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WO2001085795A1 - Anticorps de recombinaison trivalents a regions variables stabilisees par des ponts disulfure - Google Patents

Anticorps de recombinaison trivalents a regions variables stabilisees par des ponts disulfure Download PDF

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Publication number
WO2001085795A1
WO2001085795A1 PCT/EP2001/005161 EP0105161W WO0185795A1 WO 2001085795 A1 WO2001085795 A1 WO 2001085795A1 EP 0105161 W EP0105161 W EP 0105161W WO 0185795 A1 WO0185795 A1 WO 0185795A1
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Prior art keywords
recombinant antibody
construct according
antibody construct
binding
antigen
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PCT/EP2001/005161
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German (de)
English (en)
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Stefan Dübel
Andreas Schmiedl
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Universität Heidelberg
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Priority to AU60285/01A priority Critical patent/AU6028501A/en
Priority to EP01933941A priority patent/EP1287036A1/fr
Publication of WO2001085795A1 publication Critical patent/WO2001085795A1/fr

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)

Definitions

  • the present invention relates to recombinant antibody constructs, processes for their preparation, pharmaceutical compositions and diagnostic agents which contain these recombinant antibody constructs, and the use of these recombinant antibody constructs for the diagnosis and treatment of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxae.
  • Bispecific antibodies are best prepared recombinantly, since in the conventional quadroma or hybrid hybridoma technique a mixture of 10 different products occurs, from which the only correct one can only be separated with considerable effort (Milstein and Cuello, 1983).
  • bispecific antibodies are necessary as agents for new types of therapeutic procedures that could not be carried out without such molecules. They are used in particular to stimulate the immune response against tumor cells (Segal et al., 1999; Hombach et al., 1993; Manzke et al, 1999). T-lymphocytes are specifically introduced to tumor cells and often additionally activated by one of the binding arms of the bispecific agent.
  • Various molecular formats have been developed for the production of therapeutic antibodies in general and bispecific antibodies in particular (Breitling and Dübel, 1997; Carter and Merchant, 1997).
  • minibody about 90 kDa
  • scFv fragments two identical scFv fragments, which dimerize through fused CH3 domains.
  • This construct is bivalent, but monospecific.
  • the production of bispecific antibodies according to this pattern is difficult because it can lead to homologous pairings and thus to undesirable and difficult-to-separate by-products.
  • CH1 and CL (k a pp a ) domains for dimerization solved the problem of by-products in the production of bispecific minibodies, but the affinity is limited by the monovalence of the respective antigen binding sites.
  • Bispecific diabodies such as those often produced for therapy, are smaller than the pharmacokinetically optimal construct for tumor therapy in that they are composed of only two antigen-binding antibody fragments (Hollinger et al., 1993, 1996, 1999; Arndt et al., 1999 ; Helfrich ef a /., 1998).
  • dsFv-dsFv 'antibodies ie constructs from two disulphide bridge-stabilized Fv fragments, which are linked by a short linker peptide (Breitling and Dübel, 1997; Schmiedl et al., 2000) eliminates the stability problems of the diabodies , the construct also has a too low molecular weight for optimal tumor localization.
  • the present invention is therefore based on the object of providing new antibody constructs for diagnostic, therapeutic and research purposes which are intended to eliminate the disadvantages of the prior art listed above.
  • a recombinant antibody construct with at least three antigen-binding antibody fragments is provided, at least one antigen-binding antibody fragment having a disulfide bridge between the variable domains.
  • the recombinant antibody construct according to the invention is preferably at least bispecific, the number of specificities depending on the diversity of the antigen-binding antibody fragments.
  • the recombinant antibody construct according to the invention can have three different antigen-binding antibody fragments, which is therefore trispecific.
  • the valence of the recombinant antibody constructs according to the invention generally corresponds to the number of antigen-binding antibody fragments.
  • the recombinant antibody construct according to the invention preferably has a molecular weight of approximately 90 kDa.
  • the two polypeptide chains of the antigen-binding antibody fragment with at least one disulfide bridge are preferably different from one another.
  • these polypeptide chains are selected from the variable domains (hereinafter also referred to as “Fv”) of immunoglobulins, such as IgA, IgD, IgE and IgG, and biologically active fragments of these variable domains.
  • Fv variable domains
  • biological active fragment here means that these fragments have or result in an essentially identical or improved antigenic binding property, such as the naturally present Fvs.
