively. Cells expressing CD3 antibodies were subjected to two rounds of cloning in soft agar, and then expanded into roller bottle cultures. The immunoglobulin from approximately 4 litres of tissue culture supernatant from each cell line was concentrated by ammonium sulphate precipitation, dialysed extensively against PBS and then quantified as follows:
  • As the antibody was not pure, a competition assay was designed to specifically quantitate the concentration of antibody with CD3 antigen binding capacity. Human T-cell blasts were incubated with FITC labelled UCHT-1, an antibody which binds to the same epitope of the CD3 antigen as the chimaeric panel. The concentration of FITC reagent used had previously been determined to be half saturating. Unlabelled YTH 12.5 (HPLC purified) was titrated from a known starting concentration and added to wells containing T-cells and UCHT-1 FITC. The unlabelled antibody serves as a competitor for the antigen binding site. This is detected as decrease in the mean fluorescence seen when the cells are studied using FACS analysis. Thus, titration of the chimaeric antibodies from unknown starting concentrations yields a series of sigmoidal curves when mean fluorescence is plotted against antibody dilution. These can be directly compared with the standard YTH 12.5 curve.
  • Example 2 Proliferation Assays
  • The capacity of a CD3 antibody to support T-cell proliferation in solution is related to the interaction of the Fc region of the antibody with Fc receptors on accessory cells.
  • The aglycosylated chimaeric CD3 antibody prepared as described in Example 1 was compared with a panel of other chimaeric antibodies which shared the same variable region architecture but different H chain constant regions (see Example 1) for the ability to induce proliferation of human peripheral blood lymphocytes. Lymphocytes isolated from healthy donors' blood were separated on a lymphopaque gradient, washed and resuspended in IMDM containing 5% (v/v) heat-inactivated human AB serum and plated at 5×104 to 1×105 cells per well in plates containing CD3 antibodies in solution. After 3 days in culture the cells were pulse labelled with tritiated thymidine and harvested 6 hours later and the level of cellular 3H incorporation was determined by scintillation counting. The proliferation response to titrated antibody was studied in four blood donors. For a fifth donor the proliferation response was studied only at 1 μg. The results are shown in FIGS. 1 to 8.
  • For the donors studied, all ‘wild type’ antibodies led to T-cell proliferation. However the mutant, aglycosylated IgG1 isotype was never mitogenic implicating an important role for the carbohydrate side chains.
  • In a separate experiment, the ability of a panel of CD3 monoclonal antibodies in solution to stimulate T-cell mitogenesis was studied using lymphocytes isolated from the blood of 10 donors from a variety of ethnic backgrounds. All of the naturally occurring isotypes caused proliferation in the presence of 5% human AB serum, although there were T-cell donor dependent variations in the extent of the responses caused by the antibodies. In general, the γ1 and ε monoclonal antibodies were the most mitogenic, activating cells at the lowest concentration, and the γ3 and γ2 were the least active. The aglycosyl derivative of the γ1 monoclonal antibody was the only CD3 antibody which consistently failed to induce T-cell proliferation in any of the donors tested, giving responses equivalent to those of the non-activating control monoclonal antibody Campath-1H. In order to exclude endotoxin contamination as the cause of the proliferation seen with the α2 and ε preparations, it was confirmed that proliferation could be blocked by the addition of an excess of the aglycosyl CD3 mAb thus implicating the CD3 antigen in the activation process.
  • The total lack of proliferative response seen with the aglycosyl γ1 CD3 monoclonal antibody was surprising, given its position in the ECR activity hierarchy (see Example 5 below). The reason for this inactivity is probably due to its reduced affinity for FcRs rather than because aglycosylation has abolished the ability to trigger some post-binding event required for proliferation, e.g. by destroying a secondary recognition site on the monoclonal antibody. This is supported by the observation that the aglycosyl γ1 mAb could stimulate proliferation to a considerable degree if the assay was performed in IgG-free medium. There is probably a minimum thereshold level for the number of contacts or strength of interaction between T-cell and accessory cell which must be exceeded before proliferation can be initiated, and this cannot be achieved by the aglycosyl γ1 mAb in the presence of significant levels of competing immunoglobulin.
  • Example 3 The Effect of Chimaeric CD3 Antibodies in Mixed Lymphocyte Reactions
  • A series of experiments was conducted to test whether the aglycosylated antibody IgG1Ag of Example 1 had the capacity to block T-cell proliferation in a mixed lymphocyte reaction (MLR) and the results of 2 experiments are shown in FIGS. 9 to 12.
  • Peripheral blood lymphocytes were-isolated from two blood donors. The stimulator cell population was caesium irradiated. The responder population was incubated with titrated antibody for 30 minutes before the irradiated stimulator cells were added (FIGS. 9, 10 and 11). Control wells of responder cells were also incubated with irradiated responder cells at each antibody condition, to determine the specific effect of the antibody on the responder cells (FIGS. 9, 10 and 12).
  • After 5 days incubation the wells were pulse labelled with tritiated thymidine and harvested 6 hours later.
  • The aglycosylated antibody does block the mixed lymphocyte reaction; as the antibody is titrated out the blockade effect is less and the proliferation increases. The ‘wild type’ IgG1 actually has a mitogenic effect on the T-cells and so any blockade of the MLR is not seen through this response.
  • An ‘irrelevant’ aglycosylated antibody specific for the murine CD8 antigen was included as a negative control. This antibody has as expected no effect on the MLR.
  • Example 4 In Vivo Effect of IgG1 Antibodies
  • In vivo experiments were performed with chimaeric anti-human CD3 antibodies in mice which were transgenic for the human CD3 epsilon subunit including the aglycosylated antibody of Example 1 (IgG1Ag). The ability of some of the chimaeric CD3 antibodies to cause the release of TNF factor following a single injection was compared.
  • In this set of experiments serum was collected from the mice before injection and then at 90 minutes and 4 hours following intravenous injection with 10 μg of relevant CD3 antibody. Groups consisted of 5 mice and the serum collected from each group was pooled. Analysis of the level of TNF in the serum was performed in the laboratory of Professor Jean-Francois Bach, using a bioassay which measured the cytotoxic effect of sera on the L929 mouse fibroblast cells as a result of the presence of TNF.
  • The results are shown in Table 1 below:
    TABLE 1
    TNF detected in serum of hCD3 mice following
    injection with chimaeric CD3 antibodies
    Prebleed 90 minute 4 hour
    Sample TNF level TNF level TNF level
    Saline
    0 units/ml  0 units/ml 0 units/ml
    IgG1
    0 units/ml >400 units/ml   0 units/ml
    IgG2
    0 units/ml >400 units/ml   0 units/ml
    IgG1Ag
    0 units/ml 50 units/ml 0 units/ml
    IgE
    0 units/ml 50 units/ml 25 units/ml 
    YTH 12.5 0 units/ml 50 units/ml 0 units/ml
  • A significant difference is seen between the level of TNF associated with injection of the two forms of human IgG1. The aglycosylated form is associated with at least an eight-fold less release of TNF than the wild type IgG1 or with the IgG2 antibody.
  • The results of Examples 2-4 show that the aglycosylated CD3 antibody was not mitogenic to T-cells in solution indicating that the antibody had a reduced capacity to interact with Fc receptors on accessory cells. The antibody retained the immunosuppressive properties that are characteristic of CD3 antibodies. In vivo the aglycosylated antibody led to a significantly lower release of tumour necrosis factor in human CD3 transgenic mice than the parental IgG1 antibody. Thus this agent may be an ‘improved’ CD3 antibody for the purposes of immunosuppression if the decreased TNF release seen in mice is mirrored in humans.
  • Example 5 Effector Cell Retargetting Assays for the Detection of CD3 Antibodies with the Ability to Direct T-cell Killing
  • This was performed as described elsewhere (Gilliland et al., 1988, PNAS USA, 85, 4419) and measures the ability of a CD3 monoclonal antibody to cross-link activated T-cells to FcγR bearing target cells and thus to mediate target cell lysis. Briefly U937 human monocytic human cells which express the Fcy receptors I, II and III were labelled with 5 Cr sodium chromate and resuspended to 2×105 cells per ml. These cells were used as targets. Human T cell blasts, generated from human peripheral blood lymphocytes by activation with mitogenic CD3 antibody followed by culture in medium containing IL-2, were used as the effector cells. They were washed and resuspended at a concentration of 2×105 cells ml−1 prior to use in the assay. 100 μl volumes of the purified chimaeric antibody preparations were diluted in 3-fold steps in the wells of a microtitre plate. 50 μl each of the effector and target cells were then added to each well and the mixture was incubated at 37° C. for at least 4 hours. After this time 100 μl of supernatant was removed and assayed for released 51Cr. Each antibody dilution was tested in duplicate.
  • The U937 monocytic cell line expresses human Fc receptors and can be lysed by activated human T-cell blasts in the presence of CD3 monoclonal antibodies capable of cross-linking the two cell types. The results (FIGS. 13 and 14) show that when aglycosylated, the human IgG1 antibody of Example 1 is still able to cross-link T-cells to the U937 cells, albeit at a reduced level, and thus redirect T-cell cytotoxicity. This was a surprising finding since, given the published data, the effective killing mediated by aglycosyl γ1 monoclonal antibody was unexpected. There existed the possibility that removal of the carbohydrate had made this monoclonal antibody especially sticky, and so able to bind to U937 cells without interacting with FcγRs. However, it was subsequently demonstrated (results not shown) that the ability of the aglycosyl γ1 monoclonal antibody to mediate the destruction of mouse L cell targets (a mouse cell line which expresses the human FcγRI) was dependent on the expression of a transfected human FcγRI gene, thus confirming the FcγR binding activity of this monoclonal antibody. We conclude that the ECR assay is a particularly sensitive method of detecting Fc-FcR interactions. The ECR results indicate that the hierarchy of binding of the IgG chimaeric antibodies is γ2<γ3<Agγ1<γ4<γ1. If the assumption is made that the mitogenic activity of an antibody is predicted by its Fc receptor binding ability, then one would expect the above hierarchy to be displayed in the T cell proliferation assays. However, this was not the case; the order of activities in T cell proliferation experiments (1 to 3) was Agγ1<γ2<γ4<γ3<γ1. This demonstrates that the mitogenicity of an antibody cannot be predicted in a straightforward fashion from the results of assays which measure Fc-Fc receptor interactions. This view is supported by the behaviour of the epsilon chimaeric antibody which performed poorly in the ECR assay and yet consistently had the highest mitogenic activity. This suggests that antibodies can activate T cells by binding to something other than Fcy receptors (as displayed on U937 cells) on accessory cells, i.e. an inability to bind to Fcy receptors is no guarantee that an antibody will not be mitogenic.

