WO2015101588A1 - Monovalent blood brain barrier shuttle modules - Google Patents
Monovalent blood brain barrier shuttle modules Download PDFInfo
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- WO2015101588A1 WO2015101588A1 PCT/EP2014/079353 EP2014079353W WO2015101588A1 WO 2015101588 A1 WO2015101588 A1 WO 2015101588A1 EP 2014079353 W EP2014079353 W EP 2014079353W WO 2015101588 A1 WO2015101588 A1 WO 2015101588A1
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A—HUMAN NECESSITIES
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
Definitions
- the present invention relates to a blood brain barrier shuttle module that has one binding specificity that specifically binds to a blood brain barrier receptor (BBBR) and which is monovalent with respect to this binding specificity and methods of using this construct as blood barrier shuttle and in the treatment of neurological disorders.
- BBBR blood brain barrier receptor
- Brain penetration of neurological disorder drugs such alls e.g. large biotherapeutic drugs or small molecule drugs having a low brain penetration, is strictly limited by the extensive and impermeable blood brain barrier (BBB) together with the other cell component in the neurovascular unit (NVU).
- BBB blood brain barrier
- NNU neurovascular unit
- Many strategies to overcome this obstacle have been tested and one is to utilize transcytosis pathways mediated by endogenous receptors expressed on the brain capillary endothelium (blood brain barrier receptor).
- Recombinant proteins such as monoclonal antibodies or peptides have been designed against these receptors to enable receptor-mediated delivery of biotherapeutics to the brain.
- strategies to maximize brain uptake while minimizing miss-sorting within the brain endothelial cells (BECs), and the extent of accumulation within certain organelles (especially organelles that leads to degradation of the biotherapeutic) in BECs remain unexplored.
- VL variable light chain domain
- a blood brain barrier shuttle module comprising a brain effector entity, a linker and one monovalent binding entity which binds to a blood brain barrier receptor, wherein the linker couples the effector entity to the monovalent binding entity, which binds to the blood brain barrier receptor, wherein the monovalent binding entity does not comprise the variable domains of the anti-transferrin receptor antibody 8D3 (SEQ ID NO: 01 and SEQ ID NO: 02) or of the variant anti-transferrin receptor antibody 8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
- the anti-transferrin receptor antibody 8D3 has a heavy chain variable domain with the following amino acid sequence:
- the anti-transferrin receptor antibody 8D3 has a light chain variable domain with the following amino acid sequence:
- the variant anti-transferrin receptor antibody 8D3v has the same heavy chain variable domain as antibody 8D3 and a light chain variable domain with mutants LI 04V and LI 061 that has the following amino acid sequence:
- the monovalent binding entity which binds to the blood brain barrier receptor is a polypeptide.
- the monovalent binding entity which binds to the blood brain barrier receptor comprises a molecule selected from the group consisting of a blood brain barrier receptor ligand, a full length antibody, a scFv, an Fv, a scFab, and a VHH.
- the blood brain receptor is selected from the group consisting of transferrin receptor, insulin receptor, insulin-like growth factor receptor, low density lipoprotein receptor- related protein 8, low density lipoprotein receptor-related protein 1 and heparin- binding epidermal growth factor-like growth factor.
- the blood brain receptor is the transferrin receptor.
- the monovalent binding entity specifically binds to human transferrin receptor and to cynomolgus transferrin receptor.
- the monovalent binding entity which binds to the blood brain barrier receptor comprises one scFab directed to the transferrin receptor, more particularly, a scFab that specifically binds to an epitope in the transferrin receptor comprised within the amino acid sequence of SEQ ID NO: 04, 05 or 06.
- the monovalent binding entity which binds to the blood brain barrier receptor comprises one scFv directed to the transferrin receptor, more particularly, a scFv recognizing an epitope in the transferrin receptor comprised within the amino acid sequence of SEQ ID NO: 04, 05 or 06.
- the brain effector entity is selected from the group consisting of neurological disorder drugs, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed to a brain target, monoclonal antibodies directed to a brain target, peptides directed to a brain target.
- the brain target is selected from the group consisting of ⁇ -secretase 1, ⁇ (Abeta), epidermal growth factor, epidermal growth factor receptor 2, tau, phosphorylated tau, phosphorylated tau(pS422), apolipoprotein E4, alpha synuclein, oligomeric fragments of alpha synuclein, CD20, huntingtin, prion protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid precursor protein, p75 neurotrophin receptor and caspase 6.
- the brain effector entity is a polypeptide.
- the monovalent binding entity which binds to the blood brain receptor is a polypeptide and the monovalent binding entity is conjugated to the C-terminal end of the brain effector entity either directly or via a linker.
- the brain effector entity comprises a full length antibody directed to a brain target.
- the full length antibody is an IgG.
- the blood brain barrier shuttle comprises a full length IgG antibody as brain effector entity, a linker and one scFab as the monovalent binding entity which binds the blood brain barrier receptor, wherein the scFab is conjugated to the C-terminal end of the Fc- region of one of the heavy chains of the IgG antibody via the linker.
- the first heavy chain of the antibody of the blood brain barrier shuttle directed to a brain target comprises a first dimerization module and the second heavy chain of the antibody of the blood brain barrier shuttle to a brain target comprises a second dimerization module allowing heterodimerization of the two heavy chains.
- the first dimerization module of the first heavy chain of the antibody of the blood brain barrier shuttle directed to the brain target comprises knobs and the dimerization module of the second heavy chain of the antibody of the blood brain barrier shuttle directed to the brain target comprises holes according to the knobs-into-holes strategy.
- the linker is a peptidic linker.
- the peptidic linker has an amino acid sequence with a length of at least 25 amino acids.
- the peptidic linker has an amino acid sequence with a length of 30 to 50 amino acids.
- the peptidic linker is (G4S)6G2 (SEQ ID NO: 07) or (G4S)4 (SEQ ID NO: 08). The following three embodiments are directed to a blood brain barrier shuttle module wherein the brain effector entity is a polypeptide with the proviso that the brain effector entity is not a full length antibody, in particular not a full length IgG.
- the monovalent binding entity which binds to the blood brain barrier receptor comprises a CH2-
- CH3 Ig entity and one scFab (comprising a first linker), which binds to the blood brain barrier receptor, wherein the scFab is coupled to a C-terminal end of the CH2-CH3 Ig entity by a second linker.
- the blood brain barrier shuttle comprises a brain effector entity, a linker, a CH2-CH3 Ig domain, a second linker and one scFab, which binds to the blood brain barrier receptor, wherein the brain effector entity is conjugated by a first linker to an N-terminal end of the CH2- CH3 Ig domain and the scFab is conjugated to a C-terminal end of the CH2-CH3 Ig domain by a second linker.
- the CH2-CH3 Ig entity is a CH2-CH3 IgG entity.
- Further aspects of the current invention is an (isolated) nucleic acid encoding the blood brain barrier shuttle module as reported herein, a host cell comprising the (isolated) nucleic acid encoding the blood brain barrier shuttle module and a pharmaceutical formulation comprising the blood brain barrier shuttle module.
- the blood brain barrier shuttle module as reported herein can be used as a medicament, in particular it can be used for the treatment of a neurological disorder such as e.g. Alzheimer's disease.
- the blood brain barrier shuttle module as reported herein can be used to transport the brain effector entity across the blood brain barrier.
- the heavy chain of the IgG antibody of the blood brain barrier shuttle module as reported herein conjugated at its C-terminal end of the Fc-region to the scFab as monovalent binding entity, which binds to the blood brain barrier receptor has the following structure:
- variable heavy chain domain VH of the scFab antibody and IgG CHI heavy chain domain.
- a fusion polypeptide to transport a brain effector entity across the blood brain barrier comprising a CH2-CH3 Ig entity, a linker and one scFab that specifically binds to a blood brain barrier receptor, wherein the scFab is conjugated to a C-terminal end of the CH2-CH3 Ig entity by the linker, wherein scFab does not comprise the variable domains of the anti-transferrin receptor antibody 8D3 (SEQ ID NO: 01 and SEQ ID NO: 02) or of the variant anti- transferrin receptor antibody 8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
- a fusion polypeptide to transport a brain effector entity across the blood brain barrier comprising a CH2-CH3 Ig entity, a linker and one scFv that specifically binds to a blood brain barrier receptor, wherein the scFv is conjugated to a C-terminal end of the CH2-CH3 Ig entity by the linker, wherein scFv does not comprise the variable domains of the anti-transferrin receptor antibody 8D3 (SEQ ID NO: 01 and SEQ ID NO: 02) or of the variant anti- transferrin receptor antibody 8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
- the fusion polypeptide further comprises a linker at the N-terminal end of the CH2-CH3 Ig entity to conjugate the brain effector entity to the N-terminal end of the CH2-CH3 Ig entity.
- the brain effector entity is selected from the group consisting of neurological disorder drugs, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibody fragments or peptides directed to a brain target selected from the group consisting of scFv, Fv, scFab, Fab, VHH, F(ab')2.
- the scFab or the scFv that specifically binds to the blood brain barrier receptor specifically binds to the transferrin receptor.
- the linker is a peptidic linker.
- the peptidic linker has an amino acid sequence with a length of at least 15 amino acids.
- the peptidic linker has a length of 20 to 50 amino acids.
- the peptidic linker has the amino acid sequence (G4S)6G2, (SEQ ID NO: 07) or (G4S)4 (SEQ ID NO: 08).
- the CH2-CH3 Ig entity is a CH2-CH3 IgG entity.
- nucleic acid encoding the fusion polypeptide as reported herein and a host cell comprising the nucleic acid encoding the fusion polypeptide as reported herein.
- One aspect as reported herein is a conjugate comprising a fusion polypeptide as reported herein and a brain effector entity conjugated to an N-terminal end of the CH2-CH3 Ig entity of the fusion polypeptide as reported herein via a linker.
- the brain effector entity is a neurotrophic factor and the linker conjugating the neurotrophic factor to the N-terminal end of the CH2-CH3 Ig entity is a peptidic linker.
- compositions comprising the conjugate as reported herein and a pharmaceutical carrier, the use of the conjugate as reported herein, in particular the use of the conjugate for the treatment of a neurodegenerative disorder in particular Alzheimer's disease.
- the monovalent binding entity that specifically binds to a blood brain barrier receptor can be conjugated to any terminus of the light or heavy chain of the antibody either directly or via a peptidic linker.
- the monovalent binding entity is conjugated to a C-terminus of the heavy chain.
- the C-terminus of a heavy chain of an antibody can be a complete C-terminus ending with the amino acid residues PGK.
- the C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C-terminal amino acid residues have been removed.
- the C-terminus of the heavy chain is a shortened C-terminus ending with the amino acid residues PG.
- the monovalent binding entity can be conjugated to the respective antibody chain either directly or via a peptidic linker.
- the peptidic linker has the amino acid sequence GGSGGGGSGGGGSGGGGS (SEQ ID NO: 09).
- the monovalent binding entity can be an antibody scFv fragment.
- the monovalent binding entity is a scFv comprising in N- to C-terminal order a light chain variable domain-a light chain constant domain-a peptidic linker-a heavy chain variable domain-the heavy chain constant domain 1.
- the monovalent binding entity is a scFv fragment of an anti- transferrin receptor-antibody with a (G4S) 6 peptidic linker (SEQ ID NO: 10).
- the blood-brain-barrier receptor is selected from the group consisting of transferrin receptor, insulin receptor, insulin-like growth factor receptor, low density lipoprotein receptor-related protein 8, low density lipoprotein receptor-related protein 1 and heparin-binding epidermal growth factor- like growth factor.
- blood-brain-barrier receptor is a human blood-brain- barrier receptor.
- the blood-brain-barrier receptor is the transferrin receptor and the antibody does not inhibit the binding of the transferrin receptor to transferrin.
- the blood-brain-barrier receptor is the human transferrin receptor.
- the peptidic linker conjugating the monovalent binding entity to the brain effector entity is an amino acid sequence with a length of at least 15 amino acids. In one embodiment the peptidic linker has a length of 18 to 25 amino acids. In one embodiment, the brain effector entity is a full length antibody. In one embodiment the brain effector entity is a full length antibody of the subclass IgGl or IgG4.
- the monovalent binding entity is an anti-blood brain barrier receptor antibody or a blood brain barrier receptor binding fragment thereof.
- the anti-blood brain barrier receptor antibody or fragment thereof does not impair the binding of the blood brain barrier receptor to one or more of its native ligands.
- the anti-blood brain barrier receptor antibody specifically binds to human transferrin receptor in such a manner that it does not inhibit binding of the human transferrin receptor to human transferrin.
- the blood brain barrier shuttle module is effector silent.
- the brain effector entity is a full length antibody comprising an Fc-region, wherein in case the Fc-region is of the human subclass IgGl the Fc-region comprises the mutations L234A, L235A and P329G (numbering according to the EU index of Kabat), or in case the Fc-region is of the human subclass IgG4 the Fc-region comprises the mutations S228P, L235E and P329G (numbering according to the EU index of Kabat).
- acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
- An acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
- the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
- Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
- binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
- an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
- HVRs hypervariable regions
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv,
- Fab, Fab', Fab'-SH, F(ab') 2 diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
- chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
- the "class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses
- immunoglobulins e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA ls and IgA 2 .
- the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- Effective functions refer to those biological activities attributable to the Fc-region of an antibody, which vary with the antibody class. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
- An "effective amount” of an agent, e.g., a pharmaceutical formulation refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- Fc-region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc-regions and variant Fc-regions.
- a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
- the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
- numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 -3242.
- FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
- the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2- H2(L2)-FR3-H3(L3)-FR4.
- full length antibody is used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc-region as defined herein.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- a "human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
- the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
- the subgroup of sequences is a subgroup as in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3.
- the subgroup is subgroup kappa I as in Kabat et al, supra.
- the subgroup is subgroup III as in Kabat et al, supra.
- a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
- a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
- a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
- a "humanized form" of an antibody, e.g., a non- human antibody refers to an antibody that has undergone humanization.
- hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions” or “CDRs") and form structurally defined loops ("hypervariable loops"), and/or contain the antigen-contacting residues ("antigen contacts").
- CDRs complementarity determining regions
- hypervariable loops form structurally defined loops
- antigen contacts Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
- HVRs herein include
- HVR residues and other residues in the variable domain are numbered herein according to Kabat et al, supra.
- mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
- domesticated animals e.g. cows, sheep, cats, dogs, and horses
- primates e.g., humans and non-human primates such as monkeys
- rabbits e.g., mice and rats
- rodents e.g., mice and rats.
- the individual or subject is a human.
- An "isolated" antibody is one which has been separated from a component of its natural environment.
- an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
- electrophoretic e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatographic e.g., ion exchange or reverse phase HPLC
- nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
- An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
- polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
- each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
- “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures.
- native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
- VH variable heavy domain
- VL variable region
- the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
- Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
- Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
- the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office,
- the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
- the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the
- % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
- pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- treatment and grammatical variations thereof such as “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
- Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
- the term "variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
- variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
- FRs conserved framework regions
- HVRs hypervariable regions
- a single VH or VL domain may be sufficient to confer antigen-binding specificity.
- antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al, J. Immunol. 150 (1993) 880-887; Clackson, T. et al, Nature 352 (1991) 624-628).
- vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
- blood-brain barrier denotes the physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Daltons).
- the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS, and are herein collectively referred to an the blood-brain barrier or BBB.
- the BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.
- central nervous system CNS denotes the complex of nerve tissues that control bodily function, and includes the brain and spinal cord.
- BBBR blood-brain barrier receptor
- TfR transferrin receptor
- IGF-R insulin receptor
- LRP1 low density lipoprotein receptors
- LRP8 low density lipoprotein receptors
- HB-EGF heparin-binding epidermal growth factor-like growth factor
- TfR transferrin receptor
- brain effector entity denotes a molecule that is to be transported to the brain across the BBB.
- the effector entity typically has a characteristic therapeutic activity that is desired to be delivered to the brain.
- Effector entities include neurologically disorder drugs and cytotoxic agents such as e.g. polypeptides and antibodies, in particular monoclonal antibodies or fragments thereof directed to a brain target.
- the term "monovalent binding entity” denotes a molecule able to bind specifically and in a monovalent binding mode to a BBBR.
- the blood brain shuttle module and/or conjugate as reported herein are characterized by the presence of a single unit of a monovalent binding entity i.e. the blood brain shuttle module and/or conjugate of the present invention comprise exactly one unit of the monovalent binding entity.
- the monovalent binding entity includes but is not limited to polypeptides, full length antibodies, antibody fragments including Fab, Fab', Fv fragments, single-chain antibody molecules such as e.g. single chain Fab, scFv.
- the monovalent binding entity can for example be a scaffold protein engineered using state of the art technologies like phage display or immunization.
- the monovalent binding entity can also be a polypeptide.
- the monovalent binding entity comprises a CH2-CH3 Ig domain and a single chain Fab (scFab) directed to a blood brain barrier receptor.
- the scFab is coupled to the C- terminal end of the CH2-CH3 Ig domain by a linker.
- the scFab is directed to the transferrin receptor.
- the term "monovalent binding mode" denotes a specific binding to the BBBR where the interaction between the monovalent binding entity and the BBBR takes place through one single epitope.
- the monovalent binding mode prevents any dimerization/multimerization of the BBBR due to a single epitope interaction point.
- the monovalent binding mode prevents that the intracellular sorting of the BBBR is altered.
- epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
- An epitope is a region of an antigen that is bound by an antibody.
- the "transferrin receptor” is a transmembrane glycoprotein (with a molecular weight of about 180,000 Da) which is composed of two disulfide-bonded sub-units (each of apparent molecular weight of about 90,000 Da) and is involved in iron uptake in vertebrates.
- the TfR herein is human TfR comprising the amino acid sequence as reported in Schneider et al. (Nature 311 (1984) 675 - 678).
- neurological disorder denotes a disease or disorder which affects the CNS and/or which has an etiology in the CNS.
- CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.
- the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier.
- neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt- Jakob syndrome, kuru, Gerstmann-Straussler- Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, la
- Neurological disorder drug denotes a drug or therapeutic agent that treats one or more neurological disorder(s).
- Neurological disorder drugs include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS target(s), modified versions of natural ligands of one or more CNS target(s), aptamers, inhibitory nucleic acids (i.e., small inhibitory R As (siR A) and short hairpin RNAs (shRNA)), ribozymes, and small molecules, or active fragments of any of the foregoing.
- inhibitory nucleic acids i.e., small inhibitory R As (siR A) and short hairpin RNAs (shRNA)
- ribozymes i.e., ribozymes, and small molecules, or active fragments of any of the foregoing.
- Exemplary neurological disorder drugs include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the foregoing that either are themselves or specifically recognize and/or act upon (i.e., inhibit, activate, or detect) a CNS antigen or target molecule such as, but not limited to, amyloid precursor protein or portions thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins.
- a CNS antigen or target molecule such as, but not limited to, amyloid precursor protein or portions thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, gli
- imaging agent denotes a compound that has one or more properties that permit its presence and/or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds incorporating a labeled entity that permits detection.
- CNS antigen and "brain target” denote an antigen and/or molecule expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule.
- antigen and/or molecule include, without limitation: beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
- BACE1 beta-secretase 1
- Abeta amyloid beta
- EGFR epidermal growth factor receptor
- HER2 human
- binding affinity is generally determined using a standard assay, such as Scatchard analysis, or surface plasmon resonance technique (e.g. using BIACORE®).
- CH2-CH3 Ig entity refers to a protein entity derived from immunoglobulin CH2 or CH3 domains.
- the "CH2-CH3 Ig entity” comprises two "CH2-CH3" polypeptides forming a dimer.
- the immunoglobulin can be IgG,
- CH2-CH3 Ig entity derived from an IgG immunoglobulin and is referred to herein as "CH2-CH3 IgG entity".
- the term includes native sequence of CH2-CH3 domains and variant CH2-CH3 domains.
- the "CH2-CH3 Ig entity” derives from human heavy chain CH2- CH3 IgG domain which extends from Cys226, or from Pro230, to the carboxyl- terminus of the heavy chain.
- the C-terminal lysine (Lys447) of the Fc region may or may not be present.
- numbering of amino acid residues in the CH2-CH3 domain region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service,
- a “conjugate” is fusion protein of the present invention conjugated to one or more heterologous molecule(s), including but not limited to a label, neurological disorder drug or cytotoxic agent.
- the term “linker” denotes a chemical linker or a single chain peptidic linker that covalently connects different entities of the blood brain barrier shuttle module and/or the fusion polypeptide and/or the conjugate as reported herein.
- the linker connects for example the brain effector entity to the monovalent binding entity.
- the monovalent binding entity comprises a CH2-CH3 Ig entity and a scFab directed to the blood brain barrier receptor, then the linker conjugates the scFab to the C-terminal end of the CH3-CH2 Ig entity.
- the linker conjugating the brain effector entity to the monovalent binding entity (first linker) and the linker connecting the scFab to the C-terminal end of the CH2-CH3 Ig domain (second linker) can be the same or different.
- Single chain peptidic linkers comprising of from one to twenty amino acid residues joined by peptide bonds, can be used.
- the amino acids are selected from the twenty naturally-occurring amino acids.
- one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine.
- the linker is a chemical linker.
- the linker is a single chain peptidic linker with an amino acid sequence with a length of at least 25 amino acid residues, in one preferred embodiment with a length of 32 to 50 amino acid residues.
- linker is (G4S)4 (SED ID NO: 08). In one embodiment the linker is (G4S)6G2 (SEQ ID NO: 07).
- Conjugation may be performed using a variety of chemical linkers.
- the monovalent binding entity or the fusion polypeptide and the brain effector entity may be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N- maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis- azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as to
- the linker may be a "cleavable linker" facilitating release of the effector entity upon delivery to the brain.
- a "cleavable linker” facilitating release of the effector entity upon delivery to the brain.
- an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide- containing linker (Chari et al, Cancer Res. 52 (1992) 127-131; US 5,208,020) may be used.
- Covalent conjugation can either be direct or via a linker.
- direct conjugation is by construction of a polypeptide fusion (i.e. by genetic fusion of the two genes encoding the monovalent binding entity towards the BBBR and effector entity and expressed as a single polypeptide (chain)).
- direct conjugation is by formation of a covalent bond between a reactive group on one of the two portions of the monovalent binding entity against the BBBR and a corresponding group or acceptor on the brain effector entity.
- direct conjugation is by modification (i.e.
- a reactive group as non-limiting examples, a sulfhydryl group or a carboxyl group
- a molecule i.e. an amino acid
- a desired reactive group i.e. a cysteine residue
- Methods for covalent conjugation of nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, e.g., Zatsepin et al. Russ.
- Conjugation may also be performed using a variety of linkers.
- a monovalent binding entity and a effector entity may be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine
- SPDP N-succinimidyl-3
- Peptidic linkers comprised of from one to twenty amino acid residues joined by peptide bonds, may also be used.
- the amino acid residues are selected from the twenty naturally-occurring amino acids.
- one or more of the amino acid residues are selected from glycine, alanine, proline, asparagine, glutamine and lysine.
- the linker may be a "cleavable linker" facilitating release of the effector entity upon delivery to the brain.
- an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide- containing linker (Chari et al, Cancer Res. 52 (1992) 127-131; US 5,208,020) may be used.
- the invention is based, in part, on the finding that the blood brain barrier shuttle modules as reported herein can be used to deliver a brain effector entity across the blood brain barrier into the brain.
- the blood brain barrier shuttle module comprises a monovalent binding entity that specifically binds to a blood brain barrier receptor, such as the transferrin receptor.
- the blood brain barrier shuttle modules as reported herein are useful, e.g., for the diagnosis or treatment of neurological disorders, such as Alzheimer's disease, Parkinson's Disease and Alzheimer's Disease with Parkinson's Disease comorbidity.
- A. Exemplary anti-blood brain barrier receptor antibodies are useful, e.g., for the diagnosis or treatment of neurological disorders, such as Alzheimer's disease, Parkinson's Disease and Alzheimer's Disease with Parkinson's Disease comorbidity.
- the transcytosis screening was performed in an hCMEC/D3 based assay.
- the assay was performed in a pulse-chase mode.
- the hCMEC/D3 brain endothelial cells were incubated with the monovalent binding entity for 1 hour, washed thereafter and the following parameters were determined 0 hours and 4 hours post washing:
- the monovalent binding entity has to be i) taken up by the hCMEC/D3 cells (endocytosis), ii) transported outside the hCMEC/D3 cells (exocytosis), and iii) stable inside the hCMEC/D3 cells (no or low transport to the endosome for degradation).
- the monovalent binding entity is characterized in a hCMEC/D3 based assay by i) an (substantial) uptake into the hCMEC/D3 cells during a one hour loading period, ii) a release into the apical and/or basolateral compartment after the loading period and a washing step within 4 hours after the washing, and iii) a low (intracellular) degradation rate.
- the loading is at a concentration of about 2.67 ⁇ g/mL monovalent binding entity for one hour.
- the mouse anti-human transferrin-receptor antibody 128.1 (for variable region sequences see WO 93/10819 and SEQ ID NO: 11 and 12) can be taken as reference.
- the monovalent binding entity in order to be eligible as monovalent binding entity of a blood brain barrier shuttle module as reported herein has to show in the above described hCMEC/D3 based assay the following threshold values:
- the hCMEC/D3 based assay was performed as follows (this is one embodiment of all aspects as reported herein):
- hCMEC/D3 Medium and supplements for hCMEC/D3 (see WO 2006/056879 and Weksler, B.B., et al, FASEB J. 19 (2005) 1872-1874) can be obtained from Lonza.
- hCMEC/D3 cells (passages 26-29) are/can be cultured to confluence on collagen- coated coverslips (microscopy) or flasks in EBM2 medium containing 2.5 % FBS, a quarter of the supplied growth factors and fully complemented with supplied hydrocortisone, gentamycin and ascorbic acid.
- high density pore (l x lO 8 pores/cm 2 ) PET membrane filter inserts (0.4 ⁇ pore size, 12 mm diameter) are/can be used in 12-well cell culture plates. Media volumes are calculated to be 400 ⁇ , and 1600 ⁇ , for apical and basolateral chambers, respectively. Apical chambers of filter inserts are/can be coated with rat tail collagen I (7.5 ⁇ g/cm 2 ) followed by fibronectin (5 ⁇ g/mL), each incubation lasting for 1 h at RT. hCMEC/D3 cells are/can be grown to confluent monolayers ( ⁇ 2> ⁇ 10 5 cells/cm 2 ) for 10-12 days in EBM2 medium.
- Empty filters are/can be blocked in PBS containing 1 % BSA for 1 hour or overnight (o/n) before assay and then calibrated for at least 1 hour in EBM2 before the assay.
- the assay (for assay scheme see Figure 1) was performed in serum-free EBM2 medium which was otherwise reconstituted as described herein. On day of the assay, cells are serum-starved for 60 min. to deplete the natural ligand of the blood brain barrier receptor in question. Filter inserts with or without (but blocked overnight in complete medium) cells were incubated apically with monoclonal antibodies in question (monovalent binding entity) for 1 hour at 37 °C.
- the monolayers were washed at room temperature (RT) in serum-free medium apically (400 ⁇ ) and basolaterally (1600 ⁇ ) three times for 3-5 min. each.
- Pre -warmed medium was added to the apical chamber and the filters transferred to a fresh 12 well plate (blocked overnight with PBS containing 1 % BSA) containing 1600 ⁇ ⁇ pre-warmed medium.
- filters with or without cells were lysed in 500 ⁇ ⁇ RIP A buffer in order to determine specific antibody (monovalent binding entity) uptake.
- the remaining filters were incubated at 37 °C or at 4 °C and samples were collected at various time points to determine apical and/or basolateral release of the antibody (monovalent binding entity).
- the content of antibody in the samples can be quantified using a highly sensitive IgG ELISA (see Example 10).
- an anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising
- a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 41 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 42 or (16) a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 43 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 44, or
- (31) a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 73 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 74, or
- a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 87 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 88, or (39) a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 89 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 90, or
- a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 97 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 98.
- One preferred aspect as reported herein is an anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 23 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 24.
- an anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising a heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain that has the amino acid sequence of SEQ ID NO: 92.