  • these two polypeptide chains can be in any combination of the variable parts of the H and L chains from one immunoglobulin ("monoclonal") or two different immunoglobulins of one class, such as IgG, or different classes, such as IgG and IgA , occurrence.
  • the recombinant antibody construct can have one or more antigen-binding antibody fragments (hereinafter also referred to as “scFv”) which have a polypeptide chain with two peptide sequences which are different from one another and from the primary structures of the variable domains of immunoglobulins and the biologically active portions thereof are selected.
  • scFv antigen-binding antibody fragments
  • biologically active portion here means that these portions have or result in substantially identical or improved antigenic binding properties to that of naturally occurring Fvs.
  • the recombinant antibody construct according to the invention is linked to one another in accordance with the number n of antigen-binding antibody fragments present via (n-1) peptide linkers.
  • n-1 the number of antigen-binding antibody fragments present via (n-1) peptide linkers.
  • Antibody fragments two peptide linkers present can also contain, in part or in place of the variable parts of the H and L chains of immunoglobulins, at least one corresponding domain of another protein, in particular a member of the immunoglobulin superfamily, for example variable regions or parts of T cell receptors , MHC proteins (class I and II), cell surface proteins, cytokines or growth factors.
  • the recombinant antibody construct according to the present invention can further comprise at least one effector domain, which for example consists of interleukin 2, interferon- ⁇ , interferon- ⁇ , B7.1, B7.2, TNF- ⁇ , complement cascade components, toxins such as ricin, PE, DT , and RNAsen and biologically active fragments thereof are selected.
  • effector domain includes domains that catalyze or inhibit a biochemical reaction.
  • the recombinant antibody construct according to the invention can further contain at least one radioactive substance which is covalently bound by chemical reaction.
  • cytokines for example cytokines, antibodies, receptors, complement proteins or low molecular weight compounds
  • cytokines for example cytokines, antibodies, receptors, complement proteins or low molecular weight compounds
  • the destruction of which of the cells in the vicinity of the V-bodies is caused by the body's own or endogenous effectors, for example by other cells such as Immune system cells, e.g. T lymphocytes or macrophages, or by molecules of the immune system, such as complement proteins.
  • Anchor domains can also be added to the recombinant antibody construct according to the invention, which allow coupling to one of the above-mentioned effectors or effector domains.
  • anchor domains of this type are avidin, streptavidin, or biologically active derivatives or mutations thereof, biotin, streptavidin / avidin binding peptides, bacterial immunoglobulin binding molecules such as protein A, protein G, protein H, protein L, calmodulin, calmodulin binding molecules, fragments of RNAsen , non-natural sequences that bind a fragment of RNAsen, and leucine zippers.
  • polyethylene molecules can be covalently coupled to the recombinant antibody construct according to the invention.
  • Figure 4 is a graphical representation of the vector used in Example 3 to produce a bispecific antibody construct.
  • Another object of the present invention is a method for producing the recombinant antibody constructs defined above, wherein the antigen-binding antibody fragments by means of recombinant DNA technology and introduction of a disulfide bridge into at least one antigen-binding antibody fragment via suitable mutagenesis, for example "site-specific mutagenesis" or PCR Mutagenesis of the nucleic acid sequences coding for the two polypeptide chains.
  • suitable mutagenesis for example "site-specific mutagenesis” or PCR Mutagenesis of the nucleic acid sequences coding for the two polypeptide chains.
  • the antibody constructs according to the invention are recombinantly produced, for example, in E. coli, insect cell cultures, Pichia patoris, CHO cell culture cells or transgenic plants.
  • vectors are produced by means of DNA cloning according to the prior art, which, after transfection into the corresponding host cells or organisms, enable the subunits to be produced recombinantly.
  • the DNA sequences which code for the subunits of the V-bodies are constructed for this purpose by PCR mutagenesis or other methods according to the prior art such that at least one of the Fv regions involved is mutated in the recombinantly produced protein (i) of two at the contact point between VH and V * . opposite amino acid positions (one each in VH and V L ) to cysteine can be stabilized with an interface disulfide bridge (Brinkmann et al., 1993, Proc. Natl. Acad.
  • the resulting molecular mass corresponds to the advantageous minibody size.
  • the optimal arrangement (sequence) of the different VH and V * _ chain genes in relation to each other in the vector can be optimally selected individually for each construct. Possible by-products of the recombinant production which can arise can be separated off using chromatographic methods known in the art.