Claims (27)

1. An aglycosylated IgG antibody having a binding affinity for the CD3 antigen complex.
2. An aglycosylated antibody according to claim 1, which has a binding affinity for the human CD3 antigen complex.
3. An aglycosylated antibody according to claim 2, in which at least one CDR is selected from the amino acid sequences:
(a) Ser-Phe-Pro-Met-Ala, (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr- Arg-Asp-Ser-Val-Lys-Gly, (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr, (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr- Val-His, (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
4. An aglycosylated antibody according to claim 2, which has a heavy chain with at least one CDR selected from the amino acid sequences:
(a) Ser-Phe-Pro-Met-Ala, (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr- Arg-Asp-Ser-Val-Lys-Gly, (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr,
and conservatively modified variants thereof, and/or a light chain with at least one CDR selected from the amino acid sequences:
(d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr- Val-His, (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
5. An aglycosylated antibody according to claim 2, which has a heavy chain with three CDRs comprising the amino acid sequences:
(a) Ser-Phe-Pro-Met-Ala, (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr- Arg-Asp-Ser-Val-Lys-Gly, (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr,
or conservatively modified variants thereof, and a light chain with three CDRs comprising the amino acid sequences:
(d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr- Val-His, (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
or conservatively modified variants thereof, the heavy chain CDRs being arranged in the order (a), (b), (c) in the leader→constant domain direction and the light chain CDRs being arranged in the order (d), (e), (f) in the leader→constant domain direction.
6. An aglycosylated antibody according to any of claims claim 1, in which the variable domain framework regions are of or are derived from those of rat or mouse origin.
7. An aglycosylated antibody according to any of claims claim 1, in which the CDRs are of different origin to the variable framework region.
8. An aglycosylated antibody according to claim 7, in which the variable domain framework regions are of or are derived from those of human origin.
9. An aglycosylated antibody according to claim 8, in which the heavy chain variable domain framework region reading from in the leader→constant domain direction comprises
Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Ser-/CDR/- Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/-Arg-Phe-Thr-lle-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln- Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-Lys-/CDR/-Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser, CDR indicating the presence of a CDR of which at least one is (a), (b) or (c) or a conservatively modified variant thereof.
10. An aglycosylated antibody according to claim 8, in which the light chain variable domain framework region reading in the leader→constant domain direction comprises
Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser- Cys-/CDR/-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-lle-Phe-/CDR/-Gly- Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser-lle-Asp-Arg-Ser-Ser-Asn-Ser-Ala-Ser-Leu-Thr-lle- Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-/CDR/-Phe-Gly-Gly-Gly-Thr- Lys-Leu-Thr-Val-Leu-Gly-Gln-Pro-Lys-Ala-Ala-Pro-Ser-Val-Thr-Leu-Phe-Pro-Pro-Ser-Ser-Glu-Glu-Leu-Gln, CDR indicating the presence of a CDR of which at least one is (d), (e) or (f) or a conservatively modified variant thereof.
11. An aglycosylated antibody according to claim 9 having a heavy chain variable domain which comprises
Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val- Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala- Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Met-Ala-Trp- Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val- Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr- Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg- Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn- Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys- Ala-Lys-Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr- Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser.
12. An aglycosylated antibody according to claim 8 having a light chain variable domain which comprises
Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu- Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu- Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp- Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val- Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro- Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn- Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu- Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-His-Ser-Tyr-Val-Ser- Ser-Phe-Asn-Val-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr- Val-Leu-Gly-Gln-Pro-Lys-Ala-Ala-Pro-Ser-Val-Thr- Leu-Phe-Pro-Pro-Ser-Ser-Glu-Glu-Leu-Gln.
13. An aglycosylated antibody according to claim 1, in which the constant domains are of or are derived from those of rat or mouse origin.
14. An aglycosylated antibody according to claim 1, in which the CDRs are of different origin to the constant region.
15. An aglycosylated antibody according to claim 1, in which the constant domains are of or are derived from those of human origin.
16. An aglycosylated antibody according to claim 1, in which the constant region is of an IgG isotype.
17. An aglycosylated antibody according to claim 15, in which the constant region is of an IgG1 isotope.
18. An aglycosylated antibody according to claim 15, in which asparagines residue at position 297 of each constant region heavy chain is replaced by an alternative amino acid residue.
19. An aglycosylated antibody according to claim 18, in which the asparagine residue is replaced by an alanine residue.
20. An aglycosylated antibody according to claim 1, in which only one of the arms thereof has an affinity for the CD3 antigen.
21. An aglycosylated antibody according to claim 20 which is monovalent.
22. An aglycosylated antibody according to claim 21, in which one half of the antibody consists of a complete heavy chain and light chain and the other half consists of a similar but truncated heavy chain lacking the binding site for the light chain.
23. An aglycosylated antibody according to claim 1 in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier.
24. An aglycosylated antibody according to claim 1, for use in therapy.
25. The use of an aglycosylated antibody according to claim 1 for the manufacture of a medicament for use in immunosuppression.
26. The use according to claim 25, in which the medicament is for use in the treatment of recipients of a transplant.
27. A method of treating a patient having cancer or requiring immunosuppression which comprises administering to said patient a therapeutically effective amount of a ligand or an antibody or fragment thereof according to claim 1.
US11/704,940 1992-03-24 2007-02-12 Humanized anti-CD3 specific antibodies Abandoned US20070154477A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/704,940 US20070154477A1 (en) 1992-03-24 2007-02-12 Humanized anti-CD3 specific antibodies

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
GB9206422.9 1992-03-24
GB929206422A GB9206422D0 (en) 1992-03-24 1992-03-24 Antibody preparation
US07/988,925 US5585097A (en) 1992-03-24 1992-10-21 Humanized anti-CD3 specific antibodies
PCT/GB1992/001933 WO1993019196A1 (en) 1992-03-24 1992-10-21 ANTI-CD3 AGLYCOSYLATED IgG ANTIBODY
US08/478,684 US6706265B1 (en) 1992-03-24 1995-06-07 Humanized anti-CD3 specific antibodies
US10/743,423 US20040202657A1 (en) 1992-03-24 2003-12-23 Humanized anti-CD3 specific antibodies
US11/300,396 US20060165693A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/704,940 US20070154477A1 (en) 1992-03-24 2007-02-12 Humanized anti-CD3 specific antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US11/300,396 Continuation US20060165693A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies

Publications (1)

Publication Number Publication Date
US20070154477A1 true US20070154477A1 (en) 2007-07-05

Family

ID=10712757

Family Applications (11)

Application Number Title Priority Date Filing Date
US07/988,925 Expired - Lifetime US5585097A (en) 1992-03-24 1992-10-21 Humanized anti-CD3 specific antibodies
US08/478,684 Expired - Fee Related US6706265B1 (en) 1992-03-24 1995-06-07 Humanized anti-CD3 specific antibodies
US10/743,423 Abandoned US20040202657A1 (en) 1992-03-24 2003-12-23 Humanized anti-CD3 specific antibodies
US11/300,396 Abandoned US20060165693A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,278 Abandoned US20060165691A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,279 Abandoned US20060165692A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,563 Abandoned US20060088526A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/501,894 Abandoned US20060269547A1 (en) 1992-03-24 2006-08-10 Humanized anti-CD3 specific antibodies
US11/636,655 Abandoned US20070178092A1 (en) 1992-03-24 2006-12-11 Humanized anti-CD3 specific antibodies
US11/700,150 Abandoned US20070134241A1 (en) 1992-03-24 2007-01-31 Humanized anti-CD3 specific antibodies
US11/704,940 Abandoned US20070154477A1 (en) 1992-03-24 2007-02-12 Humanized anti-CD3 specific antibodies

Family Applications Before (10)