- IGIIS S SDNTYYT S WAKGRFTISKT STTVD ASILQGGVPSRFSGSGSGTDFTLTISSLQ
- VDLKIPSPTTEDTATYFCARG C AD A AT YYCQQGL S S S YVDNT ADEYGDANGFNIWGPGTLVTVSL (37) FGGGTEVWKR (38) heavy chain variable domain amino light chain variable domain amino clone acid sequence (SEQ ID NO:) acid sequence (SEQ ID NO:)
- VYMELS SLRSEDTAVYYCARS W EDEADYYCNSIDSALDHVFGG FGEWMDYWGQGTLVTVSS (61) GTKLTVL (62)
- VYMELS SLRSEDTAVYYCARE S EDEADYYCNSRDSTLIVWFG FYGWAFDYWGQGTLVTVSS (65) GGTKLTVL (66) heavy chain variable domain amino light chain variable domain amino clone acid sequence (SEQ ID NO:) acid sequence (SEQ ID NO:)
- One aspect as reported herein is a humanized anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising
- a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 33 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 34 or (12) a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 35 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 36, or
- a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 49 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 50 or (20) a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 51 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 52, or
- One preferred aspect as reported herein is a humanized anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 23 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 24.
- a humanized anti-human transferrin receptor antibody comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112.
- the antibody further comprises (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- a humanized anti-human transferrin receptor antibody comprising (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112.
- the antibody further comprises (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- the invention provides an anti-transferrin receptor antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112; (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109
- a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110
- a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112
- the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112.
- the antibody comprises a HVR- H3 comprising the amino acid sequence of SEQ ID NO: 112.
- the antibody comprises a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112 and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- the antibody comprises a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112, a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111.
- the antibody comprises (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112.
- the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- the antibody comprises (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109, (ii) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and (iii) a HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 112; and
- VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (ii) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.
- the invention provides an antibody comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 112; (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115.
- a humanized anti-transferrin receptor antibody or transferrin receptor binding fragment thereof comprising a humanized heavy chain variable domain derived from the heavy chain variable domain that has the amino acid sequence of SEQ ID NO: 91 and a humanized light chain variable domain derived from the light chain variable domain that has the amino acid sequence of SEQ ID NO: 92.
- a humanized anti-human transferrin receptor antibody comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119.
- the antibody further comprises (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- a humanized anti-human transferrin receptor antibody comprising (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119.
- the antibody further comprises (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- the invention provides an anti-transferrin receptor antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119; (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119.
- the antibody comprises a HVR- H3 comprising the amino acid sequence of SEQ ID NO: 119.
- the antibody comprises a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119 and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- the antibody comprises a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119, a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118.
- the antibody comprises (a) a HVR-Hl comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119.
- the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- the antibody comprises (a) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 116, (ii) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) a HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 119; and
- VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120, (ii) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121, and (c) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
- the invention provides an antibody comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; (c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 119; (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 122.
- One aspect as reported herein is a transferrin shuttle module comprising a full length IgG antibody as brain effector entity, a linker and one scFab as the monovalent binding entity, which binds the transferrin receptor, wherein the scFab is conjugated to the C-terminal end of the Fc-region of one of the heavy chains of the IgG antibody via the linker.
- One aspect as reported herein is a transferrin shuttle module comprising a full length IgG antibody as brain effector entity, a linker and one scFv as the monovalent binding entity, which binds the transferrin barrier receptor, wherein the scFv is conjugated to the C-terminal end of the Fc-region of one of the heavy chains of the IgG antibody via the linker.
- the monovalent binding entity comprises the HVRs of SEQ ID NO: 109, 110, 112, 113, 114 and 115, or of SEQ ID NO: 116, 117, 119, 120, 121 and 122.
- an anti-transferrin receptor antibody may incorporate any of the features, singly or in combination, as described in Sections 1-6 below:
- Kd is measured by a radiolabeled antigen binding assay (RIA).
- an RIA is performed with the Fab version of an antibody of interest and its antigen.
- solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al, J.
- MICROTITER ® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/mL of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C).
- a non-adsorbent plate (Nunc #269620) 100 pM or 26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta, L.G. et al., Cancer Res. 57 (1997) 4593-4599).
- the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour).
- Kd is measured using a BIACORE surface plasmon resonance assay.
- a BIACORE surface plasmon resonance assay For example, an assay using a BIACORE ® -2000 or a BIACORE ® -3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with immobilized antigen CM5 chips at ⁇ 10 response units (RU).
- carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions.
- EDC N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride
- NHS N-hydroxysuccinimide
- Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/mL (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ / ⁇ to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block non-reacted groups.
- an antibody provided herein is an antibody fragment.
- Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fv, and scFv fragments, and other fragments described below.
- Fab fragment-specific antibody fragment
- Fab' fragment-specific Fab
- Fab'-SH fragment-specific Fab
- F(ab') 2 fragment-specific Fab
- Fv fragment antigen Vpent antibody fragment
- scFv fragments see, e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer- Verlag, New York (1994), pp.
- Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993/01161; Hudson, P.J. et al, Nat. Med. 9 (2003) 129-134; and Holliger, P. et al, Proc. Natl. Acad.
- Triabodies and tetrabodies are also described in Hudson, P.J. et al, Nat. Med. 9 (20039 129-134).
- Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
- a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).
- Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. 3. Chimeric and Humanized Antibodies
- an antibody provided herein is a chimeric antibody.
- Certain chimeric antibodies are described, e.g., in US 4,816,567; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855).
- a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
- a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody.
- Chimeric antibodies include antigen-binding fragments thereof.
- a chimeric antibody is a humanized antibody.
- a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
- a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
- a humanized antibody optionally will also comprise at least a portion of a human constant region.
- some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
- a non-human antibody e.g., the antibody from which the HVR residues are derived
- Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L.G. et al, J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, J.C. and Fransson, J., Front. Biosci. 13
- Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom, H.R. et al, Methods in Molecular Biology 178 (2001) 1-37 and further described, e.g., in the McCafferty, J. et al,
- repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in
- Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
- Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
- the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths, A.D. et al, EMBO J. 12 (1993) 725-734.
- naive libraries can also be made synthetically by cloning non-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388.
- Patent publications describing human antibody phage libraries include, for example: US 5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.
- an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
- Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for the transferrin receptor and the other is for any other antigen.
- Bispecific antibodies may also be used to localize cytotoxic agents to cells which express the transferrin receptor. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
- Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain- light chain pairs having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A. et al, EMBO J. 10 (1991) 3655- 3659), and "knob-in-hole” engineering (see, e.g., US 5,731,168).
- Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.A. et al, J. Immunol. 148 (1992) 1547-1553; using "diabody” technology for making bispecific antibody fragments (see, e.g.,
- the antibody or fragment herein also includes a "Dual Acting Fab” or “DAF” comprising an antigen binding site that binds to the transferrin receptor as well as another, different antigen (see, US 2008/0069820, for example).
- the antibody or fragment herein also includes multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.
- the anti-transferrin receptor antibody is a bispecific antibody.
- a bivalent, bispecific antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other, wherein the first antigen or the second antigen is human transferrin receptor.
- the antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains.
- the variable light chain domain VL is replaced by the variable heavy chain domain VH of said antibody
- the variable heavy chain domain VH is replaced by the variable light chain domain VL of said antibody.
- the amino acid at position 124 (numbering according to Kabat) is substituted by a positively charged amino acid
- the constant domain CHI of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negatively charged amino acid
- the amino acid at position 124 (numbering according to Kabat) is substituted by a positively charged amino acid
- the amino acid at position CHI of the second heavy chain under b) the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negatively charged amino acid
- the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and wherein in the constant domain CHI of the first heavy chain under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index), or ii) in the constant domain CL of the second light chain under b) the amino acid at position 124 is substituted independently by lysine (K), arginine
- the amino acids at position 124 and 123 are substituted by K (numbering according to Kabat EU index).
- the amino acids at position 147 and 213 are substituted by E (numbering according to EU index of Kabat).
- the amino acids at position 124 and 123 are substituted by K
- the amino acids at position 147 and 213 are substituted by E (numbering according to Kabat EU index).
- the amino acids at position 124 and 123 are substituted by K
- the amino acids at position 147 and 213 are substituted by E
- the amino acid at position 38 is substituted by K
- the amino acid at position 39 is substituted by E
- the amino acid at position 38 is substituted by K
- the amino acid at position 39 is substituted by E (numbering according to Kabat EU index).
- bivalent, bispecific antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other, and wherein the constant domains CL and CHI of the second light chain and the second heavy chain are replaced by each other, wherein the first antigen or the second antigen is human transferrin receptor.
- the antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain und a) are isolated chains.
- variable light chain domain VL is replaced by the variable heavy chain domain VH of said antibody, and the constant light chain domain CL is replaced by the constant heavy chain domain CHI of said antibody; and within the heavy chain the variable heavy chain domain VH is replaced by the variable light chain domain VL of said antibody, and the constant heavy chain domain CHI is replaced by the constant light chain domain CL of said antibody.
- One aspect as reported herein is a bivalent, bispecific antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI of the second light chain and the second heavy chain are replaced by each other, wherein the first antigen or the second antigen is human transferrin receptor.
- the antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains.
- a multispecific antibody comprising a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) one, two, three or four single chain Fab fragments specifically binding to one to four further antigens (i.e. a second and/or third and/or fourth and/or fifth antigen, preferably specifically binding to one further antigen, i.e.
- a second antigen wherein said single chain Fab fragments under b) are fused to said full length antibody under a) via a peptidic linker at the C- or N- terminus of the heavy or light chain of said full length antibody, wherein the first antigen or one of the further antigens is human transferrin receptor.
- one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C- terminus of the heavy or light chains of said full length antibody. In one embodiment one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C- terminus of the heavy chains of said full length antibody. In one embodiment one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C- terminus of the light chains of said full length antibody.
- two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each heavy or light chain of said full length antibody.
- two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each heavy chain of said full length antibody.
- two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each light chain of said full length antibody.
- One aspect as reported herein is a trivalent, bispecific antibody comprising a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, b) a first polypeptide consisting of
- VH antibody heavy chain variable domain
- VH antibody heavy chain variable domain
- CHI antibody constant domain 1
- first polypeptide is fused with the N-terminus of its VH domain via a peptidic linker to the C-terminus of one of the two heavy chains of said full length antibody, c) a second polypeptide consisting of
- VL an antibody light chain variable domain
- VL antibody light chain variable domain
- CL antibody light chain constant domain
- said second polypeptide is fused with the N-terminus of the VL domain via a peptidic linker to the C-terminus of the other of the two heavy chains of said full length antibody, and wherein the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding site specifically binding to a second antigen, and wherein the first antigen or the second antigen is human transferrin receptor.
- VH antibody heavy chain variable domain
- VL antibody light chain variable domain
- the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introduction of a disulfide bond between the following positions:
- the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between heavy chain variable domain position 105 and light chain variable domain position 43. (numbering always according to EU index of Kabat)
- a trivalent, bispecific antibody without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragments is preferred.
- a trispecific or tetraspecific antibody comprising a) a first light chain and a first heavy chain of a full length antibody which specifically binds to a first antigen, and b) a second (modified) light chain and a second (modified) heavy chain of a full length antibody which specifically binds to a second antigen, wherein the variable domains VL and VH are replaced by each other, and/or wherein the constant domains CL and CHI are replaced by each other, and c) wherein one to four antigen binding peptides which specifically bind to one or two further antigens (i.e.
- a third and/or fourth antigen are fused via a peptidic linker to the C- or N-terminus of the light chains or heavy chains of a) and/or b), wherein the first antigen or the second antigen or one of the further antigens is human transferrin receptor.
- the antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain und a) are isolated chains.
- the trispecific or tetraspecific antibody comprises under c) one or two antigen binding peptides which specifically bind to one or two further antigens.
- the antigen binding peptides are selected from the group of a scFv fragment and a scFab fragment.
- the antigen binding peptides are scFv fragments.
- the antigen binding peptides are scFab fragments. In one embodiment the antigen binding peptides are fused to the C-terminus of the heavy chains of a) and/or b).
- the trispecific or tetraspecific antibody comprises under c) one or two antigen binding peptides which specifically bind to one further antigen.
- the trispecific or tetraspecific antibody comprises under c) two identical antigen binding peptides which specifically bind to a third antigen.
- such two identical antigen binding peptides are fused both via the same peptidic linker to the C-terminus of the heavy chains of a) and b).
- the two identical antigen binding peptides are either a scFv fragment or a scFab fragment.
- the trispecific or tetraspecific antibody comprises under c) two antigen binding peptides which specifically bind to a third and a fourth antigen.
- said two antigen binding peptides are fused both via the same peptide connector to the C-terminus of the heavy chains of a) and b). In one preferred embodiment said two antigen binding peptides are either a scFv fragment or a scFab fragment.
- a bispecific, tetravalent antibody comprising a) two light chains and two heavy chains of an antibody, which specifically bind to a first antigen (and comprise two Fab fragments), b) two additional Fab fragments of an antibody, which specifically bind to a second antigen, wherein said additional Fab fragments are fused both via a peptidic linker either to the C- or N-termini of the heavy chains of a), and wherein in the Fab fragments the following modifications were performed i) in both Fab fragments of a), or in both Fab fragments of b), the variable domains VL and VH are replaced by each other, and/or the constant domains CL and CHI are replaced by each other, or ii) in both Fab fragments of a) the variable domains VL and VH are replaced by each other, and the constant domains CL and CHI are replaced by each other, and in both Fab fragments of b) the variable domains VL and VH are replaced by each other
- said additional Fab fragments are fused both via a peptidic linker either to the C-termini of the heavy chains of a), or to the N-termini of the heavy chains of a).
- said additional Fab fragments are fused both via a peptidic linker either to the C-termini of the heavy chains of a).
- said additional Fab fragments are fused both via a peptide connector to the N-termini of the heavy chains of a).
- the constant domains CL and CHI are replaced by each other.
- the following modifications are performed: i) in both Fab fragments of a) the constant domains CL and CHI are replaced by each other.
- the constant domains CL and CHI are replaced by each other.
- the following modifications are performed: i) in both Fab fragments of b) the constant domains CL and CHI are replaced by each other.
- a bispecific, tetravalent antibody comprising: a) a (modified) heavy chain of a first antibody, which specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein to the C- terminus of said heavy chain the N-terminus of a second VH-CH1 domain pair of said first antibody is fused via a peptidic linker, b) two light chains of said first antibody of a), c) a (modified) heavy chain of a second antibody, which specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein to the C-terminus of said heavy chain the N-terminus of a second VH-CL domain pair of said second antibody is fused via a peptidic linker, and d) two (modified) light chains of said second antibody of c), each comprising a CL-CH1 domain pair, wherein the first antigen or the second antigen is human transferrin receptor.
