WO1995008577A1 - Reciblage d'anticorps - Google Patents

Reciblage d'anticorps Download PDF

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Publication number
WO1995008577A1
WO1995008577A1 PCT/GB1994/002019 GB9402019W WO9508577A1 WO 1995008577 A1 WO1995008577 A1 WO 1995008577A1 GB 9402019 W GB9402019 W GB 9402019W WO 9508577 A1 WO9508577 A1 WO 9508577A1
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WIPO (PCT)
Prior art keywords
binding
antibody
domain
target
cells
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PCT/GB1994/002019
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English (en)
Inventor
Gregory Paul Winter
Kaspar Philippe Holliger
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Medical Research Council
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Priority claimed from GB939319969A external-priority patent/GB9319969D0/en
Priority claimed from PCT/GB1993/002492 external-priority patent/WO1994013804A1/fr
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to DK94926336T priority Critical patent/DK0720624T3/da
Priority to JP7509628A priority patent/JPH09503759A/ja
Priority to DE69414870T priority patent/DE69414870T2/de
Priority to EP94926336A priority patent/EP0720624B1/fr
Priority to AU76214/94A priority patent/AU680685B2/en
Publication of WO1995008577A1 publication Critical patent/WO1995008577A1/fr
Priority to US08/621,038 priority patent/US6589527B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/124—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes based on group I or II introns
    • C12N2310/1241—Tetrahymena
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/127—DNAzymes

Definitions

  • the present invention relates to retargetting of antibodies to a site or antigen for which they have no functional specificity under normal circumstances.
  • a method is described employing an antigen-specific binding substance which possesses at least two specificities; one specificity for the target site, the other capable of binding to part of an antibody molecule.
  • antibodies with no specificity for the antigen target may be brought into proximity with the antigen via the antigen-specific binding substance.
  • This principle is advantageous for re-targeting antibodies in the circulation to sites of disease within the body, e.g. tumours or sites of viral, bacterial or parasitic infection or combinations thereof.
  • This principle may also be applied to block inappropriate immune responses exemplified by autoimmune disease or hypersensitivity reactions.
  • Retargetting can be achieved with conventional bispecific antibodies, e.g.
  • Antibodies are proteins elaborated by B- lymphocytes to play a key role in the specific arm of the vertebrate immune system. This arises from their collective capacity to bind to an enormous diversity of antigen structures, with individual antibody molecules capable of precise specificity for their cognate antigen. The bulk of the antibody population is found in abundance in the blood and interstitial fluids, with minor types located at mucosal surfaces such as the intestinal lumen.
  • ADCC antibody directed cell-mediated cytotoxicity
  • the immune system operates natural checks and balances to prevent production of antibodies with specificity for the host, so-called ' self-antigens' .
  • the simplest antibody comprises four polypeptide chains inter-connected by disulphide bonds.
  • the light chains exist in two different forms called kappa (K) and lambda ( ⁇ ) .
  • K kappa
  • ⁇ lambda
  • Each chain has a constant region (C) and a variable region
  • V V-region domain
  • the basic IgG antibody is Y-shaped; the two arms (tip of the Y, each being an 'Fab' region) contain a VH and a VL domain associated with one another. It is this pair of V-regions that differ from one antibody to another (owing to amino acid sequence variations) , and which together are responsible for recognising the antigen and providing an antigen binding site (ABS) .
  • each V-region (whether heavy chain or light chain) consists of three complementarity determining regions (CDRs) separated by four framework regions (FR)
  • CDRs complementarity determining regions
  • FR framework regions
  • the CDR' s are the most variable part of the variable regions, and they perform the critical binding function.
  • the CDR regions are derived from many potential germline sequences via a complex process involving recombination, mutation and selection.
  • Example binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al.
  • V-domains and fragments containing V-domains
  • C-domains recruit effector functions.
  • the type of effector function recruited is largely governed by the class of C-domain (the isotype; M. Bruggemann et al J. Exp. Med. 166 1351 1987; L. Riechmann et al Nature 332 323 1988; J. Greenwood et al Eur. J. Immunol. 23 1098-1104 1993) .
  • the isotype M. Bruggemann et al J. Exp. Med. 166 1351 1987; L. Riechmann et al Nature 332 323 1988; J. Greenwood et al Eur. J. Immunol. 23 1098-1104 1993.
  • antibodies which have evolved to combat pathogens, bind to antigens on the pathogen and in so doing initiate an appropriate immune response aimed at destroying the invader.
  • C-domains of the IgGl ( ⁇ l) isotype can kill cells by triggering the complement cascade at the cell surface, resulting in lysis, or through binding C-domain receptors (Fc receptors) on specialised phagocytic and killer cells through ADCC.
  • antibodies of the IgG4 isotype ( ⁇ 4) appear actively to block a response. In the context of the present application this blocking is considered to be an effector function which can be recruited to a chosen target.
  • the binding sites for complement and Fc receptors map to the CH2 domain, sequence variation between CH2 domains of the different isotypes results in different strengths of interaction with complement and Fc receptors. All isotypes except IgE require that the C-domain is correctly glycosylated.
  • an appropriate immune response can be triggered when the antibody binds to antigen. Because the type of immune response is governed by the isotype, artificially-made antibodies can be endowed with appropriate constant regions to be used therapeutically, for example to destroy tumour cells (Hale, G et al. , Lancet ii, 1394-1399 (1988)) .
  • an antibody is to be used in such a way that requires recruitment of natural effector functions, then the antibody (except for the IgE isotype) must be manufactured in eukaryotic cells in order that the protein is glycosylated.
  • the type and extent of glycosylation varies with eukaryotic cell- type and culture conditions (Borys, M.C. et al. , Biotechnology 11, 720-725 (1993)) , and this can dramatically shorten their longevity in the circulation as well as adversely influencing recruitment of effector functions.
  • bispecific antibodies comprising at least two different antigen- binding sites. These are known as bispecific antibodies and they can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)) . Again using tumour killing as an example, one antigen binding site is directed against a tumour marker whereas the other can be directed against an antigen present on an effector cell-type. Bispecific antibodies incorporating a specificity for the T-cell co-receptor CD3 have been shown to inhibit tumour growth (Titus, J. A. et al . , J. Immunol.