Application Number Title Priority Date Filing Date
US07/988,925 Expired - Lifetime US5585097A (en) 1992-03-24 1992-10-21 Humanized anti-CD3 specific antibodies
US08/478,684 Expired - Fee Related US6706265B1 (en) 1992-03-24 1995-06-07 Humanized anti-CD3 specific antibodies
US10/743,423 Abandoned US20040202657A1 (en) 1992-03-24 2003-12-23 Humanized anti-CD3 specific antibodies
US11/300,396 Abandoned US20060165693A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,278 Abandoned US20060165691A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,279 Abandoned US20060165692A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/300,563 Abandoned US20060088526A1 (en) 1992-03-24 2005-12-15 Humanized anti-CD3 specific antibodies
US11/501,894 Abandoned US20060269547A1 (en) 1992-03-24 2006-08-10 Humanized anti-CD3 specific antibodies
US11/636,655 Abandoned US20070178092A1 (en) 1992-03-24 2006-12-11 Humanized anti-CD3 specific antibodies
US11/700,150 Abandoned US20070134241A1 (en) 1992-03-24 2007-01-31 Humanized anti-CD3 specific antibodies

Country Status (12)

Country Link
US (11) US5585097A (en)
EP (1) EP0586617B1 (en)
JP (3) JP4065554B2 (en)
KR (1) KR100238497B1 (en)
AT (1) ATE155818T1 (en)
AU (1) AU671085B2 (en)
CA (1) CA2109815C (en)
DE (1) DE69221147T2 (en)
ES (1) ES2106195T3 (en)
GB (1) GB9206422D0 (en)
GR (1) GR3024489T3 (en)
WO (1) WO1993019196A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018183929A1 (en) 2017-03-30 2018-10-04 Progenity Inc. Treatment of a disease of the gastrointestinal tract with an immune modulatory agent released using an ingestible device
WO2019246312A1 (en) 2018-06-20 2019-12-26 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with an immunomodulator
WO2019246317A1 (en) 2018-06-20 2019-12-26 Progenity, Inc. Treatment of a disease or condition in a tissue originating from the endoderm
WO2020106750A1 (en) 2018-11-19 2020-05-28 Progenity, Inc. Methods and devices for treating a disease with biotherapeutics
WO2021119482A1 (en) 2019-12-13 2021-06-17 Progenity, Inc. Ingestible device for delivery of therapeutic agent to the gastrointestinal tract
EP4252629A2 (en) 2016-12-07 2023-10-04 Biora Therapeutics, Inc. Gastrointestinal tract detection methods, devices and systems