- One aspect as reported herein is a bispecific antibody comprising a) the heavy chain and the light chain of a first full length antibody that specifically binds to a first antigen, and b) the heavy chain and the light chain of a second full length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is connected to the C-terminus of the light chain via a peptidic linker, wherein the first antigen or the second antigen is human transferrin receptor.
- the antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain are isolated chains.
- bispecific antibody comprising a) a full length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) an Fv fragment specifically binding to a second antigen comprising a VH 2 domain and a VL 2 domain, wherein both domains are connected to each other via a disulfide bridge, wherein only either the VH 2 domain or the VL 2 domain is fused via a peptidic linker to the heavy or light chain of the full length antibody specifically binding to a first antigen, wherein the first antigen or the second antigen is human transferrin receptor.
- the heavy chains and the light chains under a) are isolated chains.
- the other of the VH 2 domain or the VL 2 domain is not fused via a peptide linker to the heavy or light chain of the full length antibody specifically binding to a first antigen.
- the first light chain comprises a VL domain and a CL domain and the first heavy chain comprises a VH domain, a CHI domain, a hinge region, a CH2 domain and a CH3 domain.
- the antibody as reported herein is a multispecific antibody, which requires heterodimerization of at least two heavy chain polypeptides, and wherein the antibody specifically binds to human transferrin receptor and a second non-human transferrin receptor antigen.
- the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner so that the heavy chain comprising one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure (e.g. a CH3 -engineered first heavy chain can no longer homodimerize with another CH3 -engineered first heavy chain; and a CH3 -engineered second heavy chain can no longer homodimerize with another CH3 -engineered second heavy chain).
- the heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising the CH3 domain, which is engineered in a complementary manner.
- the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions, such that the first heavy chain and the second heavy chain are forced to heterodimerize, whereas the first heavy chain and the second heavy chain can no longer homodimerize (e.g. for steric reasons).
- a multispecific antibody according to the invention which comprises a "non-crossed Fab region" derived from a first antibody, which specifically binds to a first antigen, and a "crossed Fab region” derived from a second antibody, which specifically binds to a second antigen, in combination with the particular amino acid substitutions described above for the invention.
- the CH3 domains of the multispecific antibody as reported herein can be altered by the "knob-into-holes" technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al, Protein Eng. 9 (1996) 617- 621; and Merchant, A.M., et al, Nat. Biotechnol. 16 (1998) 677-681.
- the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains.
- Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole”.
- the multispecific antibody as reported herein comprises a T366W mutation in the CH3 domain of the "knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the "hole-chain” (numbering according to Kabat EU index).
- An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the "knobs chain” and a E356C mutation or a S354C mutation into the CH3 domain of the "hole chain”.
- the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A and Y407V mutations in the other of the two CH3 domains or the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering according to Kabat EU index).
- the multispecific antibody as reported herein comprises the R409D and K370E mutations in the CH3 domain of the "knobs chain” and the D399K and E357K mutations in the CH3 domain of the
- the multispecific antibody as reported herein comprises a T366W mutation in the CH3 domain of the "knobs chain” and the T366S, L368A and Y407V mutations in the CH3 domain of the "hole chain” and additionally the R409D and K370E mutations in the CH3 domain of the "knobs chain” and the
- the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains and additionally the R409D and K370E mutations in the CH3 domain of the "knobs chain" and the D399K and
- the approach described in EP 1870459 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 -domain-interface between both, the first and the second heavy chain.
- this embodiment relates to a multispecific antibody as reported herein, wherein in the tertiary structure of the antibody the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface that is located between the respective antibody CH3 domains, wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain each comprise a set of amino acids that is located within said interface in the tertiary structure of the antibody, wherein from the set of amino acids that is located in the interface in the CH3 domain of one heavy chain a first amino acid is substituted by a positively charged amino acid and from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain a second amino acid is substituted by a negatively charged amino acid.
- the multispecific antibody according to this embodiment is herein also referred to as "CH3(+/-)-engineered multispecific antibody" (wherein the abbreviation "+/-" stands for the oppositely charged amino acids that were introduced in the respective CH
- the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.
- the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.
- the positively charged amino acid is K
- the negatively charged amino acid is E
- a multispecific antibody as reported herein the approach described in WO2013/157953 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- said multispecific antibody as reported herein in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).
- the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K
- the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).
- the amino acid Y at position 349 is substituted by E
- the amino acid Y at position 349 is substituted by D
- the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).
- the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).
- a multispecific antibody as reported herein the approach described in WO2012/058768 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- said multispecific antibody as reported herein in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index).
- a multispecific antibody as reported herein the approach described in WO 2011/143545 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- amino acid modifications in the CH3 domains of both heavy chains are introduced at positions
- WO 2011/090762 In one embodiment of a multispecific antibody as reported herein the approach described in WO 2011/090762 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- WO 2011/090762 relates to amino acid modifications according to the "knob-into-hole" technology.
- in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by W, and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted by A (numbering according to Kabat EU index).
- the approach described in WO 2009/089004 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- the amino acid K or N at position 392 is substituted by a negatively charged amino acid (in one preferred embodiment by E or D, in one preferred embodiment by D)
- the amino acid D at position 399 the amino acid E or D at position 356 or the amino acid E at position 357 is substituted by a positively charged amino acid (in one preferred embodiment K or R, in one preferred embodiment by K, in one preferred embodiment the amino acids at positions 399 or 356 are substituted by K) (numbering according to Kabat EU index).
- the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one preferred embodiment by
- the amino acid K at position 439 and/or the amino acid K at position 370 is substituted independently from each other by a negatively charged amino acid (in one preferred embodiment by E or D, in one preferred embodiment by D) (numbering according to Kabat EU index).
- a multispecific antibody as reported herein, the approach described in WO 2007/147901 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
- the amino acid K at position 253 is substituted by E
- the amino acid D at position 282 is substituted by K and the amino acid K at position 322 is substituted by D
- the amino acid D at position 239 is substituted by K
- the amino acid E at position 240 is substituted by K
- the amino acid K at position 292 is substituted by D (numbering according to Kabat EU index).
- the approach described in WO 2007/110205 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody
- the multispecific antibody is a bispecific antibody or a trispecific antibody.
- the multispecific antibody is a bispecific antibody.
- the antibody is a bivalent or trivalent antibody. In one embodiment the antibody is a bivalent antibody.
- the multispecific antibody has a constant domain structure of an IgG type antibody. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgGl, or of human subclass IgGl with the mutations L234A and L235A. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG2. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG3.
- the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 or, of human subclass IgG4 with the additional mutation S228P. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgGl or human subclass IgG4. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgGl with the mutations L234A and L235A (numbering according to Kabat EU index).
- the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgGl with the mutations L234A, L235A and P329G (numbering according to Kabat EU index). In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 with the mutations S228P and L235E (numbering according to Kabat EU index). In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 with the mutations S228P, L235E and P329G (numbering according to Kabat EU index).
- an antibody comprising a heavy chain including a CH3 domain as specified herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one embodiment of all aspects as reported herein, an antibody comprising a heavy chain including a CH3 domain, as specified herein, comprises an additional C-terminal glycine residue (G446, numbering according to Kabat EU index).
- amino acid sequence variants of the antibodies provided herein are contemplated.
- Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, Insertion and Deletion Variants
- antibody variants having one or more amino acid substitutions are provided.
- Sites of interest for substitutional mutagenesis include the HVRs and FRs.
- Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions”. More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes.
- Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE 1
- Amino acids may be grouped according to common side-chain properties:
- substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications
- An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
- Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity.
- Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom, H.R. et al. in
- affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
- a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
- HVR-directed approaches in which several HVR residues (e.g., 4-6 residues at a time) are randomized.
- HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
- CDR-H3 and CDR-L3 in particular are often targeted.
- substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
- conservative alterations e.g., conservative substitutions as provided herein
- Such alterations may, for example, be outside of antigen contacting residues in the HVRs.
- each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
- a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by
- a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
- a neutral or negatively charged amino acid e.g., alanine or polyalanine
- Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
- a crystal structure of an antigen- antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
- Variants may be screened to determine whether they contain the desired properties.
- Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
- terminal insertions include an antibody with an N-terminal methionyl residue.
- Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
- an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
- Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
- the antibody comprises an Fc-region
- the carbohydrate attached thereto may be altered.
- Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc-region (see, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32).
- the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
- modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
- antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc-region.
- the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5%> to 65%o or from 20%> to 40%>.
- the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
- Asn297 refers to the asparagine residue located at about position 297 in the Fc-region (EU numbering of Fc-region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
- Such fucosylation variants may have improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621.
- Examples of publications related to "defucosylated” or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;
- Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc-region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; US 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc-region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764. c) Fc-region variants
- one or more amino acid modifications may be introduced into the Fc-region of an antibody provided herein, thereby generating an Fc-region variant.
- the Fc-region variant may comprise a human Fc-region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
- the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
- In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
- Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
- NK cells express Fc(RIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
- FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492.
- Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in US 5,500,362 (see, e.g. Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502);
- non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI).
- Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
- ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95 (1998) 652-656.
- Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
- a CDC assay may be performed (see, for example, Gazzano-Santoro, H. et al, J. Immunol.
- FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int. Immunol. 18 (2006) 1759-1769).
- Antibodies with reduced effector function include those with substitution of one or more of Fc-region residues 238, 265, 269, 270, 297, 327 and 329 (US 6,737,056).
- Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US 7,332,581).
- an antibody variant comprises an Fc-region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region (EU numbering of residues).
- alterations are made in the Fc-region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent
- Cytotoxicity e.g., as described in US 6,194,551, WO 99/51642, and Idusogie, E.E. et al, J. Immunol. 164 (2000) 4178-4184.
- FcRn neonatal Fc receptor
- Those antibodies comprise an Fc-region with one or more substitutions therein which improve binding of the Fc-region to FcRn.
- Fc variants include those with substitutions at one or more of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (US 7,371,826).
- cysteine engineered antibodies e.g., "thioMAbs”
- one or more residues of an antibody are substituted with cysteine residues.
- the substituted residues occur at accessible sites of the antibody.
- reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
- any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc-region.
- Cysteine engineered antibodies may be generated as described, e.g., in US 7,521,541.
- an antibody provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
- the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
- Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co- polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
- PEG polyethylene glycol
- copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
- dextran polyvinyl alcohol
- Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
- the polymer may be of any molecular weight, and may be branched or non-branched.
- the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
- conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided.
- the non-proteinaceous moiety is a carbon nanotube (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605).
- the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.
- Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567.
- isolated nucleic acid encoding an anti-transferrin receptor antibody described herein is provided.
- Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
- one or more vectors e.g., expression vectors
- a host cell comprising such nucleic acid is provided.
- a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
- the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
- a method of making an anti- transferrin receptor antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
- nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
- nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
- Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
- antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
- For expression of antibody fragments and polypeptides in bacteria see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In:
- the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al, Nat. Biotech. 24 (2006) 210-215.
- Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
- Vertebrate cells may also be used as hosts.
- mammalian cell lines that are adapted to grow in suspension may be useful.
- useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F.L. et al, J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod.
- monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J.P. et al, Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells.
- Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR " CHO cells (Urlaub, G. et al, Proc. Natl. Acad.
- Anti-transferrin receptor antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
- an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, alphaLISA, Western blot, antibody or reverse phase array, etc.
- transferrin receptor in solution (cell supernatant, cell or tissue lysates, body fluids etc.) is bound by a capture antibody, which specifically binds to a first epitope on the transferrin receptor, or transferrin receptor in a certain conformation and a detection antibody coupled to a detection entity, which specifically binds to a second epitope or conformation of the transferrin receptor.
- the readout is based on the detection entity (chemiluminescence, fluorescence, energy transfer induced luminescence etc.).
- antibodies are spotted onto glass or nitrocellulose chips.
- the slides are blocked and incubated with transferrin receptor containing solution, washed to remove unbound antibodies and bound antibodies are detected with a fluorescently labeled corresponding secondary antibody.
- the fluorescence signal is measured by a fluorescence slide scanner.
- recombinant transferrin receptor, cell supernatant, cell or tissue lysates, body fluids etc. are spotted onto glass or nitrocellulose chips.
- the slides are blocked and individual arrays are incubated with an antibody against a specific epitope on the transferrin receptor. Unbound antibodies are washed off and bound antibodies are detected with a fluorescently labeled corresponding secondary antibody.
- the fluorescence signal is measured by a fluorescence slide scanner (Dernick, G., et al, J. Lipid Res. 52 (2011) 2323-2331).
- any of the anti-transferrin receptor antibodies provided herein is useful for detecting the presence of human transferrin receptor in a biological sample.
- the term "detecting" as used herein encompasses quantitative or qualitative detection.
- a biological sample comprises a cell or tissue, such as brain tissue.
- an anti-transferrin receptor antibody for use in a method of diagnosis or detection is provided.
- a method of detecting the presence of the transferrin receptor in a biological sample is provided.
- the method comprises contacting the biological sample with an anti- transferrin receptor antibody as described herein under conditions permissive for binding of the anti-transferrin receptor antibody to the transferrin receptor, and detecting whether a complex is formed between the anti-transferrin receptor antibody and the transferrin receptor.
- Such method may be an in vitro or in vivo method.
- an anti-transferrin receptor antibody is used to select subjects eligible for therapy with an anti-transferrin receptor antibody, e.g.
- the transferrin receptor is a biomarker for selection of patients.
- exemplary disorders that may be diagnosed using an antibody of the invention include neurodegeneration with brain iron accumulation type 1 (NBIA1), pure autonomic failure, Down's syndrome, complex of Guam, and several Lewy body disorders, such as diffuse Lewy body disease (DLBD), the Lewy body variant of Alzheimer's disease (LBV AD), certain forms of Gaucher' s disease, and Parkinson's Disease dementia (PDD).
- NBIA1 brain iron accumulation type 1
- DLBD diffuse Lewy body disease
- LBV AD Lewy body variant of Alzheimer's disease
- PDD Parkinson's Disease dementia
- labeled anti-transferrin receptor antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
- Exemplary labels include, but are not limited to, the radioisotopes P, C, I, H, and 13 T, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (US 4,737,456), luciferin, 2,3- dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen per
- compositions of an anti-transferrin receptor antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of lyophilized formulations or aqueous solutions.
- Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, argin
- Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.).