  • the present applicants have realised that the direct interaction between the C-region of the antibody molecule and the effector results in limited activation of the immune system, and that it would be advantageous to activate (or indeed shut down) immune responses at a given target to a much greater degree.
  • modulation may be achieved by redirecting naturally occurring antibodies to a site or target for which they do not necessarily possess specificity.
  • the present applicants have realised in addition that this principle may be brought into effect through the use of binding substances which possess two or more specificities.
  • One of many examples is a bispecific antibody which incorporates specificity for other antibodies.
  • An antibody with specificity for a tumour cell and, for example, IgGl constant regions will bind to the tumour in si tu and accumulate IgGl antibodies present in the circulation, such that IgGl-specific effector functions are called down at the tumour site.
  • Antibodies in the serum of an individual are native to that person and therefore will be functional in activating complement or ADCC.
  • the principle of indirect recruitment is beneficial over direct interaction with effector cells for several main reasons.
  • a second reason why this arrangement is beneficial relates to control of serum half-life.
  • Correctly glycosylated antibodies have fairly reliable serum clearance rates, the rate of turnover being different for different isotypes .
  • IgGl has a serum half life in the order of 21 days, whereas on the other hand, IgG3 and IgE are turned over in a matter of 1-2 days.
  • the duration of the therapeutic effect may be controlled by the half-life of the administered bispecific antibody, e.g. diabody.
  • the half-life is likely to depend on its binding affinity (and kinetics) for the targetted antibody and antigen and on the serum concentration of the antibody target.
  • this approach can be used in site- specific immunosuppression.
  • Some antibodies such as IgG4, actively prevent immune responses by blocking the epitopes. Indeed, some parasites are known exploit this property to escape immune attack (A. Capron et al Mem. Inst. Oswaldo Cruz 87 Suppl.5 1-9 1992) , their antigens inducing antibody production of the correct specificity but with C-region isotypes incapable of inducing killing.
  • This principle can be extended within the scope of the present invention to uses such as alleviation of autoimmune disorders such as rheumatoid arthritis and myasthenia gravis.
  • the bispecific antibody has specificity for the target epitope and for example, IgG4.
  • patients may need to be screened for the ability of their immunoglobulin IgG4 to recruit effector functions, since the ability to do this appears to vary between individuals (Greenwood et al, supra) .
  • binding substances other than antibodies could be incorporated into a multiply- specific substance described herein.
  • examples include lectins, Fc-binding proteins such as protein A or protein G, receptors such as Fc receptors and components from the complement system.
  • Small molecules such as peptides, nucleic acids or naturally-occurring, partially synthetic or synthetic chemicals can also be used.
  • the aforementioned can be used in any order, number and combination to create multiply-specific substances described herein for use in therapy, diagnosis and scientific research. However, the use of antibody or a fragment thereof is preferred.
  • antibody fragments such as (Fab) 2 and diabodies lacking Fc regions, for reasons explained below.
  • the term antibody is used herein (and commonly in the art) to include antibody fragments, both synthetic and naturally occurring, i.e. molecules comprising an immunoglobulin binding domain.
  • the multiply- specific substance described herein is a bispecific antibody capable of binding to an appropriate antibody isotype.
  • “Diabodies” may be particularly advantageous for the purpose since they can be readily constructed and expressed in E. coli . Diabodies of appropriate binding specificities can be readily selected using phage display (WO 94/13804) from libraries.
  • one arm of the diabody is to be kept constant, for instance, with a specificity directed against an immunoglobulin light chain, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • Fab bispecific antibody molecule
  • scFv dimers or diabodies rather than whole antibodies.
  • the presence of Fc in whole antibody may cause complications in vivo arising from direction to non-specific sites, especially to Fc receptors.
  • Diabodies can be constructed without Fc, using only variable domains, avoiding this potential problem. In vi tro, the simplicity of making bispecific diabodies, as opposed to bispecific whole antibodies, makes them the antibody form of choice.
  • One aspect of the present invention provides a method of recruiting an antibody mediated effector function to a target, the method employing a multi- specific binding substance having anti-antibody binding specificity and binding specificity for a target.
  • a multi-specific binding substance having anti-antibody binding specificity and binding specificity for a target.
  • Binding of the multi- specific binding substance to the target and to antibody allows recruitment of antibody-mediated effector function to the target .
  • the binding substance is bound to antibody and to the target where it mediates the effector function of the antibody, generally, the effector function is the natural one of the bound antibody (e.g. ADCC, complement fixation or blocking, as discussed) .
  • the antibody may be any isotype, e.g.
  • the anti-antibody binding specificity of the binding substance is for the constant region of antibodies of one or more isotypes. Use of isotype- specific anti-antibody binding specificity enables choice of effector function recruited.
  • IgG3 and IgM are particularly valuable for complement fixation and IgGl and IgG3 are particularly valuable for ADCC.
  • the multi- specific binding substance will have binding specificity for a constant region of the isotype.
  • IgM molecules are particularly useful in agglutination assays.
  • IgG4 is the most suitable isotype for blocking antibodies, since it does not in general recruit antibody directed cellular cytotoxicity or complement. It may be valuable in some cases to use an isotype which does not activate complement to too great an extent, to prevent a toxic response.
  • IgGl may be particularly suitable.
  • Mast cells may be recruited via IgE antibodies. This may make them of value for cancer cell killing, but may limit their use for other applications (WO 92/11031) .
  • Specificity directed against light chains allows recruitment of a spectrum of antibody isotypes including those which activate complement or ADCC.
  • Anti-idiotype specificity may be used.
  • idiotypes such as that which may be provided by the commonly used DP-47 V gene germline sequence may be used to recruit any antibody where that idiotype is still recognisable in the mature antibody.
  • Specificity for idiotypes of specific antibodies is useful for using the antibody displaying that idiotype in an agglutination assay.
  • diabodies a diabody molecule directed against a cell surface marker and the idiotype of the antibody would bridge one cell to the antibody.
  • a second diabody molecule would be able to bind to another antigen binding site on the antibody and to a second cell thus crosslinking them.