Families Citing this family (168)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9206422D0 (en) * 1992-03-24 1992-05-06 Bolt Sarah L Antibody preparation
US5834597A (en) * 1996-05-20 1998-11-10 Protein Design Labs, Inc. Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same
JP4503706B2 (en) * 1996-06-07 2010-07-14 ポニアード ファーマシューティカルズ,インコーポレイティド Humanized antibodies with modified glycosylation
US6528624B1 (en) * 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
GB9815909D0 (en) * 1998-07-21 1998-09-16 Btg Int Ltd Antibody preparation
US6737056B1 (en) * 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US7183387B1 (en) 1999-01-15 2007-02-27 Genentech, Inc. Polypeptide variants with altered effector function
NZ539776A (en) * 1999-01-15 2006-12-22 Genentech Inc Polypeptide variants with altered effector function
AU2154401A (en) * 1999-11-12 2001-05-30 Merck Patent Gmbh Erythropoietin forms with improved properties
US6979556B2 (en) * 2000-12-14 2005-12-27 Genentech, Inc. Separate-cistron contructs for secretion of aglycosylated antibodies from prokaryotes
DE60135498D1 (en) * 2000-12-14 2008-10-02 Genentech Inc Production of Whole Antibodies in Prokaryotic Cells
US6720165B2 (en) 2001-06-14 2004-04-13 Zyomix, Inc. Methods for making antibody fragments and compositions resulting therefrom
CA2454731C (en) * 2001-08-27 2010-11-02 Genentech, Inc. A system for antibody expression and assembly
GB2380127A (en) * 2001-09-26 2003-04-02 Isis Innovation Treatment of chronic joint inflammation
IL161412A0 (en) * 2001-10-25 2004-09-27 Genentech Inc Glycoprotein compositions
AU2003223474B2 (en) * 2002-04-25 2008-09-04 Eli Lilly And Company Method for treating anxiety and mood disorders in older subjects
AU2003287345A1 (en) * 2002-10-31 2004-06-07 Genentech, Inc. Methods and compositions for increasing antibody production
AU2011224032B2 (en) * 2003-06-13 2013-01-31 Biogen Ma Inc. Aglycosyl Anti-CD154 (CD40 Ligand) Antibodies and Uses Thereof
JP4667383B2 (en) * 2003-06-13 2011-04-13 バイオジェン・アイデック・エムエイ・インコーポレイテッド Aglycosyl anti-CD154 (CD40 ligand) antibody and use thereof
AU2004266159A1 (en) 2003-08-22 2005-03-03 Biogen Idec Ma Inc. Improved antibodies having altered effector function and methods for making the same
US20050069521A1 (en) * 2003-08-28 2005-03-31 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of interleukin-2 proteins
JP2007504245A (en) * 2003-09-05 2007-03-01 ジェネンテック・インコーポレーテッド Antibodies with altered effector functions
ES2383300T3 (en) * 2003-11-13 2012-06-20 Hanmi Holdings Co., Ltd Fc fragment of IgG for a drug vehicle and procedure for its preparation
US8110665B2 (en) 2003-11-13 2012-02-07 Hanmi Holdings Co., Ltd. Pharmaceutical composition comprising an immunoglobulin FC region as a carrier
DE602004013372T2 (en) 2003-12-30 2009-07-02 Merck Patent Gmbh IL-7 FUSION PROTEINS WITH ANTIBODY PORTIONS, THEIR PREPARATION AND THEIR USE
WO2005063808A1 (en) 2003-12-31 2005-07-14 Merck Patent Gmbh Fc-ERYTHROPOIETIN FUSION PROTEIN WITH IMPROVED PHARMACOKINETICS
US7728114B2 (en) 2004-06-03 2010-06-01 Novimmune S.A. Anti-CD3 antibodies and methods of use thereof
KR20070047327A (en) * 2004-07-26 2007-05-04 비오겐 아이덱 엠에이 아이엔씨. Anti-cd154 antibodies
WO2006028936A2 (en) * 2004-09-02 2006-03-16 Genentech, Inc. Heteromultimeric molecules
WO2006074397A2 (en) 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Cripto binding molecules
EP1866339B8 (en) 2005-03-25 2021-12-01 GITR, Inc. Gitr binding molecules and uses therefor
AU2006261127B2 (en) * 2005-06-17 2012-03-15 Merck Sharp & Dohme Corp. ILT3 binding molecules and uses therefor
US8663634B2 (en) 2005-07-11 2014-03-04 Macrogenics, Inc. Methods for the treatment of autoimmune disorders using immunosuppressive monoclonal antibodies with reduced toxicity
CN101258164B (en) 2005-08-16 2013-05-01 韩美科学株式会社 A method for the mass production of immunoglobulin fc region deleted initial methionine residues
UA92505C2 (en) * 2005-09-12 2010-11-10 Новиммюн С.А. Anti-cd3 antibody formulations
SI1966238T1 (en) * 2005-12-30 2012-08-31 Merck Patent Gmbh Interleukin-12p40 variants with improved stability
PT2270050E (en) 2005-12-30 2013-09-12 Merck Patent Gmbh Anti-cd19 antibodies with reduced immunogenicity
EP2433650A3 (en) 2006-06-06 2012-12-19 Tolerrx Inc. Administration of anti-CD3 antibodies in the treatment of autoimmune diseases
SG10201504662WA (en) * 2006-06-14 2015-07-30 Macrogenics Inc Methods For The Treatment Of Autoimmune Disorders Using Immunosuppressive Monoclonal Antibodies With Reduced Toxicity
AU2007345745C1 (en) * 2006-06-19 2013-05-23 Merck Sharp & Dohme Corp. ILT3 binding molecules and uses therefor
WO2008013918A2 (en) * 2006-07-26 2008-01-31 Myelin Repair Foundation, Inc. Cell cycle regulation and differentiation
JP5511654B2 (en) * 2007-05-31 2014-06-04 ゲンマブ エー/エス Recombinant non-glycosylated monovalent half antibody obtained by molecular manipulation
JP5932217B2 (en) 2007-07-12 2016-06-08 ジーアイティーアール, インコーポレイテッド Combination therapy using GITR binding molecules
WO2011028952A1 (en) 2009-09-02 2011-03-10 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
CA2806252C (en) 2010-07-29 2019-05-14 Xencor, Inc. Antibodies with modified isoelectric points
WO2012066058A1 (en) 2010-11-16 2012-05-24 Boehringer Ingelheim International Gmbh Agents and methods for treating diseases that correlate with bcma expression
TWI685503B (en) 2010-11-30 2020-02-21 中外製藥股份有限公司 Cell injury-inducing therapeutic agent
US9249217B2 (en) 2010-12-03 2016-02-02 Secretary, DHHS Bispecific EGFRvIII x CD3 antibody engaging molecules
WO2012122528A1 (en) 2011-03-10 2012-09-13 Hco Antibody, Inc. Bispecific three-chain antibody-like molecules
SG10201602394QA (en) 2011-03-29 2016-05-30 Roche Glycart Ag Antibody FC Variants
CA2836857C (en) 2011-05-21 2019-12-03 Macrogenics, Inc. Cd3-binding molecules capable of binding to human and non-human cd3
EP2742953B1 (en) 2011-08-11 2021-09-22 ONO Pharmaceutical Co., Ltd. Therapeutic agent for autoimmune diseases comprising pd-1 agonist
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
JP6371059B2 (en) 2011-10-31 2018-08-08 中外製薬株式会社 Antigen-binding molecules with controlled association of heavy and light chains
MA37794B1 (en) 2012-07-13 2017-07-31 Roche Glycart Ag Anti-vegf / anti-ang-2 bispecific antibodies and their use in the treatment of ocular vascular pathologies
WO2014028939A2 (en) 2012-08-17 2014-02-20 California Institute Of Technology Targeting phosphofructokinase and its glycosylation form for cancer
WO2014036562A2 (en) 2012-08-31 2014-03-06 University Of Virginia Patent Foundation Target peptides for immunotherapy and diagnostics
WO2014039675A2 (en) 2012-09-05 2014-03-13 University Of Virginia Patent Foundation Target peptides for colorectal cancer therapy and diagnostics
US20160000893A1 (en) 2012-12-13 2016-01-07 University Of Virginia Patent Foundation Target peptides for ovarian cancer therapy and diagnostics
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
US10131710B2 (en) 2013-01-14 2018-11-20 Xencor, Inc. Optimized antibody variable regions
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
CA3211863A1 (en) 2013-01-14 2014-07-17 Xencor, Inc. Novel heterodimeric proteins
WO2014113510A1 (en) 2013-01-15 2014-07-24 Xencor, Inc. Rapid clearance of antigen complexes using novel antibodies
JP2016093104A (en) * 2013-02-19 2016-05-26 国立大学法人京都大学 ANTIHUMAN CD3ε ANTIBODY OR FRAGMENT THEREOF, AND IMMUNOSUPPRESSIVE AGENT CONTAINING IT AS ACTIVE INGREDIENT
US10106624B2 (en) 2013-03-15 2018-10-23 Xencor, Inc. Heterodimeric proteins
EP2970486B1 (en) 2013-03-15 2018-05-16 Xencor, Inc. Modulation of t cells with bispecific antibodies and fc fusions
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
US10519242B2 (en) 2013-03-15 2019-12-31 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
KR101895634B1 (en) 2013-05-31 2018-09-05 한미약품 주식회사 IgG4 Fc fragment comprising modified hinge region
PT3406633T (en) 2013-07-25 2022-05-04 Cytomx Therapeutics Inc Multispecific antibodies, multispecific activatable antibodies and methods of using the same
US9770461B2 (en) 2013-08-02 2017-09-26 California Institute Of Technology Tailored glycopolymers as anticoagulant heparin mimetics
US10227370B2 (en) 2013-08-02 2019-03-12 California Institute Of Technology Heparan sulfate/heparin mimetics with anti-chemokine and anti-inflammatory activity
AP2016009475A0 (en) 2014-03-28 2016-09-30 Xencor Inc Bispecific antibodies that bind to cd38 and cd3
CN106459206A (en) 2014-04-07 2017-02-22 中外制药株式会社 Immunoactivating antigen-binding molecule
WO2015160928A2 (en) 2014-04-15 2015-10-22 University Of Virginia Patent Foundation Isolated t cell receptors and methods of use therefor
MX2016014434A (en) 2014-05-13 2017-02-23 Chugai Pharmaceutical Co Ltd T cell-redirected antigen-binding molecule for cells having immunosuppression function.
SG10201912986PA (en) 2014-05-28 2020-02-27 Agenus Inc Anti-gitr antibodies and methods of use thereof
EP3172235A2 (en) 2014-07-25 2017-05-31 Cytomx Therapeutics Inc. Anti-cd3 antibodies, activatable anti-cd3 antibodies, multispecific anti-cd3 antibodies, multispecific activatable anti-cd3 antibodies, and methods of using the same
US10259887B2 (en) 2014-11-26 2019-04-16 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
RS62332B1 (en) 2014-11-26 2021-10-29 Xencor Inc Heterodimeric antibodies that bind cd3 and cd20
BR112017011166A2 (en) 2014-11-26 2018-02-27 Xencor, Inc. heterodimeric antibodies that bind to cd3 and cd38
US10428155B2 (en) 2014-12-22 2019-10-01 Xencor, Inc. Trispecific antibodies