- sHASEGP soluble neutral-active hyaluronidase glycoproteins
- rhuPH20 HYLENEX®, Baxter International, Inc.
- Certain exemplary sHASEGPs and methods of use, including rhuPH20 are described in US 2005/0260186 and US 2006/0104968.
- a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
- Exemplary lyophilized antibody formulations are described in US 6,267,958.
- Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908
- the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
- active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
- Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
- Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
- an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
- the containers may be formed from a variety of materials such as glass or plastic.
- the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- At least one active agent in the composition is an antibody of the invention.
- the label or package insert indicates that the composition is used for treating the condition of choice.
- the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
- the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
- the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer's solution and dextrose solution.
- BWFI bacteriostatic water for injection
- any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-transferrin receptor antibody.
- Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).
- mice were immunized genetically, using a plasmid expression vector coding for full-length human or cynomolgus TfR by intradermal application of 100 ⁇ g vector DNA, followed by electroporation (2 square pulses of 1000 V/cm, duration 0.1 ms, interval 0.125 s; followed by 4 square pulses of 287.5 V/cm, duration 10 ms, interval 0.125 s.
- Mice received either 6 or 7 consecutive immunizations at days 0, 14, 28, 42, 56, 70, and 84.
- the fourth and sixth immunizations were performed with vector coding for cynomolgus TfR; vector coding for human TfR was used for all other immunizations.
- TfR lacking the helical domain extracellular domain of the human TfR beginning at Leul22, ending at Asn608, expressed in HEK293F cells as an N-terminal fusion to human Fc-region and purified by protein A affinity chromatography and size exclusion chromatography, and monoclonal antibodies were isolated by hybridoma technology, based on their ability to bind human and cynomolgus transferrin receptor expressed on the surface of stably trans fected CHO-K1 cells (see Example 4) ⁇
- New Zealand White rabbits or transgenic rabbits expressing a humanized antibody repertoire were immunized genetically, using a plasmid expression vector coding for full-length human or cynomolgus TfR, by intradermal application of 400 ⁇ g vector DNA, followed by electroporation (5 square pulses of 750 V/cm, duration 10 ms, interval 1 s.). Rabbits received 6 consecutive immunizations at days 0, 14, 28, 56, 84 and 112. The fourth and sixth immunizations were performed with vector coding for cynomolgus TfR; vector coding for human TfR was used for all other immunizations.
- Blood (10 % of estimated total blood volume) was taken at days 35, 63, 91 and 119. Serum was prepared, which was used for titer determination by ELISA (see below), and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process (see Example 2).
- PBMC peripheral blood mononuclear cells
- First immunization campaign Sterile 6-well plates (cell culture grade) covered with a confluent monolayer of CHO cells were used to deplete macrophages/monocytes through unspecific adhesion as well as non-specifically binding lymphocytes.
- each well was filled at maximum with 4 mL medium and up to 6xl0 6 PBMCs from the immunized rabbit and allowed to bind for 1 h at 37 °C in the incubator.
- the cells in the supernatant peripheral blood lymphocytes (PBLs) were used for the antigen panning step.
- PBLs peripheral blood lymphocytes
- 6-well tissue culture plates covered with a monolayer of human transferrin receptor-positive CHO cells were seeded with up to 6xl0 6 PBLs per 4 mL medium and allowed to bind for 1 h at 37 °C in the incubator.
- Non-adherent cells were removed by carefully washing the wells 1-2 times with lx PBS. The remaining sticky cells were detached by trypsin for 10 min. at 37 °C in the incubator. Trypsination was stopped with EL-4 B5 medium. The cells were kept on ice until the immune fluorescence staining.
- the anti-IgG FITC (AbD Serotec, Dusseldorf, Germany) was used for single cell sorting.
- cells from the depletion and enrichment step were incubated with the anti-IgG FITC antibody in PBS and incubated for 45 min. in the dark at 4 °C.
- the PBMCs were washed two fold with ice cold PBS.
- the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses.
- Propidium iodide in a concentration of 5 ⁇ g/mL (BD Pharmingen, San Diego, CA, USA) was added prior to the FACS analyses to discriminate between dead and live cells.
- the cultivation of the rabbit B-cells was prepared by a method similar to that described by Zubler et al. (1985). Briefly, single sorted rabbit B-cells were incubated in 96-well plates with 200 ⁇ EL-4 B5 medium containing Pansorbin Cells (1 : 100000) (Calbiochem (Merck), Darmstadt, Germany), 5 % rabbit thymocyte supernatant (charge TSN-M13 (10242), MicroCoat, Bernried, Germany) and gamma-irradiated murine EL-4-B5 thymoma cells (2.5 10 4 /well) for 7 days at 37 °C in an atmosphere of 5 % C0 2 in the incubator. The supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and were frozen at -80 °C in 100 RLT buffer (Qiagen, Hilden, Germany).
- AviTag antigen was cloned by connecting the hTfR ECD to the N-terminal hinge of a human hole Fc-region, which carried a C-terminal Avi-tag (SEQ ID NO: 99), and ligation into a mammalian expression vector. All our mammalian expression vector carries a MPSV promoter for initiation of transcription and translation, where transcription is terminated by a synthetic polyA signal sequence located downstream of the ORF. In addition the vector contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines. The correct ORF was verified by sequencing.
- This vector was used for expression in HEK293 EBNA suspension cells, by co-expression with an empty knob-Fc-region and the BirA protein, and adding 1 mM of biotin to the culture medium.
- the resulting biotinylated protein was purified by protein A affinity chromatography, followed by size exclusion chromatography.
- the cyTfR-Fc(KiH)-Avi antigen was produced respectively (SEQ ID NO: 100).
- the library used was a fully synthetic library using human frameworks of the VH1, 3, 4, and 5 families, as well as V-kappal, -2, and -3, and V-lambda3. Randomized amino-acids were in CDR-H3 and CDR-L3. Library pools were generated, using each heavy chain VH together with all different light chain libraries. Selections was carried out in solution according to the following procedure: 1. binding of approx. 10 12 phageimid particles of each library to 100 nM biotinylated TfR-avi-his for 0.5 h in a total volume of 1 mL, 2.
- binders generated against the recombinant soluble antigen often showed only weak binding on cells
- the two cell-lines TF-1, and NCI-H460 were used (both available from ATCC).
- 1 * 10 6 cells were incubated with approx. 10 12 phage particles for lh on ice to avoid receptor mediated internalization. Washing was performed by 5- 10 centrifugation steps using PBST buffer (PBS containing 1 % Tween-20). Entire cells were used to infect the TGI bacteria for phage rescue.
- binders were identified by ELISA as follows: 100 ⁇ , of 10 nM biotinylated TfR-avi-his per well were coated on neutravidin plates. Fab-containing bacterial supernatants were added and binding Fabs were detected via their FLAG- tags by using an anti-FLAG/HRP secondary antibody. ELISA-positive clones were bacterially expressed as soluble Fab fragments in 96-well format and supernatants were subjected to a kinetic screening experiment by SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing Fabs with the highest affinity constants were identified and the corresponding phageimids were sequenced.
- Fab antibodies were isolated by periplasmic preparation and purified using IMAC.
- FACS analysis Purified Fab antibodies were applied to TF-1 cells in various concentrations. Cell bound Fab antibodies were detected using a fluorescent labeled anti-Fab antibody and measurements by FACS. EC50 values were calculated.
- VL domain was amplified by PCR and cloned into a mammalian expression vector, as described above, directly upstream of a CL domain.
- VH domain was amplified by PCR and cloned directly upstream of a CH-Fc domain. The sequences of both expression vectors were determined. Production of IgG antibodies
- HEK293 EBNA suspension cells were transfected with both, the LC and HC encoding, plasmids and cultivated for 7days. The supernatant was cleared by sterile filtration. IgG concentration was determined by protein A chromatography. Determination of EC50 using the TagLite technology
- the gene of the ECD of hTfR or cyTfR, respectively, including the transmembrane domain was ligated downstream to SNAP -tag into a mammalian expression vector.
- This vector was used to transfect HEK293 EBNA suspension cells, resulting in display of the TfR with a C-terminal SNAP tag fusion ((SEQ ID NO: 101, SEQ ID NO: 102).
- the SNAP tag was specifically labeled with SNAP-Lumi4Tb (Cisbio).
- the labeling efficiency determined by measuring the emission of Terbium at 615 nm. Labeled cells were stored at -80°C.
- the VH domain was cloned into a mammalian expression vector encoding the human knob IgGl HC, which carries the L234A, L235A, and P329G mutations.
- This vector was used for expression in HEK293 EBNA suspension cells, co- expressing the LC (vector described above) and an empty hole-Fc domain carrying the L234A, L235A, P329G, H435R and Y436F mutations.
- the resulting protein was purified by protein A affinity chromatography. In cases of monomeric protein was >95 %, determined by analytical size exclusion chromatography, the protein was further purified by size exclusion chromatography.
- a cell ELISA using stably transfected CHO-K1 cells way employed Stable transfectants were obtained by transfecting CHO-K1 cells with expression plasmids containing expression cassettes for the human or cynomolgus TfR as well as for neomycin-phosphotransferase. After transfection, cells were diluted in growth medium containing 500 ⁇ g/mL G418 (Life Technologies).
- Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).
- the PCR conditions for the RbVH+RbVL were as follows: Hot start at 94 °C for 5 min.; 35 cycles of 20 sec. at 94 °C, 20 sec. at 70 °C, 45 sec. at 68 °C, and a final extension at 68 °C for 7 min.
- the PCR conditions for the HuVL were as follows: Hot start at 94 °C for 5 min.; 40 cycles of 20 sec. at 94 °C, 20 sec. at 52 °C, 45 sec. at 68 °C, and a final extension at 68 °C for 7 min.
- PCR- products coding for VH or VL were cloned as cDNA into expression vectors by the overhang cloning method (RS Haun et al, BioTechniques (1992) 13, 515-518; MZ Li et al, Nature Methods (2007) 4, 251-256).
- the expression vectors contained an expression cassette consisting of a 5' CMV promoter including intron A, and a 3' BGH poly adenylation sequence.
- the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E.coli.
- Three variants of the basic plasmid were used: one plasmid containing the rabbit IgG constant region designed to accept the VH regions while two additional plasmids containing rabbit or human kappa LC constant region to accept the VL regions.
- Linearized expression plasmids coding for the kappa or gamma constant region and VL /VH inserts were amplified by PCR using overlapping primers. Purified PCR products were incubated with T4 DNA-polymerase which generated single-strand overhangs. The reaction was stopped by dCTP addition. In the next step, plasmid and insert were combined and incubated with recA which induced site specific recombination. The recombined plasmids were transformed into E.coli. The next day the grown colonies were picked and tested for correct recombined plasmid by plasmid preparation, restriction analysis and DNA- sequencing.
- the isolated HC and LC plasmids were transiently co- transfected into HEK293 cells and the supernatants were harvested after 1 week.
- VH and VL chain amplification were as follows: Hot start at 94 °C for 5 min.; 35 cycles of 20 sec. at 94 °C, 20 sec. at 68 °C, 45 sec. at 68 °C, and a final extension at 68 °C for 7 min.
- PCR-products coding for VH or VL were cloned as cDNA into expression vectors by the overhang cloning method (RS Haun et al, BioTechniques (1992) 13, 515- 518; MZ Li et al, Nature Methods (2007) 4, 251-256).
- the expression vectors contained an expression cassette consisting of a 5' CMV promoter including intron A, and a 3' BGH poly adenylation sequence.
- the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E.coli.
- Two variants of the basic plasmid were used: one plasmid containing the human IgG constant region designed to accept the new amplified VH chain and a second plasmid containing the human kappa LC constant region to accept the VL chain.
- Linearized expression plasmids coding for the kappa or gamma constant region and VL /VH inserts were amplified by PCR using overlapping primers. Purified PCR products were incubated with T4 DNA-polymerase which generated single-strand overhangs. The reaction was stopped by dCTP addition.
- plasmid and insert were combined and incubated with recA which induced site specific recombination.
- the recombined plasmids were transformed into E.coli.
- hCMEC/D3 Medium and supplements for hCMEC/D3 (Weksler, B. B. et al, FASEB J. 19 (2005), 1872-1874) were obtained from Lonza.
- hCMEC/D3 cells (passages 26-29) were cultured to confluence on collagen-coated coverslips (microscopy) or flasks in EBM2 medium containing 2.5 % FBS, a quarter of the supplied growth factors and fully complemented with supplied hydrocortisone, gentamycin and ascorbic acid.
- Transcytosis assay of monovalent antibodies The entire assay was performed in serum-free EBM2 medium which was otherwise reconstituted as described in Example 1. Filter inserts with cells were incubated apically with monovalent antibodies (concentration: 2.67 ⁇ g/mL) for 1 hour at 37 °C following which the entire apical and basolateral media were collected. From these values, paracellular flux was calculated. The monolayers were washed at RT in serum-free medium apically (400 ⁇ ) and basolaterally (1600 ⁇ ) 3 x 3-5 min. each. All the washes were collected to monitor efficiency of removal of the unbound antibody.
- Pre-warmed medium was added to the apical chamber and the filters transferred to a fresh 12 well plate (blocked overnight with PBS containing 1 % BSA) containing 1600 ⁇ ⁇ pre-warmed medium. At this point, cells on filters were lysed in 500 ⁇ , RIPA buffer in order to determine specific antibody uptake.
- the remaining filters were incubated at 37 °C and samples collected at various time points to determine apical and/or basolateral release of antibody.
- the content of antibody in the samples was quantified using a highly sensitive IgG ELISA (see Example 3). For each time point, data were generated from three filter cell cultures.
- Sensitive IgG ELISA after transcytosis assay The entire procedure was performed at RT using an automated washer for the wash steps. A 384-well plate was coated with 30 ⁇ /well of 1 ⁇ g/mL anti-human/mouse- IgG, Fey-specific in PBS for 2 hours followed by 1 hour incubation in blocking buffer PBS containing 1 % BSA or 1 % CroteinC for human and mouse IgG assays, respectively). Serially diluted samples from the transcytosis assay and standard concentrations of the antibody used in the transcytosis assay were added to the plate and incubated for 2 hours.
- the binding experiment were carried out on a BIAcore B 4000 (GE Healthcare) equipped with CI sensorchip (GE Healthcare, cat.no. BR1005-35) pre-treated with anti-human Fab antibody (GE Healthcare, cat.no 28-9583-25) using a standard amine coupling chemistry procedure accordingly to the vendor's manual.