  • IgM molecules would be particularly suitable for this, because they have 10 antigen binding sites per molecule.
  • the multi-specific binding substance may be bi ⁇ specific. It may be a bi-specific antibody or antibody fragment (as discussed) . Preferably, it is a "diabody", ie a multimer (e.g. dimer) of polypeptides each of which have a first domain comprising a binding region of an immunoglobulin heavy chain variable region and a second domain comprising a binding region of an immunoglobulin light chain variable region, the two domains being linked but not able to associate to form an antigen binding site.
  • the linkage may be by a peptide linker of -1 to about 10 amino acids (e.g. 5) .
  • polypeptides associate into multimers wherein the first domain of one polypeptide associates with the second domain of another polypeptide to form an antigen binding site.
  • a "diabody" for use in the present invention, refers to WO 94/13804.
  • scFv dimers wherein each polypeptide comprises heavy and light chain variable region binding regions which can associate intra-molecularly to form antigen binding site (in contrast to diabodies) because the peptide limber joining the two domains in each polypeptide is long enough, and (Fab) 2 .
  • a method according to the present invention may be carried out in vitro or in vivo where it may be a method of treatment of an individual for a condition wherein recruitment of antibody mediated effector function is, or is likely to be, of benefit.
  • Administration to an individual may be using any standard technique, the criteria for selection of a technique and selection of dosages, frequency of administration etc, being well known to those skilled in the art.
  • Administration of antibody is described, for example, in Hale et al, Lancet , ii, 1394-1399 (1988) , Simmons et al, Circulation, 89, 596-603 (1994) and Riethmuller et al, Lancet, 343, 1177-1183 (1994) .
  • a multispecific binding substance such as a bispecific antibody such as a diabody, in retargetting antibodies to recruit antibody effector function to treat target cells/tissue removed from a patient.
  • bone marrow from a patient with leukaemia may be taken and the cells treated, ex vivo, with a binding substance such as a bispecific diabody directed against a marker specific for the tumour cells and an immunoglobulin IgGl constant region, together with IgGl antibody and complement.
  • Tumour cells would then be specifically lysed and the whole cells remaining may be taken and returned to the patient.
  • ADCC may be used, the binding substance (e.g. diabody) together with IgGl and a preparation of killer cells being added to the bone marrow cells to lyse the tumour cells before returning the remaining cells to the patient.
  • the recruitment of effector function may be used in a diagnostic assay for the number of cells expessing a particular marker, e.g. tumour specific antigen, present in a sample e.g. of blood.
  • the degree of lysis would reflect the number of cells present. If an anti- IgM binding substance (e.g. diabody) plus IgM were used, the increased complement lysis would increase the sensitivity to detect very small numbers of tumour cells expressing cell surface markers.
  • Mediation of effector function may be caused or allowed according to conditions under which the invention is operated.
  • vi tro mediation may be caused by addition into the medium of required components of the effector system (e.g. complement) .
  • the effector system e.g. complement
  • serum for example, either in vi tro or in vivo all necessary components for effector function may be present ab ini tio, allowing effector function to be called down upon binding of the multi- specific binding substance to the target and to antibody.
  • a further aspect of the invention provides the use of a multi-specific binding substance in the recruitment of an antibody-mediated effector function to a target, the binding substance having an anti- antibody binding specificity and binding specificity for the target.
  • the use may be made of the multi- specific binding substance in any method provided by the present invention. Use may be in the manufacture of a medicament for recruitment of antibody mediated effector function, e.g. for the treatment of a condition wherein this is, or is likely to be, of benefit (see above) .
  • compositions comprising multi- specific binding substances as disclosed, and use of such compositions, are also provided by the present invention.
  • Such pharmaceutical compositions may comprise any suitable pharmaceutically acceptable excipient.
  • Another aspect of the present invention provides a multi-specific (e.g. bispecific) binding substance e.g. "diabody” (as disclosed) having an anti-antibody binding specificity (and a binding specificity for a target) .
  • Such a multi-specific binding substance has a binding site with anti-antibody binding specificity and a binding site with binding specificity for a target, and comprises a multimer of polypeptides, each polypeptide having a first domain comprising a binding region of an immunoglobulin heavy chain variable region and a second domain comprising a binding region of an immunoglobulin light chain variable region, the binding sites being formed by association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer.
  • the first domain of each polypeptide is unable to associate with the second domain of that polypeptide to form an antigen binding site.
  • Compositions comprising such a multimer, e.g. pharmaceutical compositions which may include a pharmaceutically acceptable excipient, are also provided by the invention.
  • the diabody may be a polypeptide dimer.
  • such a multispecific binding substance finds utility in a further aspect of the present invention, namely, a general method of targeting or recruiting an antibody to a target for which the antibody has no binding specificity, either with or without associated effector function.
  • a multi-specific (e.g. bispecific) diabody may be used in agglutination assays.
  • Multispecific binding substances such as the preferred diabodies (e.g. bispecific) may be used for coagulation of cells, bacteria or viruses, by making multiple interactions, as with diagnostic assays of agglutination of red blood cells, to determine for instance, blood cell types.
  • Diabodies with one arm directed against an antibody molecule may be used in different formats to link together cells, as illustrated in Figure 2.
  • a diabody or other multi-specific binding substance may be used which has one arm directed against a cell surface antigen and another directed against IgM.
  • the multivalent nature of IgM means that two or more diabody molecules may bind to the IgM molecule and thus crosslink between different blood cells. This IgM may be added as an extra reagent or it may be possible to use the IgM present in blood samples tested to promote the agglutination.
  • One arm may be directed against a cell surface antigen and the another directed against an idiotype commonly found in antibody molecules, such as antibodies directed against elements of the DP-47 VH gene, a gene segment commonly used in human antibodies (Tomlinson et al, J. Mol . Biol . 227 776-798 (1992)) .
  • IgM molecules with this idiotype would be particularly useful .
  • One arm may be directed against isotypes other than IgM for use in agglutination assays, but since these other antibodies are smaller, they may be less effective in agglutinating cells.