SG11201705721WA (en) 2015-01-14 2017-08-30 Brigham & Womens Hospital Inc Treatment of cancer with anti-lap monoclonal antibodies
WO2016141387A1 (en) 2015-03-05 2016-09-09 Xencor, Inc. Modulation of t cells with bispecific antibodies and fc fusions
US10259882B2 (en) 2015-05-07 2019-04-16 Agenus Inc. Anti-OX40 antibodies
EP3091032A1 (en) 2015-05-08 2016-11-09 Miltenyi Biotec GmbH Humanized antibody or fragment thereof specific for cd3
MA45352A (en) 2015-08-07 2018-06-13 Univ Birmingham IDENTIFICATION OF GLYCOPEPTIDES ASSOCIATED WITH CATEGORY I HCM AS TARGETS FOR CANCER IMMUNOTHERAPY
CN114605548A (en) 2015-09-01 2022-06-10 艾吉纳斯公司 anti-PD-1 antibodies and methods of use thereof
GB201520191D0 (en) 2015-11-16 2015-12-30 Cancer Rec Tech Ltd T-cell receptor and uses thereof
EP3378488A4 (en) 2015-11-18 2019-10-30 Chugai Seiyaku Kabushiki Kaisha Method for enhancing humoral immune response
US11660340B2 (en) 2015-11-18 2023-05-30 Chugai Seiyaku Kabushiki Kaisha Combination therapy using T cell redirection antigen binding molecule against cell having immunosuppressing function
EP3383430A4 (en) 2015-12-02 2019-12-18 Agenus Inc. Antibodies and methods of use thereof
US11623957B2 (en) 2015-12-07 2023-04-11 Xencor, Inc. Heterodimeric antibodies that bind CD3 and PSMA
CN108368166B (en) 2015-12-28 2023-03-28 中外制药株式会社 Method for improving purification efficiency of polypeptide containing FC region
AU2017233658B2 (en) 2016-03-14 2023-09-21 Chugai Seiyaku Kabushiki Kaisha Cell injury inducing therapeutic drug for use in cancer therapy
CA3024508A1 (en) 2016-05-27 2017-11-30 Agenus Inc. Anti-tim-3 antibodies and methods of use thereof
CN110352070A (en) 2016-06-14 2019-10-18 Xencor股份有限公司 Bispecific checkpoint inhibitor antibody
IL263834B2 (en) 2016-06-20 2024-01-01 Kymab Ltd Anti-pd-l1 antibodies
CN109641049B (en) 2016-06-21 2023-07-07 特尼奥生物股份有限公司 CD3 binding antibodies
EP4050032A1 (en) 2016-06-28 2022-08-31 Xencor, Inc. Heterodimeric antibodies that bind somatostatin receptor 2
US10793632B2 (en) 2016-08-30 2020-10-06 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
CA3036550A1 (en) 2016-09-14 2018-03-22 Teneobio, Inc. Cd3 binding antibodies
AU2017343621B2 (en) 2016-10-11 2021-12-02 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
EP3526240A1 (en) 2016-10-14 2019-08-21 Xencor, Inc. Bispecific heterodimeric fusion proteins containing il-15/il-15ralpha fc-fusion proteins and pd-1 antibody fragments
EP3551660B1 (en) 2016-12-07 2023-09-13 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
AU2017382251A1 (en) 2016-12-21 2019-07-11 Teneobio, Inc. Anti-BCMA heavy chain-only antibodies
AU2018253176B2 (en) 2017-04-13 2023-02-02 Agenus Inc. Anti-CD137 antibodies and methods of use thereof
HUE062927T2 (en) 2017-05-01 2023-12-28 Agenus Inc Anti-tigit antibodies and methods of use thereof
US11427642B2 (en) 2017-06-20 2022-08-30 Teneoone, Inc. Anti-BCMA heavy chain-only antibodies
EP3645122A1 (en) 2017-06-30 2020-05-06 Xencor, Inc. Targeted heterodimeric fc fusion proteins containing il-15/il-15ra and antigen binding domains
WO2019004831A1 (en) 2017-06-30 2019-01-03 Academisch Ziekenhuis Leiden (H.O.D.N. Leids Universitair Medisch Centrum) Treatment of haematological malignancies
LT3661954T (en) 2017-08-03 2022-04-11 Amgen Inc. Interleukin-21 muteins and methods of treatment
SG11202000387YA (en) 2017-08-25 2020-03-30 Five Prime Therapeutics Inc B7-h4 antibodies and methods of use thereof
EP3679040B1 (en) 2017-09-08 2022-08-03 Amgen Inc. Inhibitors of kras g12c and methods of using the same
US10981992B2 (en) 2017-11-08 2021-04-20 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US11312770B2 (en) 2017-11-08 2022-04-26 Xencor, Inc. Bispecific and monospecific antibodies using novel anti-PD-1 sequences
SG11202005732XA (en) 2017-12-19 2020-07-29 Xencor Inc Engineered il-2 fc fusion proteins
CN111886250A (en) 2017-12-27 2020-11-03 特尼奥生物股份有限公司 CD 3-/heterodimer-specific antibody
KR20200110358A (en) 2018-01-12 2020-09-23 암젠 인크 Anti-PD-1 Antibodies and Methods of Treatment
TWI804572B (en) 2018-02-09 2023-06-11 日商小野藥品工業股份有限公司 Bispecific antibody
AU2019247415A1 (en) 2018-04-04 2020-10-22 Xencor, Inc. Heterodimeric antibodies that bind fibroblast activation protein
JP2021520829A (en) 2018-04-18 2021-08-26 ゼンコア インコーポレイテッド TIM-3 targeted heterodimer fusion protein containing IL-15 / IL-15RA Fc fusion protein and TIM-3 antigen binding domain
CA3097593A1 (en) 2018-04-18 2019-10-24 Xencor, Inc. Pd-1 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and pd-1 antigen binding domains and uses thereof
WO2019226658A1 (en) 2018-05-21 2019-11-28 Compass Therapeutics Llc Multispecific antigen-binding compositions and methods of use
WO2019231326A1 (en) 2018-05-31 2019-12-05 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n.LUMC) Teipp neoantigens and uses thereof
MX2021001083A (en) 2018-07-31 2021-03-31 Amgen Res Munich Gmbh Dosing regimen for bcma-cd3 bispecific antibodies.
AU2019314999A1 (en) 2018-08-03 2021-02-11 Amgen Inc. Antibody constructs for CLDN18.2 and CD3
CN112703204A (en) 2018-09-28 2021-04-23 安进公司 Antibodies to soluble BCMA
WO2020072821A2 (en) 2018-10-03 2020-04-09 Xencor, Inc. Il-12 heterodimeric fc-fusion proteins
NL2021789B1 (en) 2018-10-10 2020-05-14 Academisch Ziekenhuis Leiden Binding proteins specific for HA-1H and uses thereof
EP3889179A4 (en) 2018-11-01 2022-10-12 Shandong New Time Pharmaceutical Co., Ltd. Bispecific antibody and use thereof
JP2022523946A (en) 2019-03-01 2022-04-27 ゼンコア インコーポレイテッド Heterodimer antibody that binds to ENPP3 and CD3
US20220227887A1 (en) 2019-06-11 2022-07-21 Ono Pharmaceutical Co., Ltd. Immunosuppressant
EA202290054A1 (en) 2019-06-14 2022-03-25 Тенеобио, Инк. POLYSPECIFIC ANTIBODIES CONTAINING ONLY HEAVY CHAINS THAT BIND TO CD22 AND CD3
BR112022002761A2 (en) 2019-08-12 2022-08-09 Aptevo Res & Development Llc 4-1BB AND OX40 BINDING PROTEINS AND RELATED COMPOSITIONS AND METHODS, 4-1BB ANTIBODIES, OX40 ANTIBODIES
JP2022545741A (en) 2019-08-30 2022-10-28 アジェナス インコーポレイテッド ANTI-CD96 ANTIBODY AND METHODS OF USE THEREOF
EP3819007A1 (en) 2019-11-11 2021-05-12 Amgen Research (Munich) GmbH Dosing regimen for anti-bcma agents
US20230093169A1 (en) 2020-01-22 2023-03-23 Amgen Research (Munch) Gmbh Combinations of antibody constructs and inhibitors of cytokine release syndrome and uses thereof
EP4186564A1 (en) 2020-04-29 2023-05-31 Teneoone, Inc. Multispecific heavy chain antibodies with modified heavy chain constant regions
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
CN115697491A (en) 2020-05-17 2023-02-03 阿斯利康(英国)有限公司 SARS-COV-2 antibodies and methods of selection and use thereof
US20220017636A1 (en) 2020-05-19 2022-01-20 Amgen Inc. Mageb2 binding constructs
AU2021320870A1 (en) 2020-08-06 2023-04-06 Bioverativ Usa Inc. Inflammatory cytokines and fatigue in subject with a complement mediated disease
KR20230045613A (en) 2020-08-10 2023-04-04 아스트라제네카 유케이 리미티드 SARS-COV-2 Antibodies for Treatment and Prevention of COVID-19
IL300666A (en) 2020-08-19 2023-04-01 Xencor Inc Anti-cd28 compositions
NL2026614B1 (en) 2020-10-02 2022-06-03 Academisch Ziekenhuis Leiden T cell receptors directed against bob1 and uses thereof
KR20230098334A (en) 2020-11-06 2023-07-03 암젠 리서치 (뮌헨) 게엠베하 Polypeptide constructs that selectively bind to CLDN6 and CD3
AR124017A1 (en) 2020-11-06 2023-02-01 Amgen Res Munich Gmbh POLYPEPTIDES WITH ENHANCED CLIPPING PROFILE
EP4243936A1 (en) 2020-11-10 2023-09-20 Amgen Inc. Methods for administering a bcma x cd3 binding molecule
JP2023551907A (en) 2020-12-01 2023-12-13 アプティーボ リサーチ アンド デベロップメント エルエルシー Tumor-associated antigens and CD3 binding proteins, related compositions, and methods
KR20230156079A (en) 2021-03-09 2023-11-13 젠코어 인코포레이티드 Heterodimeric antibody binding to CD3 and CLDN6
EP4305065A1 (en) 2021-03-10 2024-01-17 Xencor, Inc. Heterodimeric antibodies that bind cd3 and gpc3
TW202304508A (en) 2021-03-31 2023-02-01 美商百歐維拉提夫美國公司 Reducing surgery-associated hemolysis in cold agglutinin disease patients
WO2022263357A1 (en) 2021-06-14 2022-12-22 Argenx Iip Bv Anti-il-9 antibodies and methods of use thereof
WO2023056362A1 (en) 2021-09-30 2023-04-06 Seagen Inc. B7-h4 antibody-drug conjugates for the treatment of cancer
TW202342095A (en) 2021-11-05 2023-11-01 英商阿斯特捷利康英國股份有限公司 Composition for treatment and prevention of covid-19
WO2023091968A1 (en) 2021-11-17 2023-05-25 Disc Medicine, Inc. Methods for treating anemia of kidney disease
NL2030990B1 (en) 2022-02-17 2023-09-01 Academisch Ziekenhuis Leiden T cell receptors directed against jchain and uses thereof
NL2031118B1 (en) 2022-03-01 2023-09-07 Academisch Ziekenhuis Leiden T cell receptors directed against transcription factor wt1 and uses thereof
WO2023209177A1 (en) 2022-04-29 2023-11-02 Astrazeneca Uk Limited Sars-cov-2 antibodies and methods of using the same
WO2023219510A1 (en) 2022-05-13 2023-11-16 Academisch Ziekenhuis Leiden (H.O.D.N. Leids Universitair Medisch Centrum) Treatment of haematological malignancies
NL2032130B1 (en) 2022-06-10 2023-12-18 Academisch Ziekenhuis Leiden T cell receptors directed against melanoma-associated antigen and uses thereof
WO2023245048A1 (en) 2022-06-15 2023-12-21 Bioverativ Usa Inc. Anti-complement c1s antibody formulation
US20240025978A1 (en) 2022-06-24 2024-01-25 Bioverativ Usa Inc. Methods for treating complement-mediated diseases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5225539A (en) * 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5968509A (en) * 1990-10-05 1999-10-19 Btp International Limited Antibodies with binding affinity for the CD3 antigen