- sample antibody was immobilized applying a contact time of 60 seconds and a flow rate of 10 ⁇ / ⁇ in phosphate buffer saline pH 7.4, 0.05 % Tween 20 at 25 °C.
- Recombinant His6-tagged human transferrin receptor Recombinant His6-tagged human transferrin receptor
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020508049A (en) | 2017-02-17 | 2020-03-19 | デナリ セラピューティクス インコーポレイテッドDenali Therapeutics Inc. | Engineered transferrin receptor binding polypeptide |
CN108864283B (en) * | 2017-05-12 | 2021-10-29 | 中国药科大学 | Single-chain antibody of brain-targeted transferrin receptor and application thereof |
AU2018304173A1 (en) | 2017-07-17 | 2020-01-30 | Janssen Biotech, Inc. | Antigen binding regions against fibronectin type III domains and methods of using the same |
CA3072051A1 (en) * | 2017-08-10 | 2019-02-14 | Denali Therapeutics Inc. | Affinity-based methods for using transferrin receptor-binding proteins |
EP3904389A1 (en) | 2017-10-02 | 2021-11-03 | Denali Therapeutics Inc. | Fusion proteins comprising enzyme replacement therapy enzymes |
AU2021286655A1 (en) * | 2020-06-12 | 2023-01-05 | Nkarta, Inc. | Genetically modified natural killer cells for CD70-directed cancer immunotherapy |
EP4228674A1 (en) * | 2020-10-15 | 2023-08-23 | Bioincept LLC | Systems, compositions and methods of determining viability of embryos using the same |
KR20230135087A (en) * | 2021-01-29 | 2023-09-22 | 암젠 인크 | Materials and methods for monitoring cancer by administering anti-MCL1 antibodies |
EP4351648A1 (en) * | 2021-06-10 | 2024-04-17 | The Texas A&M University System | Treatment for down syndrome-related accelerated aging |
WO2023277627A1 (en) * | 2021-07-02 | 2023-01-05 | 주식회사 아임뉴런 | Blood-brain barrier permeable fusion protein and uses thereof |
WO2023128702A1 (en) * | 2021-12-31 | 2023-07-06 | 주식회사 아임뉴런 | Blood-brain barrier permeable fusion protein and uses thereof |
KR102575322B1 (en) * | 2021-12-31 | 2023-09-08 | 주식회사 아임뉴런 | Blood-brain barrier penetrating fusion protein and use thereof |
Citations (103)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4737456A (en) | 1985-05-09 | 1988-04-12 | Syntex (U.S.A.) Inc. | Reducing interference in ligand-receptor binding assays |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
EP0404097A2 (en) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof |
WO1993001161A1 (en) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Process for preparing sertraline intermediates |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
WO1993008829A1 (en) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions that mediate killing of hiv-infected cells |
WO1993010819A1 (en) | 1991-11-26 | 1993-06-10 | Alkermes, Inc. | Process for the preparation of transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
WO1993016185A2 (en) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Biosynthetic binding protein for cancer marker |
WO1994029351A2 (en) | 1993-06-16 | 1994-12-22 | Celltech Limited | Antibodies |
WO1994029350A2 (en) | 1993-06-14 | 1994-12-22 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Recombinant disulfide-stabilized polypeptide fragments having binding specificity |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
US5624821A (en) | 1987-03-18 | 1997-04-29 | Scotgen Biopharmaceuticals Incorporated | Antibodies with altered effector functions |
US5648237A (en) | 1991-09-19 | 1997-07-15 | Genentech, Inc. | Expression of functional antibody fragments |
WO1997030087A1 (en) | 1996-02-16 | 1997-08-21 | Glaxo Group Limited | Preparation of glycosylated antibodies |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
WO1998050431A2 (en) | 1997-05-02 | 1998-11-12 | Genentech, Inc. | A method for making multispecific antibodies having heteromultimeric and common components |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
WO1998058964A1 (en) | 1997-06-24 | 1998-12-30 | Genentech, Inc. | Methods and compositions for galactosylated glycoproteins |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
WO1999022764A1 (en) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Methods and compositions comprising glycoprotein glycoforms |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
WO1999051642A1 (en) | 1998-04-02 | 1999-10-14 | Genentech, Inc. | Antibody variants and fragments thereof |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
WO2000061739A1 (en) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
US6194551B1 (en) | 1998-04-02 | 2001-02-27 | Genentech, Inc. | Polypeptide variants |
WO2001029246A1 (en) | 1999-10-19 | 2001-04-26 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing polypeptide |
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
WO2002031140A1 (en) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cells producing antibody compositions |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
US20020164328A1 (en) | 2000-10-06 | 2002-11-07 | Toyohide Shinkawa | Process for purifying antibody |
WO2003011878A2 (en) | 2001-08-03 | 2003-02-13 | Glycart Biotechnology Ag | Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity |
US20030115614A1 (en) | 2000-10-06 | 2003-06-19 | Yutaka Kanda | Antibody composition-producing cell |
US6602684B1 (en) | 1998-04-20 | 2003-08-05 | Glycart Biotechnology Ag | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
US20030157108A1 (en) | 2001-10-25 | 2003-08-21 | Genentech, Inc. | Glycoprotein compositions |
WO2003084570A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM |
WO2003085107A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Cells with modified genome |
WO2003085119A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US20040110282A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost |
US20040109865A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Antibody composition-containing medicament |
WO2004056312A2 (en) | 2002-12-16 | 2004-07-08 | Genentech, Inc. | Immunoglobulin variants and uses thereof |
US20040132140A1 (en) | 2002-04-09 | 2004-07-08 | Kyowa Hakko Kogyo Co., Ltd. | Production process for antibody composition |
US20050014934A1 (en) | 2002-10-15 | 2005-01-20 | Hinton Paul R. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2005035778A1 (en) | 2003-10-09 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE |
WO2005035586A1 (en) | 2003-10-08 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Fused protein composition |
US20050119455A1 (en) | 2002-06-03 | 2005-06-02 | Genentech, Inc. | Synthetic antibody phage libraries |
US20050123546A1 (en) | 2003-11-05 | 2005-06-09 | Glycart Biotechnology Ag | Antigen binding molecules with increased Fc receptor binding affinity and effector function |
WO2005053742A1 (en) | 2003-12-04 | 2005-06-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicine containing antibody composition |
WO2005100402A1 (en) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anti-p-selectin antibodies |
US20050260186A1 (en) | 2003-03-05 | 2005-11-24 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
US20050266000A1 (en) | 2004-04-09 | 2005-12-01 | Genentech, Inc. | Variable domain library and uses |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
US20060025576A1 (en) | 2000-04-11 | 2006-02-02 | Genentech, Inc. | Multivalent antibodies and uses therefor |
WO2006029879A2 (en) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anti-ox40l antibodies |
WO2006044908A2 (en) | 2004-10-20 | 2006-04-27 | Genentech, Inc. | Antibody formulation in histidine-acetate buffer |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
WO2006056879A1 (en) | 2004-11-29 | 2006-06-01 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Human blood brain barrier model |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
WO2007044323A2 (en) * | 2005-10-07 | 2007-04-19 | Armagen Technologies, Inc. | Fusion proteins for blood-brain barrier delivery |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
US20070160598A1 (en) | 2005-11-07 | 2007-07-12 | Dennis Mark S | Binding polypeptides with diversified and consensus vh/vl hypervariable sequences |
WO2007110205A2 (en) | 2006-03-24 | 2007-10-04 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
EP1870459A1 (en) | 2005-03-31 | 2007-12-26 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
WO2007147901A1 (en) | 2006-06-22 | 2007-12-27 | Novo Nordisk A/S | Production of bispecific antibodies |
WO2008022349A2 (en) * | 2006-08-18 | 2008-02-21 | Armagen Technologies, Inc. | Agents for blood-brain barrier delivery |
US20080069820A1 (en) | 2006-08-30 | 2008-03-20 | Genentech, Inc. | Multispecific antibodies |
US7371826B2 (en) | 1999-01-15 | 2008-05-13 | Genentech, Inc. | Polypeptide variants with altered effector function |
WO2008077546A1 (en) | 2006-12-22 | 2008-07-03 | F. Hoffmann-La Roche Ag | Antibodies against insulin-like growth factor i receptor and uses thereof |
US20090002360A1 (en) | 2007-05-25 | 2009-01-01 | Innolux Display Corp. | Liquid crystal display device and method for driving same |
US7521541B2 (en) | 2004-09-23 | 2009-04-21 | Genetech Inc. | Cysteine engineered antibodies and conjugates |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
WO2009080253A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080252A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080254A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080251A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009089004A1 (en) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
WO2010112193A1 (en) | 2009-04-02 | 2010-10-07 | Roche Glycart Ag | Multispecific antibodies comprising full length antibodies and single chain fab fragments |
WO2010115589A1 (en) | 2009-04-07 | 2010-10-14 | Roche Glycart Ag | Trivalent, bispecific antibodies |
WO2010129304A2 (en) | 2009-04-27 | 2010-11-11 | Oncomed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
WO2010136172A1 (en) | 2009-05-27 | 2010-12-02 | F. Hoffmann-La Roche Ag | Tri- or tetraspecific antibodies |
WO2010145793A1 (en) | 2009-06-18 | 2010-12-23 | F. Hoffmann-La Roche Ag | Bispecific, tetravalent antigen binding proteins |
WO2010145792A1 (en) | 2009-06-16 | 2010-12-23 | F. Hoffmann-La Roche Ag | Bispecific antigen binding proteins |
WO2011090762A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Heterodimer binding proteins and uses thereof |
WO2011143545A1 (en) | 2010-05-14 | 2011-11-17 | Rinat Neuroscience Corporation | Heterodimeric proteins and methods for producing and purifying them |
WO2012058768A1 (en) | 2010-11-05 | 2012-05-10 | Zymeworks Inc. | Stable heterodimeric antibody design with mutations in the fc domain |
WO2012075037A1 (en) * | 2010-11-30 | 2012-06-07 | Genentech, Inc. | Low affinity blood brain barrier receptor antibodies and uses therefor |
WO2012143379A1 (en) * | 2011-04-20 | 2012-10-26 | Roche Glycart Ag | Method and constructs for the ph dependent passage of the blood-brain-barrier |
WO2013096291A2 (en) | 2011-12-20 | 2013-06-27 | Medimmune, Llc | Modified polypeptides for bispecific antibody scaffolds |
WO2013157953A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
WO2014033074A1 (en) | 2012-08-29 | 2014-03-06 | F. Hoffmann-La Roche Ag | Blood brain barrier shuttle |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2303461C9 (en) * | 2002-08-06 | 2007-12-27 | Глаксо Груп Лимитед | Antibodies against myelin-associated glycoprotein (mag) |
CN1713920A (en) * | 2002-08-30 | 2005-12-28 | 比奥雷克西斯药物公司 | Modified transferrin fusion proteins |
CN1788017B (en) * | 2003-02-10 | 2013-04-24 | to-BBB控股股份有限公司 | Differentially expressed nucleic acids in the blood-brain barrier under inflammatory conditions |
CN101061140B (en) * | 2004-09-17 | 2015-07-01 | 多曼蒂斯有限公司 | Compositions monovalent for CD40L binding and methods of use |
ZA200804337B (en) * | 2005-11-28 | 2009-11-25 | Genmab As | Recombinant monovalent antibodies and methods for production thereof |
WO2007143711A2 (en) * | 2006-06-07 | 2007-12-13 | Wisconsin Alumni Research Foundation | Blood-brain barrier targeting antibodies |
CN101245107B (en) * | 2007-02-14 | 2010-10-13 | 中国人民解放军军事医学科学院生物工程研究所 | Antihuman transferrin acceptor human source antibody and uses thereof |
US20100098693A1 (en) * | 2008-10-07 | 2010-04-22 | Pardridge William M | Compositions and methods for blood-brain barrier delivery of organophosphatases |
KR20110124368A (en) * | 2009-04-07 | 2011-11-16 | 로슈 글리카트 아게 | Bispecific anti-erbb-2/anti-c-met antibodies |
EP2504030A4 (en) * | 2009-11-24 | 2013-06-26 | Alderbio Holdings Llc | Antagonists of il-6 to raise albumin and/or lower crp |
FR2953841B1 (en) * | 2009-12-16 | 2011-12-30 | Centre Nat Rech Scient | ANTIBODIES AGAINST THE TRANSFERRIN RECEPTOR AND THEIR USES FOR THE IMMUNOTHERAPY OF TUMORS THAT DEPEND ON IRON |
KR20190112175A (en) * | 2010-12-01 | 2019-10-02 | 앨더바이오 홀딩스 엘엘씨 | Anti-ngf compositions and use thereof |
WO2012088247A2 (en) * | 2010-12-22 | 2012-06-28 | Medimmune, Llc | Anti-c5/c5a/c5adesr antibodies and fragments |
WO2012087835A2 (en) | 2010-12-22 | 2012-06-28 | Boston Biomedical Research Institute | Compositions and methods for enhancing protein folding |
WO2012130831A1 (en) * | 2011-03-29 | 2012-10-04 | Roche Glycart Ag | Antibody fc variants |
CN103534272B (en) | 2011-05-09 | 2016-05-11 | 株式会社英仙蛋白质科学 | Antibody that can specific recognition TfR |
US9499608B2 (en) * | 2011-06-08 | 2016-11-22 | Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University | Bispecific monoclonal antibody therapeutics against West Nile virus with improved CNS penetration |
EP2999716A2 (en) | 2013-05-20 | 2016-03-30 | F. Hoffmann-La Roche AG | Anti-transferrin receptor antibodies and methods of use |
-
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- 2014-12-29 PL PL14830531T patent/PL3092251T3/en unknown