  • nucleic acids which are at least partially those for the invention contain recombinant antibody constructs or antigen-binding antibody fragments encoding nucleic acid sequences, as well as suitable methods known in the art for the expression of the recombinant antibody constructs or antigen-binding antibody fragments.
  • the method according to the invention provides antibody constructs which, for example, enable a bivalent binding to a tumor or effector and thus a much higher off-rate during dissociation. This results in a longer localization in the target tissue and also does without any dimerization domains. As a result, excellent binding is achieved at a suitable molecular weight, preferably about 90 kDa, which cannot be achieved in the same way with any of the constructs previously described in the prior art.
  • the molecular design described here therefore combines for the first time the previously separately found positive properties of previous constructs:
  • the antibody constructs according to the invention in particular do not contain constant domains, since these domains are no longer necessary for dimerization, and the constructs according to the invention are sufficiently stabilized through the use of //7terc/.a/t.-disulfide bridges.
  • the antibody constructs according to the invention are therefore also referred to below as "V-bodies", since they may be composed exclusively of variable regions except for short spacer peptides.
  • the method according to the invention enables the construction of the V bodies from human V regions. This enables the entire construct to be made from exclusively human protein fragments - this makes one Avoid immune reaction (e.g. HAMA) against the therapeutic agent, as occurs in constructs that achieve their bivalence or bispecificity by fusing the antigen-binding domains to heterologous fusion partners (such as streptavidin, zipper motifs, protein A fusions, etc.).
  • HAMA e.g. antigen-binding domains
  • heterologous fusion partners such as streptavidin, zipper motifs, protein A fusions, etc.
  • Variations of the V-bodies consist of 2, 3 or 4 polypeptide chains, different degrees of disulfide stabilization being used (Fig. 1 (C), 2, 3).
  • Trispecific antibodies can be produced without any additional effort compared to bispecific antibodies if the recombinant expression vectors are constructed in such a way that two polypeptide chain constructs are used, for example, each from an scFv and a variable domain of a dsFv fragment (see FIGS. 2 and 3, each above right).
  • the present invention further relates to a pharmaceutical composition which contains the recombinant antibody construct according to the invention in a pharmaceutically effective amount and optionally a pharmaceutically acceptable carrier and / or diluent.
  • the pharmaceutical composition of the invention can be used, for example, to prevent or treat cancer, i.e. systemic and solid tumors, metastases and metastasis, autoimmune diseases, allergies, immunological overreactions, infections or noxious agents are used.
  • Another object of the present invention is a diagnostic agent which contains the recombinant antibody construct according to the invention.
  • This diagnostic agent can be used for in vitro and / ⁇ u / Vo diagnostics, for example for the detection of cancer, autoimmune diseases, allergies, immunological overreactions, infections or noxious substances.
  • trispecific antibodies can be produced without special Trimerization motifs must be used in addition to the antigen-binding regions of the antibodies. This is used to produce therapeutic agents that can bind several tumor markers on one cell, or several different epitopes of a tumor marker. A combination of a tumor marker and a tissue-specific antibody can also be used. This increases the tumor specificity significantly compared to monovalent or multivalent-monospecific binding, and greatly reduces the burden on the patient due to the previously very common localization of the antibodies in non-specific tissues (both by cross-reactions and by expression of the tumor marker on other tissues). Apparent affinity is also improved by the increased avidity.
  • the remaining binding site of the trispecific antibodies serves to strengthen the immune response against the tumor, in particular by binding to CD3 or CD28 of the T lymphocytes. Activation of the natural killer (NK) cells against the tumor by binding to CD16 is also possible. Alternatively, this antibody fragment can also activate complement cascades.
  • NK natural killer
  • V-bodies are assembled in the periplasm of E. coli which contain a binding site for human CD3 and two binding sites for a tumor marker, e.g. MUC1, erbB2 or similar, or differentiation markers, e.g. CD19.
  • the proteins are purified from the periplasm and used intravenously to bring T lymphocytes to the tumor. It is also possible to produce the proteins in eukaryotic cell lines (e.g. CHO or baculovirus) or transgenic plants.
  • scFv single-chain Fv fragments
  • dsFv disulfide bridge-stabilized Fv fragment
  • phOx 4-ethoxymethylene-2- phenyl-2-oxazolin-5-one
  • the C-termini of the variable domains of their heavy chains were combined with the N-termini of the variable domains of their light chains with a flexible // n comprising 18 amino acids / cer peptide, which is composed of the first amino acids of the CH1 domain and the linear ⁇ -tubulin epitope EEGEFSEAR (Yol-Tag) of the monoclonal antibody Yol1 / 34 (Kilmartin et al., 1982; Breitling and Little, 1986 ; Schmiedl et al., 2000a).