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8607679D0 (en) * 1986-03-27 1986-04-30 Winter G P Recombinant dna product
DE3883899T3 (en) * 1987-03-18 1999-04-22 Sb2 Inc CHANGED ANTIBODIES.
CA1339198C (en) * 1988-02-12 1997-08-05 Gregory Paul Winter Antibodies to the antigen campath-1
FI105320B (en) * 1988-04-04 2000-07-31 Oncogen Procedure for Preparation of Antibody Heteroconjugates for Use in Regulating Lymphocyte Activity and Diagnosis
ATE159982T1 (en) * 1988-09-15 1997-11-15 Univ Columbia ANTIBODIES WITH MODIFIED CARBOHYDRATE CONTENT AND METHOD OF PRODUCTION AND USE
GB8905669D0 (en) * 1989-03-13 1989-04-26 Celltech Ltd Modified antibodies
WO1991001752A1 (en) * 1989-07-27 1991-02-21 Fred Hutchinson Cancer Research Center Immunosuppressive monoclonal antibodies, hybridomas and methods of transplantation
GB8928874D0 (en) * 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
JP2711163B2 (en) * 1990-01-12 1998-02-10 新日本製鐵株式会社 Method for producing high corrosion resistant low alloy linepipe steel with excellent corrosion resistance
GB9020282D0 (en) * 1990-09-17 1990-10-31 Gorman Scott D Altered antibodies and their preparation
JPH06503194A (en) * 1990-11-27 1994-04-07 パラスペクティブス インコーポレイテッド Object image creation system in alternating geometry
EP0940468A1 (en) * 1991-06-14 1999-09-08 Genentech, Inc. Humanized antibody variable domain
US5932448A (en) * 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
GB9206422D0 (en) * 1992-03-24 1992-05-06 Bolt Sarah L Antibody preparation
AU2004263538B2 (en) * 2003-08-08 2009-09-17 Immunomedics, Inc. Bispecific antibodies for inducing apoptosis of tumor and diseased cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5225539A (en) * 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5968509A (en) * 1990-10-05 1999-10-19 Btp International Limited Antibodies with binding affinity for the CD3 antigen