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- 2016-07-06 US US15/203,057 patent/US10364292B2/en active Active
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Patent Citations (110)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4737456A (en) | 1985-05-09 | 1988-04-12 | Syntex (U.S.A.) Inc. | Reducing interference in ligand-receptor binding assays |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
US5648260A (en) | 1987-03-18 | 1997-07-15 | Scotgen Biopharmaceuticals Incorporated | DNA encoding antibodies with altered effector functions |
US5624821A (en) | 1987-03-18 | 1997-04-29 | Scotgen Biopharmaceuticals Incorporated | Antibodies with altered effector functions |
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
EP0404097A2 (en) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US6417429B1 (en) | 1989-10-27 | 2002-07-09 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
WO1993001161A1 (en) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Process for preparing sertraline intermediates |
US5648237A (en) | 1991-09-19 | 1997-07-15 | Genentech, Inc. | Expression of functional antibody fragments |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
WO1993008829A1 (en) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions that mediate killing of hiv-infected cells |
WO1993010819A1 (en) | 1991-11-26 | 1993-06-10 | Alkermes, Inc. | Process for the preparation of transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
WO1993016185A2 (en) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Biosynthetic binding protein for cancer marker |
WO1994029350A2 (en) | 1993-06-14 | 1994-12-22 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Recombinant disulfide-stabilized polypeptide fragments having binding specificity |
WO1994029351A2 (en) | 1993-06-16 | 1994-12-22 | Celltech Limited | Antibodies |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
WO1997030087A1 (en) | 1996-02-16 | 1997-08-21 | Glaxo Group Limited | Preparation of glycosylated antibodies |
WO1998050431A2 (en) | 1997-05-02 | 1998-11-12 | Genentech, Inc. | A method for making multispecific antibodies having heteromultimeric and common components |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
WO1998058964A1 (en) | 1997-06-24 | 1998-12-30 | Genentech, Inc. | Methods and compositions for galactosylated glycoproteins |
WO1999022764A1 (en) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Methods and compositions comprising glycoprotein glycoforms |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
WO1999051642A1 (en) | 1998-04-02 | 1999-10-14 | Genentech, Inc. | Antibody variants and fragments thereof |
US6194551B1 (en) | 1998-04-02 | 2001-02-27 | Genentech, Inc. | Polypeptide variants |
US6602684B1 (en) | 1998-04-20 | 2003-08-05 | Glycart Biotechnology Ag | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US7332581B2 (en) | 1999-01-15 | 2008-02-19 | Genentech, Inc. | Polypeptide variants with altered effector function |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US7371826B2 (en) | 1999-01-15 | 2008-05-13 | Genentech, Inc. | Polypeptide variants with altered effector function |
WO2000061739A1 (en) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
WO2001029246A1 (en) | 1999-10-19 | 2001-04-26 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing polypeptide |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
US20060025576A1 (en) | 2000-04-11 | 2006-02-02 | Genentech, Inc. | Multivalent antibodies and uses therefor |
WO2002031140A1 (en) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cells producing antibody compositions |
US20030115614A1 (en) | 2000-10-06 | 2003-06-19 | Yutaka Kanda | Antibody composition-producing cell |
US20020164328A1 (en) | 2000-10-06 | 2002-11-07 | Toyohide Shinkawa | Process for purifying antibody |
WO2003011878A2 (en) | 2001-08-03 | 2003-02-13 | Glycart Biotechnology Ag | Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity |
US20030157108A1 (en) | 2001-10-25 | 2003-08-21 | Genentech, Inc. | Glycoprotein compositions |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
US20040109865A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Antibody composition-containing medicament |
US20040132140A1 (en) | 2002-04-09 | 2004-07-08 | Kyowa Hakko Kogyo Co., Ltd. | Production process for antibody composition |
US20040110704A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells of which genome is modified |
US20040110282A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost |
WO2003085119A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa |
WO2003085107A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Cells with modified genome |
WO2003084570A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM |
US20050119455A1 (en) | 2002-06-03 | 2005-06-02 | Genentech, Inc. | Synthetic antibody phage libraries |
US20050014934A1 (en) | 2002-10-15 | 2005-01-20 | Hinton Paul R. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
WO2004056312A2 (en) | 2002-12-16 | 2004-07-08 | Genentech, Inc. | Immunoglobulin variants and uses thereof |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US20050260186A1 (en) | 2003-03-05 | 2005-11-24 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
WO2005035586A1 (en) | 2003-10-08 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Fused protein composition |
WO2005035778A1 (en) | 2003-10-09 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE |
US20050123546A1 (en) | 2003-11-05 | 2005-06-09 | Glycart Biotechnology Ag | Antigen binding molecules with increased Fc receptor binding affinity and effector function |
WO2005053742A1 (en) | 2003-12-04 | 2005-06-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicine containing antibody composition |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
US20050266000A1 (en) | 2004-04-09 | 2005-12-01 | Genentech, Inc. | Variable domain library and uses |
WO2005100402A1 (en) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anti-p-selectin antibodies |
WO2006029879A2 (en) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anti-ox40l antibodies |
US7521541B2 (en) | 2004-09-23 | 2009-04-21 | Genetech Inc. | Cysteine engineered antibodies and conjugates |
WO2006044908A2 (en) | 2004-10-20 | 2006-04-27 | Genentech, Inc. | Antibody formulation in histidine-acetate buffer |
WO2006056879A1 (en) | 2004-11-29 | 2006-06-01 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Human blood brain barrier model |
EP1870459A1 (en) | 2005-03-31 | 2007-12-26 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
WO2007044323A2 (en) * | 2005-10-07 | 2007-04-19 | Armagen Technologies, Inc. | Fusion proteins for blood-brain barrier delivery |
US20070160598A1 (en) | 2005-11-07 | 2007-07-12 | Dennis Mark S | Binding polypeptides with diversified and consensus vh/vl hypervariable sequences |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
WO2007110205A2 (en) | 2006-03-24 | 2007-10-04 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
WO2007147901A1 (en) | 2006-06-22 | 2007-12-27 | Novo Nordisk A/S | Production of bispecific antibodies |
WO2008022349A2 (en) * | 2006-08-18 | 2008-02-21 | Armagen Technologies, Inc. | Agents for blood-brain barrier delivery |
US20080069820A1 (en) | 2006-08-30 | 2008-03-20 | Genentech, Inc. | Multispecific antibodies |
WO2008077546A1 (en) | 2006-12-22 | 2008-07-03 | F. Hoffmann-La Roche Ag | Antibodies against insulin-like growth factor i receptor and uses thereof |
US20090002360A1 (en) | 2007-05-25 | 2009-01-01 | Innolux Display Corp. | Liquid crystal display device and method for driving same |
WO2009080253A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080252A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080254A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009080251A1 (en) | 2007-12-21 | 2009-07-02 | F. Hoffmann-La Roche Ag | Bivalent, bispecific antibodies |
WO2009089004A1 (en) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
WO2010112193A1 (en) | 2009-04-02 | 2010-10-07 | Roche Glycart Ag | Multispecific antibodies comprising full length antibodies and single chain fab fragments |
WO2010115589A1 (en) | 2009-04-07 | 2010-10-14 | Roche Glycart Ag | Trivalent, bispecific antibodies |
WO2010129304A2 (en) | 2009-04-27 | 2010-11-11 | Oncomed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
WO2010136172A1 (en) | 2009-05-27 | 2010-12-02 | F. Hoffmann-La Roche Ag | Tri- or tetraspecific antibodies |
WO2010145792A1 (en) | 2009-06-16 | 2010-12-23 | F. Hoffmann-La Roche Ag | Bispecific antigen binding proteins |
WO2010145793A1 (en) | 2009-06-18 | 2010-12-23 | F. Hoffmann-La Roche Ag | Bispecific, tetravalent antigen binding proteins |
WO2011090762A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Heterodimer binding proteins and uses thereof |
WO2011090754A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Polypeptide heterodimers and uses thereof |
WO2011143545A1 (en) | 2010-05-14 | 2011-11-17 | Rinat Neuroscience Corporation | Heterodimeric proteins and methods for producing and purifying them |
WO2012058768A1 (en) | 2010-11-05 | 2012-05-10 | Zymeworks Inc. | Stable heterodimeric antibody design with mutations in the fc domain |
WO2012075037A1 (en) * | 2010-11-30 | 2012-06-07 | Genentech, Inc. | Low affinity blood brain barrier receptor antibodies and uses therefor |
WO2012143379A1 (en) * | 2011-04-20 | 2012-10-26 | Roche Glycart Ag | Method and constructs for the ph dependent passage of the blood-brain-barrier |
WO2013096291A2 (en) | 2011-12-20 | 2013-06-27 | Medimmune, Llc | Modified polypeptides for bispecific antibody scaffolds |
WO2013157953A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
WO2013157954A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
WO2014033074A1 (en) | 2012-08-29 | 2014-03-06 | F. Hoffmann-La Roche Ag | Blood brain barrier shuttle |
Non-Patent Citations (103)
Title |
---|
"Remington's Pharmaceutical Sciences", 1980 |
ALMAGRO, J.C.; FRANSSON, J., FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633 |
ATWELL, S. ET AL., J. MOL. BIOL., vol. 270, 1997, pages 26 - 35 |
BACA, M. ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684 |
BOADO R J ET AL: "Selective targeting of a TNFR decoy receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 146, no. 1-2, 1 March 2010 (2010-03-01), pages 84 - 91, XP026929529, ISSN: 0168-1656, [retrieved on 20100125], DOI: 10.1016/J.JBIOTEC.2010.01.011 * |
BOADO RUBEN J ET AL: "Drug targeting of erythropoietin across the primate blood-brain barrier with an IgG molecular Trojan horse", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, US, vol. 333, no. 3, 1 June 2010 (2010-06-01), pages 961 - 969, XP009145098, ISSN: 0022-3565, DOI: 10.1124/JPET.109.165092 * |
BOADO, R.J. ET AL., BIOTECHNOL. BIOENG., vol. 102, 2009, pages 1251 - 1258 |
BRENNAN, M. ET AL., SCIENCE, vol. 229, 1985, pages 81 - 83 |
BRUGGEMANN, M. ET AL., J. EXP. MED., vol. 166, 1987, pages 1351 - 1361 |
CARTER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285 - 4289 |
CHARI ET AL., CANCER RES., vol. 52, 1992, pages 127 - 131 |
CHARLTON, K.A.: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 245 - 254 |
CHEN, Y. ET AL., J. MOL. BIOL., vol. 293, 1999, pages 865 - 881 |
CHOTHIA, C.; LESK, A.M., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
CHOWDHURY, P.S., METHODS MOL. BIOL., vol. 207, 2008, pages 179 - 196 |
CLACKSON, T. ET AL., NATURE, vol. 352, 1991, pages 624 - 628 |
CLYNES, R. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656 |
CRAGG, M.S. ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052 |
CRAGG, M.S.; M.J. GLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743 |
CUNNINGHAM, B.C.; WELLS, J.A., SCIENCE, vol. 244, 1989, pages 1081 - 1085 |
DALL'ACQUA, W.F. ET AL., METHODS, vol. 36, 2005, pages 43 - 60 |
DERNICK, G. ET AL., J. LIPID RES., vol. 52, 2011, pages 2323 - 2331 |
DUNCAN, A.R.; WINTER, G., NATURE, vol. 322, 1988, pages 738 - 740 |
FELGENHAUER, KLIN. WSCHR., vol. 52, 1974, pages 1158 - 1164 |
FELLOUSE, F.A., PROC. NATL. ACAD. SCI. USA, vol. 101, 2004, pages 12467 - 12472 |
FLATMAN, S. ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87 |
GAZZANO-SANTORO, H. ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163 - 171 |
GERNGROSS, T.U., NAT. BIOTECH., vol. 22, 2004, pages 1409 - 1414 |
GRAHAM, F.L. ET AL., J. GEN VIROL., vol. 36, 1977, pages 59 - 74 |
GRIFFITHS, A.D. ET AL., EMBO J., vol. 12, 1993, pages 725 - 734 |
GRUBER, M ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368 - 5374 |
GUYER, R.L. ET AL., J. IMMUNOL., vol. 117, 1976, pages 587 - 593 |
HELLSTROM, 1. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 7059 - 7063 |
HELLSTROM, I. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 1499 - 1502 |
HOLLIGER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448 |
HOOGENBOOM, H.R. ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 178, 2001, pages 1 - 37 |
HOOGENBOOM, H.R. ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 178, 2002, pages 1 - 37 |
HOOGENBOOM, H.R.; WINTER, G., J. MOL. BIOL., vol. 227, 1992, pages 381 - 388 |
HUDSON, P.J. ET AL., NAT. MED., vol. 9, 2003, pages 129 - 134 |
HUST MICHAEL ET AL: "Single chain Fab (scFab) fragment", BMC BIOTECHNOLOGY, BIOMED CENTRAL LTD. LONDON, GB, vol. 7, no. 1, 8 March 2007 (2007-03-08), pages 14, XP021023594, ISSN: 1472-6750, DOI: 10.1186/1472-6750-7-14 * |
IDUSOGIE, E.E. ET AL., J. IMMUNOL., vol. 164, 2000, pages 4178 - 4184 |
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH |
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION 91-3242 |
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest", vol. 1-3, 1991, NIH PUBLICATION 91-3242 |
KAM, N.W. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 11600 - 11605 |
KANDA, Y. ET AL., BIOTECHNOL. BIOENG., vol. 94, 2006, pages 680 - 688 |
KASHMIRI, S.V. ET AL., METHODS, vol. 36, 2005, pages 25 - 34 |
KIM, J.K. ET AL., J. IMMUNOL., vol. 24, 1994, pages 2429 - 2434 |
KINDT, T.J. ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91 |
KLIMKA, A. ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260 |
KOBAYASHI, H. ET AL., NUCLEAR MEDICINE & BIOLOGY, vol. 25, 1998, pages 387 - 393 |
KOSTELNY, S.A. ET AL., J. IMMUNOL., vol. 148, 1992, pages 1547 - 1553 |
LEE, C.V. ET AL., J. IMMUNOL. METHODS, vol. 284, 2004, pages 119 - 132 |
LEE, C.V. ET AL., J. MOL. BIOL., vol. 340, 2004, pages 1073 - 1093 |
LI, H. ET AL., NAT. BIOTECH., vol. 24, 2006, pages 210 - 215 |
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745 |
MARKS, J.D. ET AL., J. MOL. BIOL., vol. 222, 1992, pages 581 - 597 |
MARKS, J.D.; BRADBURY, A., METHODS IN MOLECULAR BIOLOGY, vol. 248, 2003, pages 161 - 175 |
MATHER, J.P. ET AL.: "Annals N.Y. Acad. Sci.", vol. 383, 1982, pages: 44 - 68 |
MATHER, J.P., BIOL. REPROD., vol. 23, 1980, pages 243 - 252 |
MCCAFFERTY, J. ET AL., NATURE, vol. 348, 1990, pages 552 - 554 |
MEISSNER, P. ET AL., BIOTECHNOL. BIOENG., vol. 75, 2001, pages 197 - 203 |
MERCHANT, A.M. ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 677 - 681 |
MERCHANT, A.M. ET AL., NATURE BIOTECH., vol. 16, 1998, pages 677 - 681 |
MILSTEIN, C.; CUELLO, A.C., NATURE, vol. 305, 1983, pages 537 - 540 |
MORRISON, S.L. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855 |
MZ LI ET AL., NATURE METHODS, vol. 4, 2007, pages 251 - 256 |
OKAZAKI, A. ET AL., J. MOL. BIOL., vol. 336, 2004, pages 1239 - 1249 |
OSBOURN, J. ET AL., METHODS, vol. 36, 2005, pages 61 - 68 |
PADLAN, E.A., MOL. IMMUNOL., vol. 28, 1991, pages 489 - 498 |
PETKOVA, S.B. ET AL., INT. IMMUNOL., vol. 18, 2006, pages 1759 - 1769 |
PLUECKTHUN, A.: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315 |
PORTOLANO, S. ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887 |
PRESTA, L.G. ET AL., CANCER RES., vol. 57, 1997, pages 4593 - 4599 |
PRESTA, L.G. ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 2632 |
QUEEN, C. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033 |
RAJAGOPAL, V. ET AL., PROT. ENG., 1997, pages 1453 - 59 |
RAVETCH, J.V.; KINET, J.P., ANNU. REV. IMMUNOL., vol. 9, 1991, pages 457 - 492 |
RIDGWAY, J.B. ET AL., PROTEIN ENG., vol. 9, 1996, pages 617 - 621 |
RIECHMANN, I. ET AL., NATURE, vol. 332, 1988, pages 323 - 329 |
RIPKA, J. ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 249, 1986, pages 533 - 545 |
ROSOK, M.J. ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618 |
RS HAUN ET AL., BIOTECHNIQUES, vol. 13, 1992, pages 515 - 518 |
RUBEN J BOADO ET AL: "Engineering and Expression of a Chimeric Transferrin Receptor Monoclonal Antibody for Blood-Brain Barrier Delivery in the Mouse", BIOTECHNOLOGY AND BIOENGINEERING, WILEY & SONS, HOBOKEN, NJ, US, vol. 102, no. 4, 1 March 2009 (2009-03-01), pages 1251 - 1258, XP002692095, ISSN: 0006-3592, [retrieved on 20080919], DOI: 10.1002/BIT.22135 * |
SAMBROOK, J. ET AL.: "Molecular cloning: A laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS |
SCHMIDT, M. ET AL., ONCOGENE, vol. 18, 1999, pages 1711 - 1721 |
SCHNEIDER ET AL., NATURE, vol. 311, 1984, pages 675 - 678 |
SHIELDS, R.L. ET AL., J. BIOL. CHEM., vol. 276, 2001, pages 6591 - 6604 |
SIDHU, S.S. ET AL., J. MOL. BIOL., vol. 338, 2004, pages 299 - 310 |
SILACCI ET AL., PROTEOMICS, vol. 5, 2005, pages 2340 - 2350 |
SIMS, M.J. ET AL., J. IMMUNOL., vol. 151, 1993, pages 2296 - 2308 |
TRAUNECKER, A. ET AL., EMBO J., vol. 10, 1991, pages 3655 - 3659 |
TUTT, A. ET AL., J. IMMUNOL., vol. 147, 1991, pages 60 - 69 |
URLAUB, G. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 4220 |
WEKSLER, B. B. ET AL., FASEB J., vol. 19, 2005, pages 1872 - 1874 |
WEKSLER, B.B. ET AL., FASEB J., vol. 19, 2005, pages 1872 - 1874 |
WILLIAM M PARDRIDGE: "Drug transport across the blood-brain barrier", JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM, vol. 32, no. 11, 29 August 2012 (2012-08-29), pages 1959 - 1972, XP055118945, ISSN: 0271-678X, DOI: 10.1038/jcbfm.2012.126 * |
WINTER, G. ET AL., ANN. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455 |
WRIGHT, A.; MORRISON, S.L., TIBTECH, vol. 15, 1997, pages 26 - 32 |
Y. JOY YU ET AL: "Developing Therapeutic Antibodies for Neurodegenerative Disease", NEUROTHERAPEUTICS, vol. 10, no. 3, 3 April 2013 (2013-04-03), pages 459 - 472, XP055107976, ISSN: 1933-7213, DOI: 10.1007/s13311-013-0187-4 * |
YAMANE-OHNUKI, N. ET AL., BIOTECH. BIOENG., vol. 87, 2004, pages 614 - 622 |
YAZAKI, P.; WU, A.M.: "Methods in Molecular Biology", vol. 248, 2004, HUMANA PRESS, pages: 255 - 268 |
ZATSEPIN ET AL., RUSS. CHEM. REV., vol. 74, 2005, pages 77 - 95 |
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EP3150636A1 (en) * | 2015-10-02 | 2017-04-05 | F. Hoffmann-La Roche AG | Tetravalent multispecific antibodies |
EP3150637A1 (en) * | 2015-10-02 | 2017-04-05 | F. Hoffmann-La Roche AG | Multispecific antibodies |
JP2018533930A (en) * | 2015-10-02 | 2018-11-22 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific T cell activation antigen binding molecule |
JP2018533928A (en) * | 2015-10-02 | 2018-11-22 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-CEAXCD3 T cell activation antigen binding molecule |
CN107849137A (en) * | 2015-10-02 | 2018-03-27 | 豪夫迈·罗氏有限公司 | The anti-CEAXCD3 T cell activations antigen binding molecules of bispecific |
JP2018535196A (en) * | 2015-10-02 | 2018-11-29 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Multispecific antibody |
JP2018535936A (en) * | 2015-10-02 | 2018-12-06 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Bispecific anti-human A-beta / human transferrin receptor antibody and method of use thereof |
JP2018536629A (en) * | 2015-10-02 | 2018-12-13 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-human CD20 / human transferrin receptor antibody and method of use |
JP2019500007A (en) * | 2015-10-02 | 2019-01-10 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Tetravalent multispecific antibody |
US20170129962A1 (en) * | 2015-10-02 | 2017-05-11 | Hoffmann-La Roche Inc. | Multispecific antibodies |
WO2017055388A3 (en) * | 2015-10-02 | 2017-05-11 | F. Hoffmann-La Roche Ag | Bispecific t cell activating antigen binding molecules |
TWI819458B (en) * | 2015-10-02 | 2023-10-21 | 瑞士商赫孚孟拉羅股份公司 | Bispecific anti-human cd20/human transferrin receptor antibodies and methods of use |
WO2017055393A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Anti-cd3xtim-3 bispecific t cell activating antigen binding molecules |
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WO2017055318A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Cd33xcd3 bispecific t cell activating antigen binding molecules |
WO2017055389A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Bispecific anti-ceaxcd3 t cell activating antigen binding molecules |
WO2017055539A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Multispecific antibodies |
JP2020090524A (en) * | 2015-10-02 | 2020-06-11 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-human cd20/human transferrin receptor antibodies and use methods thereof |
US10766967B2 (en) | 2015-10-02 | 2020-09-08 | Hoffmann-La Roche Inc. | Bispecific T cell activating antigen binding molecules |
JP2020158485A (en) * | 2015-10-02 | 2020-10-01 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Bispecific anti-human a-beta/human transferrin receptor antibodies and methods of use |
WO2017055395A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Anti-cd3xrob04 bispecific t cell activating antigen binding molecules |
US11603411B2 (en) | 2015-10-02 | 2023-03-14 | Hoffmann-La Roche Inc. | Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use |
WO2017055385A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Anti-cd3xgd2 bispecific t cell activating antigen binding molecules |
US10941205B2 (en) | 2015-10-02 | 2021-03-09 | Hoffmann-La Roche Inc. | Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use |
TWI723050B (en) * | 2015-10-02 | 2021-04-01 | 瑞士商赫孚孟拉羅股份公司 | Bispecific anti-human a-beta/human transferrin receptor antibodies and methods of use |
JP7239321B2 (en) | 2015-10-02 | 2023-03-14 | エフ. ホフマン-ラ ロシュ アーゲー | Tetravalent multispecific antibody |
JP7239320B2 (en) | 2015-10-02 | 2023-03-14 | エフ. ホフマン-ラ ロシュ アーゲー | Multispecific antibody |
WO2017055392A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Anti-cd3xcd44v6 bispecific t cell activating antigen binding molecules |
CN108137678B (en) * | 2015-10-02 | 2021-10-15 | 豪夫迈·罗氏有限公司 | Bispecific anti-human a-beta/human transferrin receptor antibodies and methods of use |
EP3913000A1 (en) * | 2015-10-02 | 2021-11-24 | F. Hoffmann-La Roche AG | Bispecific anti-cd19xcd3 t cell activating antigen binding molecules |
CN107849137B (en) * | 2015-10-02 | 2021-11-26 | 豪夫迈·罗氏有限公司 | Bispecific anti-CEAXCD 3T cell activating antigen binding molecules |
TWI747843B (en) * | 2015-10-02 | 2021-12-01 | 瑞士商赫孚孟拉羅股份公司 | Bispecific anti-human cd20/human transferrin receptor antibodies and methods of use |
JP2021191281A (en) * | 2015-10-02 | 2021-12-16 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-human cd20/human transferrin receptor antibody, and method of using the same |
CN108290958B (en) * | 2015-10-02 | 2021-12-28 | 豪夫迈·罗氏有限公司 | Multispecific antibodies |
JP7000473B2 (en) | 2015-10-02 | 2022-02-04 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-human CD20 / human transferrin receptor antibody and usage |
JP7005487B2 (en) | 2015-10-02 | 2022-02-04 | エフ.ホフマン-ラ ロシュ アーゲー | Bispecific anti-human A-beta / human transferrin receptor antibody and its usage |
WO2017055391A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Bispecific t cell activating antigen binding molecules binding mesothelin and cd3 |
WO2017055537A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Tetravalent multispecific antibodies |
WO2017055314A1 (en) * | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Bispecific anti-cd19xcd3 t cell activating antigen binding molecules |
JP7044700B2 (en) | 2015-10-02 | 2022-03-30 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-CEAXCD3 T cell activating antigen binding molecule |
CN108290959B (en) * | 2015-10-02 | 2022-06-03 | 豪夫迈·罗氏有限公司 | Tetravalent multispecific antibodies |
AU2016333510B2 (en) * | 2015-10-02 | 2022-08-25 | F. Hoffmann-La Roche Ag | Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use |
CN107949574A (en) * | 2015-10-02 | 2018-04-20 | 豪夫迈·罗氏有限公司 | Bispecific T cell activation antigen binding molecules |
TWI780680B (en) * | 2015-10-02 | 2022-10-11 | 瑞士商赫孚孟拉羅股份公司 | Bispecific anti-human a-beta/human transferrin receptor antibodies and methods of use |
AU2016333512B2 (en) * | 2015-10-02 | 2022-11-17 | F. Hoffmann-La Roche Ag | Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use |
WO2017055542A1 (en) | 2015-10-02 | 2017-04-06 | F. Hoffmann-La Roche Ag | Bispecific anti-human cd20/human transferrin receptor antibodies and methods of use |
JP7228641B2 (en) | 2015-10-02 | 2023-02-24 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use |
US11013801B2 (en) | 2015-12-09 | 2021-05-25 | Hoffmann-La Roche Inc. | Treatment method |
US10596257B2 (en) | 2016-01-08 | 2020-03-24 | Hoffmann-La Roche Inc. | Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies |
EP3432927A4 (en) * | 2016-03-24 | 2019-11-20 | Gensun Biopharma Inc. | Trispecific inhibitors for cancer treatment |
US11111308B2 (en) | 2016-12-26 | 2021-09-07 | Jcr Pharmaceuticals Co., Ltd. | Anti-human transferrin receptor antibody capable of penetrating blood-brain barrier |
WO2018162517A1 (en) | 2017-03-10 | 2018-09-13 | F. Hoffmann-La Roche Ag | Method for producing multispecific antibodies |
WO2019157224A1 (en) | 2018-02-07 | 2019-08-15 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for therapeutic protein delivery |
US11753455B2 (en) | 2018-06-21 | 2023-09-12 | Novo Nordisk A/S | Compounds for treatment of obesity |
WO2020260326A1 (en) | 2019-06-27 | 2020-12-30 | F. Hoffmann-La Roche Ag | Novel icos antibodies and tumor-targeted antigen binding molecules comprising them |
US11780920B2 (en) | 2020-06-19 | 2023-10-10 | Hoffmann-La Roche Inc. | Antibodies binding to CD3 and CD19 |
US11827702B2 (en) | 2021-09-01 | 2023-11-28 | Biogen Ma Inc. | Anti-transferrin receptor antibodies and uses thereof |
WO2023218431A1 (en) | 2022-05-13 | 2023-11-16 | BioNTech SE | Rna compositions targeting hiv |
WO2024026488A2 (en) | 2022-07-29 | 2024-02-01 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a modified transferrin receptor locus |
WO2024026494A1 (en) | 2022-07-29 | 2024-02-01 | Regeneron Pharmaceuticals, Inc. | Viral particles retargeted to transferrin receptor 1 |
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KR20160105799A (en) | 2016-09-07 |
RU2016130707A (en) | 2018-02-12 |
JP6557664B2 (en) | 2019-08-07 |
RU2694659C2 (en) | 2019-07-16 |
KR20210108491A (en) | 2021-09-02 |
US20170051071A1 (en) | 2017-02-23 |
BR112016015589A2 (en) | 2017-10-31 |
EP3851452A1 (en) | 2021-07-21 |
CA2932547C (en) | 2023-05-23 |
US20200071413A1 (en) | 2020-03-05 |
RU2016130707A3 (en) | 2018-08-15 |
US10364292B2 (en) | 2019-07-30 |
MX2020013058A (en) | 2021-03-02 |
ES2864160T3 (en) | 2021-10-13 |
CN105873947A (en) | 2016-08-17 |
CA2932547A1 (en) | 2015-07-09 |
CN111057147A (en) | 2020-04-24 |
AR099019A1 (en) | 2016-06-22 |
CN111057151B (en) | 2022-05-03 |
MX2016008190A (en) | 2016-10-21 |
EP3092251A1 (en) | 2016-11-16 |
EP3092251B1 (en) | 2021-01-20 |
CN111057147B (en) | 2023-11-10 |
KR102381685B1 (en) | 2022-04-01 |
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