  • One of the variable domains of the anti-phox dsFv fragment was fused to the C-termini of the light chains.
  • a short peptide of the human 62 kDa proto-oncogene product c-myc which contains the linear epitope EEKLISEEDL of the monoclonal antibody Myd-9E10 (Evan et al., 1985), is located near the C-termini of both polypeptide chains before a 6xHis- sweeps the fusion proteins scFv 215 (Yol) -10-V H (phOx) and scFv 215 (Yol) -10-V L (phOx).
  • the vector pOPE111-215HphOx / 215LphOx was generated to produce the V-body described above. It codes for the two fusion proteins scFv 215 (Yol) -10-V H (phOx) and scFv 215 (Yol) - 10-V * _ (phOx) described above. The two gene fragments were each separated, with formation of ORFs behind pelB- / eacfer sequences from Erwinia carotovora (Lei et al., 1987) for the secretion of the translation products in the cloned periplasmic space.
  • the vector pOPE111 also has a ColE1 origin of replication and the b-lactamase gene as a selection marker for ampicillin resistance.
  • the expression of the encoded V-body constructs is determined using a synthetic / ac promoter P / A1 / 04 which can be induced by IPTG / 03 and two strong ribosome binding sites (RBS).
  • a 50 mL overnight culture of E. coli cells transformed with pOPE111-215HphOx / 215LphOx was 1/20 in dYT medium (16 g / L Bacto-Trypton, 10 g / L yeast extract, 5 g / L NaCI) with 100 mM Giucose and 100 ⁇ g / mL ampicillin diluted and cultivated at 37 ° C. and 230 rpm to an OD 6 oo of 0.6 before the promoter was induced by adding 20 mM isopropyl-bD-thiogalactopyranoside. After 3 h of incubation at 22 ° C. and 230 rpm, the bacteria were cooled on ice for 10 min and pelleted by centrifugation at 4 ° C. and 5000 ⁇ g.
  • periplasmic extracts For the preparation of periplasmic extracts, the pelleted bacteria were cooled in 1/10 volume (based on the starting volume of the culture) of shock solution (50 mM Tris / HCl, pH 8.0, 20% (w / v) sucrose, 1 mM EDTA) resuspended, incubated for 20 min with occasional shaking on ice and centrifuged again for 10 min at 6200 xg and 4 ° C. The periplasmic extract in the supernatant was centrifuged for a further 30 min at 30,000 ⁇ g and 4 ° C., dialyzed against PBS and analyzed with SDS-PAGE, immunoblot and ELISA.
  • shock solution 50 mM Tris / HCl, pH 8.0, 20% (w / v) sucrose, 1 mM EDTA
  • the cell pellet was resuspended in 1/10 volume (based on the initial volume of the culture) 5 mM MgSO 4 , incubated for 20 min with occasional shaking on ice and also centrifuged for 30 min at 30,000 xg and 4 ° C.
  • the extract thus obtained was also dialyzed against PBS and analyzed with SDS-PAGE, immunoblot and ELISA.
  • a column was loaded with 2 ml / liter bacterial culture Ni-NTA-Sepharose and equilibrated in SSP (PBS, pH 7.5, 10 mM imidazole, 1 M NaCl).
  • SSP SSP
  • the dialyzed periplasmic extracts were pooled, adjusted to the same buffer conditions and added to the column.
  • the proteins were 10 Sepharose volumes elution buffer (PBS, pH 7, 5, 500 mM imidazole, 1 M NaCl) competitively eluted.
  • the collected fractions were analyzed by SDS-PAGE (Laemmli, 1975) and Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b). Fractions containing protein were pooled.
  • the antibody fragments obtained via IMAC were dialyzed against 30 mM Tris / HCl, pH 8.0 and at 0.5 mL / min on the column equilibrated in the same buffer (Mono Q HR / 5R; Amersham Pharmacia, Freiburg; Schmiedl et al., 2000b). After washing the column with 5 mL 30 mM Tris / HCl, pH 8.0, the proteins were competitively eluted using a two-phase NaCI gradient. For this purpose, the salt concentration was first increased to 20 mM over 20 mL, then over 1 mL to 1 M NaCl.
  • the column was rinsed with 5 ml of Mono Q elution buffer (30 mM Tris / HCl, pH 8.0, 1 M NaCl) and readjusted to Mono-Q buffer.
  • Mono Q elution buffer (30 mM Tris / HCl, pH 8.0, 1 M NaCl) and readjusted to Mono-Q buffer.
  • the collected fractions were analyzed by SDS-PAGE (Laemmli, 1975) and Immunoblot (Towbin et al., 1989; Schmiedl et al., 2000b). Fractions containing protein were pooled and their content determined with Bradford's solution.
  • Fp b-galactosidase-215 is a fusion protein of the bacterial b-galactosidase with a peptide which contains the mAb 215 epitope.
  • b-Galactosidase fused with a comparable peptide served as a control.
  • the recombinant b-galactosidase fusion proteins were previously obtained by expression in E. coli (Kontermann er al., 1995).
  • Phoxylated BSA was prepared by incubating a 20-fold molar excess of 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (phOx) with BSA in 0.1 M NaCO 3 (pH 8.5) for two hours. Uncoupled 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one was removed by repeated dialysis against PBS. Unmodified BSA served as a control.
  • the antibody mAb Myc1-9E10 (Calbiochem, Schwalbach; 1/1000 in MPBST) was used in combination with HRP-conjugated goat anti-mouse immunoglobulins (Dianova, Hamburg; 1/2000 in MPBST) to detect bound antibody fragments and with 100 mLl well TMB substrate (10 mL 100 mM sodium acetate, pH 6.0, 25 ⁇ L TMB solution (40 mg / mL in DMSO), 8 ⁇ L 30% (v / v) H 2 O 2 ) was detected. After the color reaction had been stopped by adding 50 ⁇ Uwell 1 MH 2 SO, the analysis was carried out using an ELISA reagent at 450 nm.