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4252629A2 (en) 2016-12-07 2023-10-04 Biora Therapeutics, Inc. Gastrointestinal tract detection methods, devices and systems
WO2018183929A1 (en) 2017-03-30 2018-10-04 Progenity Inc. Treatment of a disease of the gastrointestinal tract with an immune modulatory agent released using an ingestible device
EP4108183A1 (en) 2017-03-30 2022-12-28 Biora Therapeutics, Inc. Treatment of a disease of the gastrointestinal tract with an immune modulatory agent released using an ingestible device
WO2019246312A1 (en) 2018-06-20 2019-12-26 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with an immunomodulator
WO2019246317A1 (en) 2018-06-20 2019-12-26 Progenity, Inc. Treatment of a disease or condition in a tissue originating from the endoderm
WO2020106750A1 (en) 2018-11-19 2020-05-28 Progenity, Inc. Methods and devices for treating a disease with biotherapeutics
WO2020106704A2 (en) 2018-11-19 2020-05-28 Progenity, Inc. Ingestible device for delivery of therapeutic agent to the gastrointestinal tract
WO2020106757A1 (en) 2018-11-19 2020-05-28 Progenity, Inc. Ingestible device for delivery of therapeutic agent to the gastrointestinal tract
WO2020106754A1 (en) 2018-11-19 2020-05-28 Progenity, Inc. Methods and devices for treating a disease with biotherapeutics
WO2021119482A1 (en) 2019-12-13 2021-06-17 Progenity, Inc. Ingestible device for delivery of therapeutic agent to the gastrointestinal tract