  • the target may be any antigen e.g. of bacterial, viral, fungal, protozoal origin or antigen on the surface of cells (e.g. cancer cells) , enabling recruitment of the natureal antibody encoded effector functions to the targets displaying those antigens (e.g. bacteria, viruses, parasites or tumor cells) by way of a multi- specific binding substance which has binding specificity for the antigen and anti-antibody binding specificity.
  • antigens e.g. bacteria, viruses, parasites or tumor cells
  • Figure 1 illustrates the use of a bispecific diabody to redirect an antibody such as IgGl or IgM to a cell surface marker, triggering complement.
  • Figure 2 illustrates the agglutination of red blood cells using a bispecific diabody directed against a blood cell antigen and an antibody, such as IgM with two or more identical epitopes.
  • One diabody molecule binds to the blood cell antigen and to the IgM molecule.
  • a second diabody molecule binds to the same IgM molecule and then binds to an antigen on a second blood cell, thus crosslinking and aggregating the blood cells.
  • Example 1 Preparation and characterisation of bispecific anti -2-phenyloxazol -5-one, anti -mouse lambda light chain diabody
  • a clone encoding a bispecific diabody directed against 2-phenyl-5-oxazolone and the mouse 1 light chain with a zero amino acid linker was prepared from DNA encoding an antibody against 2-phenyl-5-oxazolone derived from hybridoma NQ11 (anti-2-phenyloxazol-5-one; C. Berek et al Nature 316 412-418, 1985; P.
  • LS136 is a murine hybridoma directed against mouse antibody 1 light chains. It has been cloned in a diabody format using a 5 residue linker in the orientation VH- GGGGS-VL in the phagemid vector pUC119SfiNotmyc. The linker sequence was incorporated into the primer
  • VkCbaLink5BstEII and primer4 (Table 1) used to amplify the 5' end of VK.Primer 4 also introduces a Sad restriction site at the 5' end of the VK. A restriction site for BstEII was incorporated 5' of the linker sequence of primer VkCbaLink5BstEII and primer4 and also at the 3' end of VHlFOR-2 (E.S. Ward, D. Gussow, A.D. Griffiths, P.T. Jones and G. Winter,
  • LS136 VH and VL domain DNA was amplified by PCR from cDNA using primers pairs VH3Aba and VH1FOR-2, and VkCbaLink5BstEII and VK4FOR (T. Clackson, H.R. Hoogenboom, A.D. Griffiths and G. Winter, Nature 352, 624-628 1991) respectively using standard conditions and reamplified by using VH3AbaSfi and VHlfor-2 (for VH) and primer 4 (P. Holliger et al, supra) and Vk4foNot (for Vk) .
  • the product of the VH PCR reaction was digested with restriction enzymes Sfil and BstEII, and the product of the Vk PCR reaction was digested with restriction enzymes NotI and BstEII.
  • the VH and the VL domain DNA was simultaneously ligated into Sfil/NotI digested pUCll9SfiNotmyc in a molar ratio 3:3:1 (VH:VL:pUC119SfiNotmyc or pCantab6) and the resulting ligation mix used to transform E. coli TGI cells.
  • the VH and VL domain DNA was also ligated into Sfi/Not digested pCANTAB6 vector in the same way and transformed into E. coli HB2151 cells.
  • Recombinants were screened for inserts of correct size using primers LMB2 and LMB3 for recombinants in the vector pUC119SfiNotmyc or LMB3 and fdSeq for recombinants in the vector pCANTAB6.
  • Soluble diabody was expressed by growth of the pUC119SfiNotmyc clone at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/100 ⁇ g mL "1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown 3 hours 22°C.
  • the cells were centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold PBS/lmM EDTA and left on ice, 60 minutes.
  • the cell suspension was centrifuged (lOOOg for 10 minutes) and the diabody-containing supernatant used in ELISA as below. 50 ⁇ L periplasmic supernatant and 50 ⁇ L 3%
  • BSA/PBS was added to ELISA wells coated with mouse IgM ⁇ or mouse IgG2a ⁇ (both from Sigma) (lO ⁇ g mL" 1 in PBS) , blocked with 3% BSA/PBS.
  • a standard ELISA protocol was followed (H.R. Hoogenboom et al. , Nucl. Acids Res. 19, 4133-4137 1991) using detection of the myc-tag with the monoclonal antibody 9E10, and horseradish peroxidase conjugated anti mouse IgG (for IgM ⁇ ) and biotinylated anti mouse k chain and peroxidase-biotin-streptavidin complex (both Amersham) (for IgG2a ⁇ l) .
  • ELISA readings after 10 minutes were greater than 1.0.
  • the two antibody specificities LS136 (anti-mouse ⁇ antibody light chain) and NQ11 (anti-phOx) were combined in the bispecific diabody format fusing the VH and VL with a 5 amino acid linker VH-GGGGS-VL or directly with 0 linker in the orientation VH--VL in the phagemid vector pUC119SfiNotmyc.
  • the linker sequence was incorporated into the primers 4 and 3 (Table 1) used to amplify the 5' end of Vk and into the primers 7 and 6 (Table 1) used to amplify the 3' end of VH.
  • a restriction site for BstEII was incorporated 5' of the linker sequence of primer 3 and a restriction site for Sad was incorporated 5' of the linker sequence of primer 6. This would allow the assembled VH-linker and linker-VL fragments to be cloned in a 3-way ligation reaction into the expression vector pUC19LS136/5 BstEII/SacI.
  • VHNQll was amplified with primers 2 and 7 (Table 1) , the VkNQll was amplified with the primers 1 and 4 using scFvNQll cloned into fdDOG-1 as template.
  • the product of the VH PCR reaction was digested with restriction enzymes Ascl and Sad, and the product of the VL PCR reaction was digested with restriction enzymes Ascl and BstEII.
  • a vector fragment of diabody LS136/5 was cut with BstEII/ SacI and the VH and the VL domain DNA were simultaneously ligated with it in a molar ratio 3:3:1 (VH:VL:pUC119-LS136/5) .
  • the resulting ligation mix used to transform E. coli TGI cells. Recombinants were screened for inserts of correct size using primers LMB2 and LMB3 for PCR amplification of recombinant colonies.
  • VHNQll was amplified with primers 2 and 6 (Table 1) , the VkNQll was amplified with the primers 1 and 3 using scFvNQll cloned into fdDOG-1 as template.
  • the product of the VH PCR reaction was digested with restriction enzymes Ascl and SacI, and the product of the VL PCR reaction was digested with restriction enzymes Ascl and BstEII.
  • a vector fragment of diabody LS136/5 (see above) was cut with BstEII/SacI and the VH and the VL domain DNA were simultaneously ligated with it in a molar ratio 3:3:1 (VH:VL:pUC119-LS136/5) .
  • the resulting ligation mix used to transform E. coli TGI cells.
  • Recombinants were screened for inserts of correct size using primers LMB2 and LMB3 for PCR amplification of recombinant colonies.
  • Soluble diabody was expressed by growth at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL' 1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown 3 hours 22°C.
  • the cells were centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold PBS/lmM EDTA and left on ice, 60 minutes.
  • the cell suspension was centrifuged (lOOOg for 10 minutes) and the diabody- containing supernatant used in ELISA for ⁇ light chain as above or for phOx as in example 1 of WO 94/13804.
  • ELISA signals of greater than 1.0 were obtained after 10 min.
  • Example 2 Preparation and characterisation of bispecific anti -hen egg lysozyme, anti -mouse lambda light chain diabody, and demonstration of complement lysis
  • a clone encoding a bispecific diabody directed against hen egg lysozyme (HEL) and the mouse ⁇ light chain with a five and a zero amino acid linker was prepared from DNA encoding a single chain Fv antibody fragment against hen egg lysozyme (HEL) derived from the V genes from the anti-HEL antibody HyHELlO (T.B. Lavoie, W.B. Drohan and S.J. Smith-Gill J. Immunol . 148 503-513 1992; gift of Sandra Smith-Gill) and from DNA derived from a hybridoma LS136 directed against a mouse lambda light chain using the methodology essentially as described in example 1 and P. Holliger et al (1993 supra) .
  • a bivalent diabody directed against the mouse lambda light chain described essentially as in example 1 was used as an intermediate step.
  • VH and VL domains of the diabody were prepared and digested exactly as described in example 1 of WO 94/13804.
  • the VH and VL domain DNA was simultaneously ligated into Sfil/Not I digested pCANTAB5-E (Pharmacia) in a molar ratio of 3:3:1 and the resulting ligation mix used to transform E. coli HB2151 cells. Recombinants were screened for inserts of the correct size using primers fdSeq and LMB3.
  • Soluble diabody was expressed by growth at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL "1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown for 3 hours at 22°C.
  • the cells were centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold
  • BSA/PBS was added to ELISA wells coated with mouse IgM ⁇ or mouse IgG2a ⁇ (both from Sigma) (lO ⁇ g mL "1 in PBS) , blocked with 3% BSA/PBS.
  • a standard ELISA protocol was followed (H.R. Hoogenboom et al . , Nucl . Acids Res. 19, 4133-4137 1991) using detection of the E-tag with the monoclonal anti-E tag antibody conjugated to HRP (Ray Mernaugh, Pharmacia) ELISA readings after 10 minutes were greater than 1.0.
  • the two antibody specificities LS136 (anti-mouse ⁇ antibody light chain) and HyHELlO (anti-lysozyme) were combined in the bispecific diabody format fusing the VH and VL domains with a 5 amino acid linker VH- GGGGS-VL or directly with 0 linker in the orientation VH--VL in the phagemid vector pCANTAB5-E (Pharmacia) .
  • the linker sequence was incorporated into the primers 3 and 4 (Table 1) used to amplify the 5' end of Vk and into the primers 6 and 7 (Table 1) used to amplify the 3' end of VH.
  • a restriction site for BstEII was incorporated 5' of the linker sequence of primers 3 and 4 and a restriction site for SacI was incorporated 5' of the linker sequence of primer 6 and 7. This would allow the assembled VH-linker and linker-VL fragments to be cloned in a 3-way ligation reaction into the expression vector pCANTAB5-E LS136/5 BstEII/SacI.
  • VHHyHELlO was amplified with primers 2 and 7 (Table 1) and VkHyHELlO was amplified with the primers 1 and 4 for the 5 amino acid linker diabody LS136/HyHEL10/5 using scFvHyHELlO cloned into pUC119 as template.
  • the product of the VH PCR reaction was digested with restriction enzymes Ascl and SacI, and the product of the VL PCR reaction was digested with restriction enzymes Ascl and BstEII.
  • a vector fragment of diabody LS136/5 (see above) was cut with BstEII/ SacI and the VH and the VL domain DNA were simultaneously ligated with it in a molar ratio 3:3:1 (VH:VL:pCANTAB5-E LS136/5) .
  • the resulting ligation mix used to transform E. coli HB2151 cells.
  • Recombinants were screened for inserts of correct size using primers fdSeq and LMB3 for PCR amplification of recombinant colonies.
  • VHHyHELlO was amplified with primers 2 and 6 (Table 1) , the VkHyHELlO was amplified with the primers 1 and 3 using scFvHyHELlO cloned into pUC119 as template.
  • the product of the VH PCR reaction was digested with restriction enzymes Ascl and SacI, and the product of the VL PCR reaction was digested with restriction enzymes Ascl and BstEII.
  • a vector fragment of diabody LS136/5 was cut with BstEII/ SacI and the VH and the VL domain DNA were simultaneously ligated with it in a molar ratio 3:3:1 (VH:VL:ppCANTAB5-E-LS136/5) .
  • the resulting ligation mix used to transform E. coli HB2151 cells.
  • Recombinants were screened for inserts of correct size using primers fdSeq and LMB3 for PCR amplification of recombinant colonies.
  • Soluble diabody was expressed by growth at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL '1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown 3 hours 22°C.
  • the cells were centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold PBS/lmM EDTA and left on ice, 60 minutes.
  • the cell suspension was centrifuged (lOOOg for 10 minutes) and the diabody- containing supernatant used in ELISA for ⁇ light chain as above or for hen egg lysozyme as in P. Holliger et al (Proc. Natl. Acad. Sci. USA 90 6444-6448) , 1993.
  • ELISA signals of greater than 1.0 were obtained after 10 min.
  • Soluble diabody was expressed by growth at 37°C.
  • the column was washed with 10 column volumes of PBS, 5 column volumes of 0.5M NaCl/O.lmM Tris, pH8.5 and protein was eluted with lOOmM triethylamine into ice-cold 1M Tris, pH7.5 and dialysed extensively against PBS/0.2mM EDTA.
  • the ability of the LS136/HyHEL10/5 diabody to retarget antibodies and utilise their effector functions was determined using a complememt lysis assay.
  • RBC's Human red blood cells
  • Complement lysis assay Red blood cells coated with lOmg/ml HEL were washed three times in complement fixation diluent (Oxoid, Basingstoke) and 50 ⁇ l of a 1% suspension added to wells of a 96 well microtitre plate. Dilutions of the purified diabody LS136/HyHEL10/5 (from lmg/ml to lOng/ml; 50 ⁇ l) were added and incubated for 20 min at room temperature. The cells were pelleted by a centrifugation at 2000rpm for 5 minutes and the supernatant was discarded.
  • the cells were resuspended in dilutions of an immunoglobulin IgM with a lambda light chain (IgM ⁇ ) that is not specific for an antigen in the assay (Myeloma MOPC 104E) and incubated for 20 minutes at room temperature.
  • the cells were again pelleted by a centrifugation at 2000rpm for 5 min and the supernatant was discarded. Now the cell pellet was washed once with complement fixation diluent and the cells were pelleted again and resuspended in a 1 in 20 dilution of guinea pig complement (prepared from guinea pig serum after agglutination of red blood cells and incubated for 30 minutes at 37°C.
  • guinea pig complement prepared from guinea pig serum after agglutination of red blood cells and incubated for 30 minutes at 37°C.
  • the complement assay was also performed by simply mixing antigen-coated red blood cells, diabody and IgM ⁇ in a volume of 150 ⁇ l of guinea pig complement, diluted 1/5 in complement fixation diluent. Efficient hemolysis was again observed after incubation at 37°C for 30 minutes. In the absence of diabody this assay set up resulted in some background hemolysis.
  • the diabody is effective in retargetting antibody effector functions of antibodies not specific for the antigen to cells with the antigen on their surface.
  • Example 3 Preparation and characterisation af an anti -CEA, anti -mouse lambda light chain diabody and demonstration of complement mediated lysis of a tumour cell .
  • a clone encoding a bispecific diabody directed against carcinoembryonic antigen (CEA) and the mouse ⁇ light chain with a five amino acid linker was prepared from DNA encoding the variable regions derived from the murine anti-CEA antibody MFE23 which binds the tumour specific antigen carcinoembryonic antigen (CEA) and from the DNA derived from a hybridoma LS136 directed against a mouse lambda light chain using the methodology essentially as described in example 1 and P. Holliger et al (1993 supra) .
  • a bivalent diabody directed against the mouse lambda light chain described in examples 1 and 2 was used as an intermediate step in the construction.
  • the MFE23 anti-CEA scFv clone described in PCT/GB93/02492 was first mutated to remove an internal BstEII site in the VL domain by in vitro mutagenesis using oligonucleotide CEA23-BstE (Table 1) and the Sculptor kit (Amersham International) .
  • VHMFE23 was amplified with primers 2 and 7 (Table 1) and VkMFE23 was amplified with primers 1 and 4 for the 5 aminoacid linker diabody LS136/MFE23/5 using the mutated MFE23 anti-CEA scFv as template.
  • VH PCR reaction was digested with restriction enzymes Ascl and SacI
  • the product of the VL PCR reaction was digested with restriction enzymes Ascl and BstEII.
  • Vector pCANTAB-5E DNA encoding the diabody LS136/5 was cut with BstEII/ SacI and the VH and the VL domain DNA was simultaneously ligated with it in a molar ratio 3:3:1 (VH:VL:pCANTAB5-E LS136/5) .
  • the resulting ligation mix was used to transform E. coli HB2151 cells.
  • Recombinants were screened for inserts of correct size using primers fdSeq and LMB3 for PCR amplification of recombinant colonies.
  • the Sfil-NotI fragment encoding the diabody was then subcloned into the vector pUC119 SfiNot-hismyc for expression.
  • Soluble diabody was expressed by growth at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL "1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown for 3 hours 22°C.
  • the cells are centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold PBS/lmM EDTA and left on ice, 60 minutes.
  • the cell suspension was centrifuged (lOOOg for 10 minutes) and the diabody- containing supernatant used in ELISA for ⁇ light chain as in examples 1 and 2 or for CEA as described by A.D. Griffiths et al (EMBO J. 12_725-734, 1993) .
  • ELISA signals of greater than 1.0 were obtained after 10 min.
  • Soluble diabody was expressed by growth at 37°C.
  • Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL" 1 ampicillin are induced by adding IPTG to a final concentration of ImM IPTG and grown for 24 hours at 22°C.
  • the cells were centrifuged (lOOOg for 10 min) and the cell pellet resuspended and supernatant filtered through a O.l ⁇ m filter and concentrated by cross-flow filtration (filter cutoff lOkD) .
  • the concentrate was purified using immobilised metal affinity chromatography (IMAC) using nickel-NTA agarose (Qiagen cat. no. 30210) using the manufacturer's instructions and dialysed extensively against PBS/EDTA.
  • IMAC immobilised metal affinity chromatography
  • Complement lysis assay The ability of the LS136/MFE23/5 diabody to retarget antibodies and utilise their effector functions is determined using a complement lysis assay using Chromium ( 51 Cr) release.
  • 2xl0 6 LS 174T target cells (ATCC CL 188, US pat. 4,288,236) are harvested after desorption and washed with RPMI 1640 medium containing 10% fetal calf serum. After centrifugation of the cells the pellet is labelled with 51 Cr (200 ⁇ Ci) for 1 hour at 37°C. After 2 washes in RPMI 1640 medium the target cells (5000 cells per assay) are aliquotted into culture wells.
  • Dilutions of the purified diabody LS136/MFE23/5 (from lmg/ml to lOng/ml; 50 ⁇ l) are added and incubated for 20 min at room temperature.
  • the cells are pelleted by a centrifugation at 2000rpm for 5 minutes and the supernatant discarded.
  • the cells are resuspended in dilutions of an immunoglobulin IgM with a lambda light chain (IgM ⁇ ) that is not specific for an antigen in the assay (Myeloma MOPC 104E) and incubated for 20 minutes at room temperature.
  • the cells are again pelleted by a centrifugation at 2000rpm for 5 min and the supernatant discarded.
  • the cell pellet is washed once with complement fixation diluent and the cells are pelleted again and resuspended in a 1 in 20 dilution of guinea pig complement (prepared from guinea pig serum after agglutination of red blood cells) and incubated for 30 minutes at 37°C.
  • Cell debris is pelleted by a centrifugation at 4000rpm for 5 minutes and the supernatant was transferred to another microtitre plate
  • the cells are spun and half the supernatant (lOO ⁇ l) is collected and chromium release is determined in a gamma counter. Each sample point is done in triplicate and the percentage of specific lysis is calculated as:
  • Spontaneous release is measured from target cells in assay medium alone and maximum release is measured after lysis of an equivalent number of target cells in 1M HC1.
  • Example 4 Lysis of a tumour cell by antibody directed cell -mediated cytotoxicity directed by a diabody directed against CEA and a mouse lambda light chain
  • ADCC is a natural antibody encoded effector function brought about by binding of antibody Fc region to Fc receptors .
  • Cells coated by antibodies are killed through lysis by a range of mononuclear cells.
  • Mononuclear cells were isolated from Balb/c mose spleen on Ficoll gradient and grown for 3 days in RPMI (Russel Park Memorial Institute) /10% Fetal calf serum (FCS) at 37 0 C in tissue culture flasks pretreated with a mitogenic anti-CD3 antibody (e.g. 2C11 at 50 ⁇ g/ml in PBS for 24h and washed 4 times with PBS to remove unbound antibody) . Then they were transferred to untreated flasks for 3-7 days for expansion in RPMI/5% FCS and 10 units /ml recombinant interleukin 2 (IL-2) at 37°C.
  • RPMI Ressel Park Memorial Institute
  • FCS Fetal calf serum
  • 2xl0 6 LS 174T target cells (ATCC CL 188, US pat. 4,288,236) are harvested after desorption and washed with RPMI 1640 medium containing 10% fetal calf serum. After centrifugation of the cells the pellet is labelled with 51 Cr (200 ⁇ Ci) for 1 hour at 37°C. After 2 washes in RPMI 1640 medium the target cells (5000 cells per assay) are aliquotted into culture wells. Dilutions of the purified diabody LS136/MFE23/5 (from lmg/ml to lOng/ml; 50 ⁇ l) are added and incubated for 20 min at room temperature.
  • the cells are pelleted by a centrifugation at 2000rpm for 5 minutes and the supernatant discarded.
  • the cells are resuspended in dilutions of an immunoglobulin IgGl with a lambda light chain (IgGl ⁇ ) that is not specific for an antigen in the assay (Myeloma 3C52'CL: anti-4-hydroxy-3-phenylacetyl (NIP)) and incubated for 20 minutes at room temperature.
  • IgGl ⁇ lambda light chain
  • K-cells were washed to remove IL-2 and are then added to give effector:target (K-cells:LS174T) ratios between 50:1 and 10:1 and incubated for 4h at 37°C.
  • the cells were spun and half the supernatant (lOO ⁇ l) is collected and chromium ( 51 Cr) release is determined in a gamma counter. Each sample point is done in triplicate and the percentage of specific lysis is calculated as:
  • Spontaneous release is measured from target cells in assay medium alone and maximum release is measured after lysis of an equivalent number of target cells in 1M HCl.
  • the degree of lysis is found to titrate with both dilutions of the LS136/MFE23/5 diabody and of the Myeloma 3C52'CL IgGl ⁇ . No lysis (apart from background lysis) is observed leaving out either the diabody or the IgGl ⁇ or using a phOx-BSA coated red blood cell control in place of the tumour cell .
  • the diabody can retarget the ADCC activity triggered by the IgGl ⁇ antibody to a tumour cell encoding an antigen to which one arm of the diabody is directed.
  • Example 5 In vivo retargetting of antibody to mediate turnover lysis .
  • the bispecific diabody LS136/MFE23/5 is useful for treatment of a xenografted CEA + adenocarcinoma LS174T in nude mice.
  • Nude mice lack T-cells and allow the growth of xenografted human tumors. They do however have normal B- cells and normal serum Ig levels and they show normal T- independent immune responses e.g. some antibody responses.
  • diabody is expressed and purified as described in Ex 3. and additionally purified on Pharmacia Superdex7TM 16/60 seizing column to remove endotoxin (LPS) .
  • Balb/c nude mice are injected (for example i.v.) with a significant number of LS174T tumour cells (e.g. 5000) on day one are treated with single or muliple i.v. injections of the desired amount of diabody (e.g. lOO ⁇ g) in phosphate buffered saline (PBS) at a later point in time.
  • LS174T tumour cells e.g. 5000
  • PBS phosphate buffered saline
  • serum Ig is in excess to the diabody and consequently the great majority of Ig will only complex with one diabody.
  • more than one diabody complexes with any species of serum Ig in order to have advantages high avidity binding to the target antigen. This may be achieved by incubation with serum Ig prior to injection.
  • a convenient amount of serum from the mouse e.g. lOO ⁇ l, total serum Ig ⁇ concentrations in naiveBalb/c mouse is ⁇ lmg/ml
  • a convenient amount of serum from the mouse e.g. lOO ⁇ l, total serum Ig ⁇ concentrations in naiveBalb/c mouse is ⁇ lmg/ml
  • diabody e.g. lOO ⁇ g
  • PBS phosphate buffered saline
  • the bispecifc LS136/MFE23/5 diabody targets ⁇ light chain bearing Ig. which amounts to ⁇ 5% of total serum Ig.
  • Ig ⁇ can be greatly boosted by immunisation with certain antigens that elicit T-cell independent responses e.g. dextran.
  • the efficiency of treatment regimes may be increased if Ig ⁇ levels are boosted in such a way prior to administration of the diabody.

Abstract

On recible des anticorps pour les diriger sur une cible pour laquelle ils n'ont aucune spécificité fonctionnelle dans des conditions normales. On utilise une substance de liaison multispécifique dotée d'une spécificité de liaison à l'égard de la cible et à l'égard d'un anti-anticorps. La substance de liaison peut comprendre un site de fixation immunoglobuline antigène et peut être un 'dianticorps' (anticorps à double spécificité). En fonction de l'anticorps lié, on utilise des fonctions effectrices telles que le Complément, la cytotoxicité cellulaire dirigée par de anticorps (ADCC), et le blocage immun pour agir sur la cible. On peut citer à titre d'exemples de cibles des cellules humaines. Des applications in vivo et in vitro servent d'illustration, y compris la lyse des cellules tumorales et l'agglutination des globules rouges.
PCT/GB1994/002019 1993-09-22 1994-09-16 Reciblage d'anticorps WO1995008577A1 (fr)

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JP7509628A JPH09503759A (ja) 1993-09-22 1994-09-16 抗体のリターゲッティング
DE69414870T DE69414870T2 (de) 1993-09-22 1994-09-16 Antikoerper mit geaenderter zielrichtung
EP94926336A EP0720624B1 (fr) 1993-09-22 1994-09-16 Reciblage d'anticorps
AU76214/94A AU680685B2 (en) 1993-09-22 1994-09-16 Retargeting antibodies
US08/621,038 US6589527B1 (en) 1993-09-22 1996-03-22 Retargetting antibodies

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GB939319969A GB9319969D0 (en) 1993-09-22 1993-09-22 Binding proteins iv
GBPCT/GB93/02492 1993-12-03
PCT/GB1993/002492 WO1994013804A1 (fr) 1992-12-04 1993-12-03 Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
GB9412166.2 1994-06-17
GB9412166A GB9412166D0 (en) 1993-09-22 1994-06-17 Retargetting antibodies
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WO1998003670A1 (fr) * 1996-07-23 1998-01-29 Tanox Pharma B.V. Induction de la tolerance aux lymphocytes t au moyen d'une molecule soluble pouvant bloquer simultanement deux mecanismes d'action costimulants
WO1998008875A1 (fr) * 1996-08-28 1998-03-05 Viva Diagnostika Diagnostische Produkte Gmbh Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie
WO1998044001A1 (fr) 1997-03-27 1998-10-08 Commonwealth Scientific And Industrial Research Organisation Reactifs polyvalents presentant une avidite elevee et une specificite multiple
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US6660842B1 (en) 1994-04-28 2003-12-09 Tripep Ab Ligand/receptor specificity exchangers that redirect antibodies to receptors on a pathogen
US6933366B2 (en) 1996-12-27 2005-08-23 Tripep Ab Specificity exchangers that redirect antibodies to bacterial adhesion receptors
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US7318926B2 (en) 2003-02-06 2008-01-15 Tripep Ab Glycosylated specificity exchangers
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US7122646B2 (en) 1992-12-04 2006-10-17 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
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US5869232A (en) * 1994-04-28 1999-02-09 Tripep Ab Antigen/antibody specificity exchanger
US6245895B1 (en) 1994-04-28 2001-06-12 Tripep Ab Antigen/antibody specificity exchanger
US6469143B2 (en) 1994-04-28 2002-10-22 Tripep Ab Antigen/antibody specificity exchanger
US6660842B1 (en) 1994-04-28 2003-12-09 Tripep Ab Ligand/receptor specificity exchangers that redirect antibodies to receptors on a pathogen
US7019111B2 (en) 1994-04-28 2006-03-28 Tripep Ab Glycosylated ligand/receptor specificity exchangers specific for bacterial adhesion receptors
US8012754B2 (en) 1995-04-20 2011-09-06 Genentech, Inc. Antibody compositions
US6040137A (en) * 1995-04-27 2000-03-21 Tripep Ab Antigen/antibody specification exchanger
WO1998003670A1 (fr) * 1996-07-23 1998-01-29 Tanox Pharma B.V. Induction de la tolerance aux lymphocytes t au moyen d'une molecule soluble pouvant bloquer simultanement deux mecanismes d'action costimulants
WO1998008875A1 (fr) * 1996-08-28 1998-03-05 Viva Diagnostika Diagnostische Produkte Gmbh Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie
US6933366B2 (en) 1996-12-27 2005-08-23 Tripep Ab Specificity exchangers that redirect antibodies to bacterial adhesion receptors
EP1997514A1 (fr) 1997-03-27 2008-12-03 Avipep Pty Limited Polyvalent à forte avidité et réactifs poly-spécifiques
WO1998044001A1 (fr) 1997-03-27 1998-10-08 Commonwealth Scientific And Industrial Research Organisation Reactifs polyvalents presentant une avidite elevee et une specificite multiple
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US7335359B2 (en) 2003-02-06 2008-02-26 Tripep Ab Glycosylated specificity exchangers
US8658179B2 (en) 2003-02-06 2014-02-25 Chrontech Pharma Ab Glycosylated specificity exchangers
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US7332166B2 (en) 2003-02-06 2008-02-19 Tripep Ab Glycosylated specificity exchangers
US8303956B2 (en) 2003-02-06 2012-11-06 Chrontech Pharma Ab Glycosylated specificity exchangers
US7534435B2 (en) 2003-02-06 2009-05-19 Tripep Ab Glycosylated specificity exchangers
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US10307481B2 (en) 2005-07-25 2019-06-04 Aptevo Research And Development Llc CD37 immunotherapeutics and uses thereof
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US9101609B2 (en) 2008-04-11 2015-08-11 Emergent Product Development Seattle, Llc CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
WO2011044133A3 (fr) * 2009-10-05 2011-08-25 Opsonic Therapeutics Inc. Molécules adaptatrices de haute affinité pour rediriger la spécificité d'anticorps
WO2011044133A2 (fr) * 2009-10-05 2011-04-14 Opsonic Therapeutics Inc. Molécules adaptatrices de haute affinité pour rediriger la spécificité d'anticorps
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