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Abstract

Anticorps de recombinaison, procédé de fabrication desdits anticorps, compositions pharmaceutiques et moyens diagnostiques contenant ces anticorps de recombinaison, et utilisation de ces anticorps de recombinaison pour le diagnostic et le traitement de cancers, de maladies auto-immunes, d'allergies, d'hyperréactions immunologiques, d'infections ou de troubles pathologiques.
PCT/EP2001/005161 2000-05-05 2001-05-07 Anticorps de recombinaison trivalents a regions variables stabilisees par des ponts disulfure WO2001085795A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU60285/01A AU6028501A (en) 2000-05-05 2001-05-07 Trivalent antibody constructs having variable regions that are stabilized by disulfide bridges
EP01933941A EP1287036A1 (fr) 2000-05-05 2001-05-07 Anticorps de recombinaison trivalents a regions variables stabilisees par des ponts disulfure

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DE10021678A DE10021678A1 (de) 2000-05-05 2000-05-05 Antikörperkonstrukte mit variablen Regionen
DE10021678.1 2000-05-05

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Cited By (12)

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EP2014680A1 (fr) * 2007-07-10 2009-01-14 Friedrich-Alexander-Universität Erlangen-Nürnberg Dérivés d'anticorps bi-spécifiques ou tri-spécifiques trivalents, recombinants, à simple chaîne
WO2010035012A1 (fr) * 2008-09-26 2010-04-01 Ucb Pharma S.A. Produits biologiques
US8629246B2 (en) 2007-09-26 2014-01-14 Ucb Pharma S.A. Dual specificity antibody fusions
WO2016087416A1 (fr) * 2014-12-03 2016-06-09 F. Hoffmann-La Roche Ag Anticorps multispécifiques
US9879095B2 (en) 2010-08-24 2018-01-30 Hoffman-La Roche Inc. Bispecific antibodies comprising a disulfide stabilized-Fv fragment
US9890204B2 (en) 2009-04-07 2018-02-13 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
US10323099B2 (en) 2013-10-11 2019-06-18 Hoffmann-La Roche Inc. Multispecific domain exchanged common variable light chain antibodies
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10640555B2 (en) 2009-06-16 2020-05-05 Hoffmann-La Roche Inc. Bispecific antigen binding proteins
US10793621B2 (en) 2011-02-28 2020-10-06 Hoffmann-La Roche Inc. Nucleic acid encoding dual Fc antigen binding proteins

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US9879095B2 (en) 2010-08-24 2018-01-30 Hoffman-La Roche Inc. Bispecific antibodies comprising a disulfide stabilized-Fv fragment
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10793621B2 (en) 2011-02-28 2020-10-06 Hoffmann-La Roche Inc. Nucleic acid encoding dual Fc antigen binding proteins
US10323099B2 (en) 2013-10-11 2019-06-18 Hoffmann-La Roche Inc. Multispecific domain exchanged common variable light chain antibodies
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
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