Also Published As

Publication number Publication date
GR3024489T3 (en) 1997-11-28
EP0586617B1 (en) 1997-07-23
ATE155818T1 (en) 1997-08-15
US20060165691A1 (en) 2006-07-27
US20070134241A1 (en) 2007-06-14
ES2106195T3 (en) 1997-11-01
AU671085B2 (en) 1996-08-15
JP2004000249A (en) 2004-01-08
CA2109815C (en) 2003-02-04
JP4113467B2 (en) 2008-07-09
CA2109815A1 (en) 1993-09-30
GB9206422D0 (en) 1992-05-06
US20060088526A1 (en) 2006-04-27
US20060165693A1 (en) 2006-07-27
KR100238497B1 (en) 2000-01-15
JP4113890B2 (en) 2008-07-09
DE69221147D1 (en) 1997-09-04
AU2766892A (en) 1993-10-21
US5585097A (en) 1996-12-17
DE69221147T2 (en) 1998-01-15
US20070178092A1 (en) 2007-08-02
WO1993019196A1 (en) 1993-09-30
JP4065554B2 (en) 2008-03-26
JP2006089501A (en) 2006-04-06
EP0586617A1 (en) 1994-03-16
US6706265B1 (en) 2004-03-16
US20060165692A1 (en) 2006-07-27
US20040202657A1 (en) 2004-10-14
US20060269547A1 (en) 2006-11-30
JPH07500017A (en) 1995-01-05

Similar Documents

Publication Publication Date Title
US5585097A (en) Humanized anti-CD3 specific antibodies
US7993641B2 (en) Methods of treatment using anti-CD3 antibodies
EP1711207B1 (en) Interferon alpha antibodies and their uses
EP2221316A1 (en) Anti-CD19 antibody therapy for autoimmune disease
KR20100015804A (en) Binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind cd154 and uses thereof

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION