WO2014044793A2 - Peptides de liaison à cd22 - Google Patents

Peptides de liaison à cd22 Download PDF

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WO2014044793A2
WO2014044793A2 PCT/EP2013/069569 EP2013069569W WO2014044793A2 WO 2014044793 A2 WO2014044793 A2 WO 2014044793A2 EP 2013069569 W EP2013069569 W EP 2013069569W WO 2014044793 A2 WO2014044793 A2 WO 2014044793A2
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peptide
binding
mrna
antibody
cell
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PCT/EP2013/069569
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WO2014044793A3 (fr
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Oliver Schmetzer
Antonio Pezzutto
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Max-Delbrück-Centrum für Molekulare Medizin
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Publication of WO2014044793A3 publication Critical patent/WO2014044793A3/fr

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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to synthetic non-antibody-derived CD22-binding peptides for use as antibody mimetics, as active agents or as targeting agents for immune conjugates.
  • the peptides of the present invention demonstrate high specificity and affinity to CD22 without requiring any structural component of an antibody or antibody-like affinity reagent.
  • the peptides are defined by their unique physical and functional properties, in particular that the net charge and dipole moment per atom of the peptides of the invention correlate with a specific and strong CD22-binding function.
  • the peptides are produced by a novel process of manufacture, comprising a novel form of in vitro mRNA display, in which negative selection against CD22-depleted human protein mixtures removes binding candidates with affinity to non-CD22 proteins.
  • the novel method imparts an unexpectedly high level of specificity to the peptides as described herein.
  • the peptides of the present invention bind CD22 protein or fragments thereof and can be used as affinity reagent, or as an antibody mimetic, as immune conjugates, for targeting active substances to CD22- expressing cells, or directly as active substances, especially for the treatment of B-cell-related medical disorders.
  • CD22 (or cluster of differentiation-22), is a molecule belonging to the siglec family. It is known to be expressed in B cells and is found on the surface of mature B cells. Generally speaking, CD22 is a regulatory molecule involved in regulation of the immune system and potentially the development of autoimmune diseases. B cells are known to be involved in a number of diseases, such as lymphomas, leukemias, transplant rejection, and autoimmune disorders. Reagents developed to target B cells are known in the art and have proven effective in treating B cell related disorders. Examples of such reagents are affinity reagents, such as antibodies, directed to B cell specific epitopes, such as CD20 or CD19.
  • mAb Monoclonal Antibodies
  • Immunoconjugates show promising clinical results in the treatment of various B cell related diseases.
  • Major drawbacks of mAb are however high costs during development and therapy, long development periods, complex chemistry and potential side-effects.
  • Application of immunoconjugates, comprising an affinity reagent (e. g. antibody) conjugated to an active substance (e. g. cytotoxin) is restricted in clinical practice to highly-pre-treated patients, for example in cases where low-toxicity during palliative care of lymphoma is required.
  • CD22 is a relevant target antigen as it is expressed by B-NHL-cells and is rapidly endocytosed.
  • Antibody-conjugates targeting CD22 have shown promising results (Inotuzumab-ozogamicin), but side effects limit the dose to a few milligrams. These side effects are likely present due to unspecific binding of the antibody to endothelial cells via Fc- and complement-receptors.
  • Antibodies are communication platforms that not only bind antigens but also connect such antigens to various receptors, which explain the side effects to some extent.
  • CD22 is rapidly endocytosed but not degraded in lysosomes. Depending on the nature of ligands, they can accumulate intracellularly or return to the surface again, still bound to the recycling CD22 molecules. While glycans and some natural ligands are released in the endocytosed vesicles, at least some antibodies (e.g. HIB22) are not released. However, Fc-receptor binding to the antibody-CD22 surface complex might also prevent
  • the vesicles in which CD22 is recycled are related to early endosomes, where changes in redox- potential e.g. reduction rapidly occur, but lowering of the pH and the action of peptidases is not performed efficiently. While glycan ligands are of low affinity and released by a discrete lowering of pH to pH 5, antibodies remain bound to the CD22 until pH 3 is reached. Drugs coupled with redox- sensitive linkers are easily released, while standard-linkers need digestion of the antibody, which occurs mainly in late endosomes of the BCR-Fc-receptor endosomal pathway.
  • mRNA display techniques are known in the art, which enable the generation of large numbers of sequences or sequence modifications and subsequent testing for binding properties.
  • WO 2004/106368 discloses modified proteins of the ubiquitin super family, which comprise a ubiquitin-like folding motif and show modified binding affinities through sequence modification.
  • Phage and mRNA display techniques are disclosed, in addition to the possibility of producing such ubiquitin- derived peptides binding CD22 as a target.
  • no examples or sequences of such CD22- peptides are disclosed.
  • Methods for in vitro evolution are also known in the art, for example as described in US 2007/015181 , which discloses methods and systems for the identification and manufacture of proteins or nucleic acids through coupling to mRNA. Methods are disclosed therein, which enable the selection of peptides with particular binding properties, which are identified over multiple of rounds of selection, thereby introducing "evolution" of the sequence during selection for particular binding targets. The possibility of producing such peptides binding CD22 as a target is disclosed. However, no examples or sequences of such CD22-peptides are disclosed.
  • Rothe et al. (FASEB Journal, 20,10, 1599-1610) and Rothe et al. (Biological Chemistry, 389, 4, 433- 439) disclose in vitro and ribosome display methods, respectfully, for the provision of proteins with high affinity for their corresponding targets.
  • CD22-binding reagents are described, for example BL- 22, which is an anti-CD22 immunotoxin fusion protein between a murine anti-CD22 disulfide-linked Fv (dsFv) antibody fragment and a modified version of the bacterial exotoxin PE38.
  • the prior art teaches the use of antibodies, antibody fragments or modifications of existing antibody sequences to provide CD22-binding reagents.
  • the prior art has failed to provide novel, in particular antibody-independent, approaches towards de novo synthesis and selection of peptides that show effective binding against CD22.
  • the technical problem underlying the invention was the provision of alternative or improved means for binding or targeting B cells, for example for use in the treatment of B-cell related disorders.
  • a further technical problem was to provide alternative or improved means to commonly known antibodies (Abs) that do not exhibit the disadvantages associated with Abs.
  • An additional technical objective of the invention was to improve cellular uptake and intra-cellular release of anti-B-cell agents via providing an improved B-cell specific targeting and carrier molecule, capable of being coupled to and transporting active agents into B cells.
  • an object of the invention is to provide a synthetic non-antibody-derived CD22-binding peptide, characterized in that said peptide exhibits
  • a) is selected from a sequence of SEQ ID No. 8, 1 , 3, 4, 5, 6, 7, 10, 1 1 , 14, 15, 16, 17, 18, 19, 20, 22, 25, 26, 27, 28, 31 , 32, 33, 34, 35, 36, 37, 38, 41 , 42, 44, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , or
  • b) exhibits more than 80%, preferably more than 90%, more preferably more than 95% sequence identity to a sequence of a) and exhibits specific binding to CD22 or a fragment thereof.
  • the specific binding to CD22 is determined in a preferred embodiment by the absence of, or negligible, binding to a CD22-depleted human protein mixture.
  • the peptides of the present invention are defined by their unique and advantageous absence of cross- reaction against other human proteins due to the negative selection of cross-reacting proteins during development by screening and removing peptides with affinity to CD22-depleted (devoid) human protein mixtures.
  • the non-antibody derived nature of the peptides can be understood in one embodiment as an absence of amino acid sequences corresponding to structural elements of an antibody or antibody fragment.
  • Such structural elements of an antibody or antibody fragment are preferably sequences with sufficient homology to antibody or analogous affinity reagent structures, which are capable of providing the corresponding function of said antibody structure, such as Fab region sequences, Fc region sequences, heavy chain variable (VH) or constant (CH) domain sequences, light chain variable (VL) or constant (CL) domain sequences, hinge region sequences, sequences of antibody fragments, variable fragments (Fv), single-chain variable fragments (scFv), di-scFvs, bi-scFvs, diabodies, or for example Ig heavy chain sequences according to ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , from IgA, IgD, IgE, IgG, IgM and/or IgY molecules, lambda ( ⁇ ), kappa ( ⁇ ) or io
  • the invention utilised a starting RNA library covering all possible 12-mer peptides 10-fold consisting of 10 ⁇ 16 different sequences.
  • the RNA was translated 1 :1 and peptide-RNA adducts were created by mRNA display. RNA adducts were positively selected to bind to target protein CD22 and negatively selected against total human protein devoid of CD22.
  • the isolated RNA was reverse transcribed and went in a new round of selection. After 5 generations of in vitro evolution 650 sequences remained, after 6 generations 99 remained.
  • Peptides were synthesized after their sequences were identified by deep sequencing. The identified peptides have been applied in flow cytometry, MACS, ELISAs, FRET, microscopy, cell culture and histochemistry-based assays to bind and/or detect CD22. The best binding peptides show binding to soluble CD22, CD22+ cell lines, primary lymphoma cells and splenocytes. The peptides are rapidly endocytosed in Burkitt cells faster than an antibody.
  • the unique method of producing the peptides of the present invention a group of peptide sequences have been developed that all exhibit unexpected shared functional and physical characteristics. It was entirely surprising that the CD22 binding peptides, which exhibited the most effective binding to their target antigen (as shown in a number of binding assays) exhibit neutral or positive charge (a net charge at pH 7 of 0 or greater than 0) and a dipole moment per atom of greater than 0.2.
  • the peptide sequences of the application are preferably defined by these unitary characteristics.
  • the CD22-binding peptide of the invention exhibits a net charge at pH 7 of between 0 and 1 1 . In a preferred embodiment the CD22-binding peptide of the invention exhibits a dipole moment per atom of between 0.2 and 1 .3.
  • the peptides of the invention exhibit a net charge at pH 7 of a value between and including 0 and 1 1 and a dipole moment per atom of a value between and including 0.2 and 1 .3.
  • Peptides of these physical properties are listed in table 2.
  • the peptides of the invention exhibit a net charge at pH 7 of a value between and including 1 and 1 1 and a dipole moment per atom of a value between and including 0.3 and 1 .3. Peptides of these physical properties are listed in table 3. In a preferred embodiment the peptides of the invention exhibit a net charge at pH 7 of a value between and including 2 and 1 1 and a dipole moment per atom of a value between and including 0.4 and 1 .3. Peptides of these physical properties are listed in table 4.
  • the peptides of the invention exhibit a net charge at pH 7 of a value between and including 3 and 1 1 and a dipole moment per atom of a value between and including 0.5 and 1 .3.
  • Peptides of these physical properties are listed in table 5.
  • the peptides of the present invention are preferably defined by the unique combination of net charge and dipole moment per atom. It was entirely surprising that a large majority of peptides developed by the in vitro evolution method of the present invention showed net charge and dipole moment per atom values falling into particular preferred ranges. It was unexpected that the most effective binders would exhibit a net charge at pH 7 of a value between and including 0 and 1 1 and a dipole moment per atom of a value between and including 0.2 and 1.3. The most effective binders are associated with the surprising combination of increased net charge and increased dipole moment per atom values. Such "increased" net charge and dipole per atom values can be seen in the tables 2, 3, 4 and 5, preferably tables 3, 4, 5, more preferably tables 4 and 5. Considering the disclosures of the prior art, which find no general relationship between charge and dipole moment, the physical properties of the peptides as claimed represent unique unifying properties.
  • the invention relates to a synthetic non-antibody-derived CD22-binding peptide, characterized in that the peptide exhibits a net charge at pH 7 of 0 or greater than 0, preferably of or between 0 and 1 1 , and a dipole moment per atom of greater than 0.2, preferably of or between 0.2 and 0.8,
  • a) is selected from a sequence of SEQ ID No. 5, 8, 10, 1 1 , 19, 32, 33, 38, 44, or b) exhibits more than 80%, preferably more than 90%, more preferably more than 95% sequence identity to a sequence of a) and exhibits specific binding to CD22 or a fragment thereof.
  • Peptides selected from this group exhibit CD22-binding properties as evidenced via various in vitro and in vivo assays.
  • the invention relates also to synthetic non-antibody-derived CD22-binding as described herein obtainable or obtained by a method of in vitro mRNA display, comprising
  • each mRNA molecule comprising a nucleotide sequence encoding a peptide variant
  • RT-PCR reverse transcriptase PCR
  • the target antigen is CD22 or a fragment thereof.
  • the CD22-binding peptide as described herein is obtainable by a method of in vitro mRNA display, wherein the steps a) - g) of the method are repeated for 3 to 10, preferably 5, or more preferably 6 repetitions.
  • the CD22-binding peptide as described herein is obtainable by a method of in vitro mRNA display, wherein short non-peptide-encoding sequences are removed via
  • the present method of in vitro mRNA display has been adjusted from methods commonly known in the art to produce a unique set of CD22-binding peptides.
  • the in vitro evolution encompassed in the present method provides selection via binding to CD22, while binding to non-CD22 human proteins was reduced by negative selection rounds.
  • the negative selection on target antigen-depleted total- human-protein beads represents a novel development in the field and provides specific selection of the desired peptides.
  • MAbs can only be negatively selected against the protein of the immunised animal but not with human protein, which provides the likelihood of further side effects arising. This disadvantage is avoided by the present invention.
  • the synthetic non-antibody-derived CD22-binding peptide of the present invention is characterised in that the peptide is synthesized and/or modified with, or comprises of, means that increase in vivo half life of the peptide, preferably means for preventing protease degradation, such as comprising D-amino acids or exhibiting a retro-inverse sequence. Means for increasing in vivo half life are known in the art and are encompassed in the present invention. In respect to the particular peptides of the invention, it was a surprising and beneficial finding that the reverse-sequences exhibit equally effective CD22 binding. In one embodiment the synthetic non-antibody-derived CD22-binding peptide of the preceding claims is characterised in that the peptide is PEGylated.
  • the invention further relates to recombinant multimeric peptides comprising more than one non- antibody-derived CD22-binding peptide as described herein, characterised in that the peptide monomers are separated by one or more peptide-spacers. Multimers of the peptides lead to enhanced binding in a surprising fashion. Multimers may bind multiple sites of the target protein or, in cell-based binding assays, multiple target protein molecules, thereby providing enhanced targeting of the peptide to CD22-expressing cells.
  • the multimeric peptides of the present invention preferably comprise proline and/or serine containing spacers, which are used to prevent misfolding, more preferably a spacer containing proline, serine and lysine is used to increase the amount of conjugated drug-molecules during reaction with primary amines.
  • the invention relates to synthetic non-antibody-derived CD22-binding peptide or multimer thereof as described herein for use as a medicament for the treatment of B-cell-related medical disorders, especially B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and rheumatic disorders.
  • B-cell-related medical disorders especially B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and rheumatic disorders.
  • the invention further relates to an immune conjugate comprising the non-antibody-derived CD22- binding peptide or multimer thereof as described herein and an additional active agent, whereby the binding peptide and active agent are conjugated preferably by a covalent cleavable linker.
  • the immune conjugates of the present invention show unexpectedly positive results in targeting conjugating molecules to CD22-expressing cells. Similar to antibody immune conjugates, the peptides of the present invention show the ability to effectively deliver bioactive substances to CD22- marked cells, although in a more efficient manner than traditional antibody-based approaches.
  • the immune conjugates of the invention allow lower dosing of active agent (such as anti-B-cell drugs) via the more specific and stronger binding interaction between the inventive peptides in comparison to antibodies known in the art.
  • One embodiment of the invention relates to an immune conjugate as described herein, whereby the linker undergoes and/or is designed to undergo cleavage (release of agent from peptide) after cell internalization, preferably in a B-cell, more preferably in a B-cell endosome, most preferably in a B- cell-endosome involved in carbohydrate- or iron-metabolism.
  • the peptides of the invention were conjugated to Vinblastin and tested in a xeno-tumor-model.
  • the peptide-conjugates led to a growth reduction of 50% or more.
  • the activity of peptide-Taxol-conjugates in a test system is about 3 to 4 logs higher as compared to antibodies, therefore much lower doses are needed to treat patients.
  • the LC50 of the peptide-Taxol conjugates is in the range of ng/ml as compared to ug/ml for antibody-conjugates.
  • Cleavable linkers have reasonable stability during systemic circulation but can be cleaved under certain intracellular conditions, such as in an acidic environment.
  • the entire molecule is processed in the lysosome by exposing the cytotoxic agent to a strong acidic environment and lots of digestive enzymes.
  • disulfide linkers is envisaged and exploits the observation that the intracellular concentration of thiols, such as glutathione and cysteine are much higher than those in plasma.
  • Disulfide linkers can be selectively cleaved in the cytosol due to a more reductive intracellular environment and were originally designed to be used with the cytotoxin, maytansinoid (ex: thiol containing DM1 or DM4).
  • hydrazone linkers have been designed to be selectively cleaved within the intracellular compartment of lysosomes (lower pH compared to the systemic blood circulation). Hydrazones have typically been linked to antibody thiol groups generated through interchain disulfide bone reduction.
  • One embodiment of the invention relates to an immune conjugate as described herein, whereby the linker comprises a disulfide bond, a carbonylester, an acid-sensitive group, a selenide, a
  • carbohydrate a carbohydrate-mimetic, a ferrocene, a ruthenium compound and/or a glutathione- derivate.
  • PMPI crosslinker N-[p-Maleimidophenyl]isocyanate, preferably in combination with Traut's reagent (2-lminothiolane), and/or
  • the peptides of the present invention showed unexpected advantages with respect to cellular delivery of conjugated active substances, in addition to improved release of active agent after internalisation, in comparison to the linkers applied previously.
  • the linkers and methods of conjugation herein were associated with stable in vivo half-lives, effective internalisation via endocytosis and subsequent release of active agent due to optimal intracellular cleavage conditions.
  • the conjugates of the present invention were internalised surprisingly faster than the antibody and antibody fragment conjugates described previously. Internalisation rates can be measured using fluorescent protein (e. g. GFP) coupled to the peptides and subsequent analysis of fluorescence in cell cultures in vitro. Release of active agent after internalisation can be similarly assessed using fluorescence microscopy, whereby time lapse approaches and quantitative and semi-quantitative approaches are well known and may be applied as required.
  • fluorescent protein e. g. GFP
  • One embodiment of the invention relates to an immune conjugate as described herein, whereby more than one active agent are linked to the non-antibody-derived antigen-binding peptide via a branching polymer.
  • Branching may occur in a branching polymer by the replacement of a substituent, e.g., a hydrogen atom, on a monomer subunit, by another covalently bonded chain of that polymer. Branching may result from the formation of carbon-carbon or various other types of covalent bonds, such as ester and amide bonds.
  • Polysaccharides preferably degradation resistant, may also be used as branching polymers.
  • One embodiment of the invention relates to an immune conjugate as described herein, whereby the active agent exhibits an anti-proliferative effect on B cells, either in vivo or in vitro.
  • Anti-B-cell agents are known in the art and may be incorporated using the conjugation techniques described herein.
  • Immune conjugates of the invention comprise those, wherein the active agent exhibits an anti- proliferative effect on B cells, either in vivo or in vitro, selected from
  • anti-oxidants selected from flavones, ellag acid derivatives, tannins, fruit or tea derived compounds or ginger-like compounds,
  • cytostatics cytostatics, growth factors, cytokines and chemokines, toxic proteins, recombinant peptide multimers comprising an endosomal-release protein domain, or c) a pseudomonas toxin or a cytolethal distending toxin combined with HlV-tat- sequences, penetratin or a domain from the tetanus toxoid.
  • the invention therefore relates to a synthetic non-antibody-derived CD22-binding peptide, multimer thereof, or immune conjugate comprising said peptide as described herein for use as a medicament in the treatment of a B-cell-related medical disorder, wherein said B-cell-related medical disorder is preferably selected from the group consisting of B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and/or rheumatic disorders.
  • the invention also relates to the method as described herein used for generated the peptides as described.
  • the method comprises comprising
  • each mRNA molecule comprising a nucleotide sequence encoding a peptide variant
  • RT-PCR reverse transcriptase PCR
  • the target antigen is CD22 or a fragment thereof.
  • the method preferably comprises removal of short non-peptide-encoding sequences via
  • the invention further relates to the in vitro use of a synthetic non-antibody-derived CD22-binding peptide or multimer thereof as described herein for binding, identification and/or sorting of CD22 and/or CD22+ cells and/or cell lines.
  • the in vitro use may comprise
  • the in vitro use as described herein may be characterised in that the diagnostic application comprises diagnosis of a B-cell-related medical disorder, selected from the group consisting of B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and/or rheumatic disorders.
  • a B-cell-related medical disorder selected from the group consisting of B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and/or rheumatic disorders.
  • the medical use as described herein therefore comprises a method of treatment of a B cell related disorder, preferably as described herein, comprising administration of the synthetic non-antibody- derived CD22-binding peptide, multimer thereof, or immune conjugate comprising said peptide as described herein in a therapeutically effective amount to a subject in need of said treatment.
  • the peptides of the present invention are characterised by unitary physical and functional properties, preferably a combination of net charge and dipole moment per atom falling within particular preferred ranges.
  • the preferred peptides although differing in sequence, are also preferably bound by the unitary characteristics of the functional binding properties (specific CD22 binding), absence of antibody-related or antibody-derived sequences and/or the physical characteristics described herein.
  • the molecular dipole moment is a measure of the separation of positive and negative electrical charges in a system of charges, that is, a measure of the charge system's overall polarity. In the present invention this relates to a measure of the polarity of the peptides.
  • proteins and peptides can be considered macro-zwitterions due to the presence of a significant number of charged residues at neutral pH. Their electrostatic properties can be considered important for stability and function.
  • the dipole moment of a peptide can be measured by deflection of a molecular beam in an inhomogeneous electric field, or simulated using results of ab initio calculations and Monte-Carlo simulations.
  • Computational methods for examination of the electrostatic properties of proteins are known in the art, for example rapid numerical implementations of the Poisson- Boltzmann equation of electrostatics (PBE) (Honig.B. and NichollsA, 1995, Science, 268, 1 144-1 149), coupled with a computer algorithm to colour the solvent-accessible molecular surface or isopotential surfaces by electrostatic potential, as implemented by computer algorithms such as Delphi and GRASP.
  • APBS Adaptive Poisson-Boltzmann Solver
  • the peptides of the present invention are preferably defined by a unique combination of net charge and dipole moment per atom. It was entirely surprising that a large majority of peptides developed by the in vitro evolution method of the present invention showed net charge and dipole moment per atom values falling into particular preferred ranges. It was unexpected that the most effective binders would exhibit a net charge at pH 7 of a value between and including 0 and 1 1 and a dipole moment per atom of a value between and including 0.2 and 1.3. The most effective binders are associated with the surprising combination of increased net charge and increased dipole moment per atom values. Considering the disclosures of the prior art, which find no general relationship between charge and dipole moment, the physical properties of the peptides as claimed represent unique unifying properties.
  • COOH-terminal residue have a net charge of -1 , and all other residues have a zero net charge. If a residue side chain is incomplete, and it is a charged residue, the entire residue charge is assigned to the last sidechain atom present; otherwise, the atomic charges of all incomplete sidechains are set to zero. Residues completely missing from the ATOM records are ignored. The next step is the actual calculation of the electrostatic and geometric properties. The net charge on the protein is simply the sum of all the partial atomic charges.
  • the numbers of positive (Lys and Arg) and negative (Glu and Asp) residues are also determined.
  • the net charge and dipole moment per atom of the best binding peptides of the present invention differ from average values significantly.
  • the net dipole moment per atom of the preferred peptides is on average higher than standard average values.
  • the net charge of the preferred peptides is also on average higher than standard values.
  • Most peptides from databases on average generally have a negative charge, whereas the peptides of the present invention show unusually a positive net charge, either being neutral or exhibiting a positive charge.
  • the molecule is to some extent negatively charged and shows itself a strong dipole moment, especially around the ligand binding domain. Hence polar peptides might show improved binding and kinetics.
  • the realisation of this potential mechanism for the claimed binding properties is a novel and surprising development over the priori to date. That a peptide could be developed based on said properties, that exhibits the advantageous binding properties demonstrated herein, is a surprising and entirely unexpected development of the prior art.
  • the net charge of a peptide is a measure of the charge of the ionisable groups of the peptide.
  • the overall or net charge on a peptide is preferably the sum of the charges of every ionizable group in the peptide.
  • the net charge Z of a peptide at a certain pH can be estimated by calculating
  • ⁇ /,- are the number, and pKa, the pKa values, of the N-terminus and the side chains of Arginine, Lysine, and Histidine.
  • the y ' -index pertains to the C-terminus and the Aspartic Acid, Glutamic Acid, Cysteine, Tyrosine amino acids.
  • Table 1 Preferred sequences of the invention.
  • the peptides described below are preferred sequences of the present invention.
  • the peptide number refers to the experimental data provided in Figure 3; distinct SEQ ID numbers are provided. These peptides showed the desired binding characteristics according to selection via the in vitro evolution method described herein. The binding affinity of the peptides is additionally listed in the right column.
  • HHHHHHGFQHFPFIVLPAPLTIPVTTPGAIRSLTMAE 2 136 0,0059 0,4026 +++ RAG KAH
  • Table 4 Peptides of a net charge at pH 7 of a value between and including 2 and 1 1 and a dipole moment per atom of a value between and including 0.4 and 1 .3.
  • Sequence variants of the claimed peptides that maintain the said properties of the invention are also included in the scope of the invention. Protein modifications which occur through substitutions are also included within the scope of the invention. Substitutions as defined herein as replacements of amino acids made to the amino acid sequence of the protein without a change in the number of amino acids, preferably producing a protein which contains a different amino acid sequence than the primary protein without significantly altering the function of the protein. Additions are also considered to fall within the scope of the invention, whereby additional amino acids, either within the provided sequences or at the N or C termini of the peptides, fall under the scope of the invention when the functional and physical properties of the peptides are significantly unchanged.
  • substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function. This is particularly true when the modification relates to a "conservative" amino acid substitution, which is the substitution of one amino acid for another of similar properties.
  • Such "conserved" amino acids can be natural or synthetic amino acids which because of size, charge, polarity and conformation can be substituted without significantly affecting the structure and function of the protein. Frequently, many amino acids may be substituted by conservative amino acids without deleteriously affecting the protein's function.
  • the non-polar amino acids Gly, Ala, Val lie and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin, Asn and Met; the negatively charged amino acids Lys, Arg and His; the positively charged amino acids Asp and Glu, represent groups of conservative amino acids.
  • This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups.
  • Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids.
  • Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexyl alanine which are neutral non-polar analogs; citulline and methionine sulfoxide which are neutral non-polar analogs, phenylglycine which is an aromatic neutral analog; cysteic acid which is a positively charged analog and ornithine which is a negatively charged amino acid analog.
  • this list is not exhaustive, but merely exemplary of the substitutions that are well known in the art.
  • multimers of the inventive peptides are understood as an aspect of the present invention.
  • the invention therefore also relates to DNA sequences that encode the peptides of the invention, comprising either single or multiple copies of any of the sequences provided herein.
  • Protein modifications or mutations may also occur through deletions.
  • Deletions as defined herein are modifications made to the nucleic acid or amino acid sequence of the protein which produce a protein containing at least one amino acid less than the primary amino acid sequence of the protein, preferably without significantly altering the function, which is preferably CD22-binding.
  • diethyldithiocarbamate (a reduced form of Disulfiram) and its structural analogues).
  • the covalent bond should preferably be stable in vivo in order to last until the immunoconjugate reaches the cell.
  • the drug should preferably be able to be separated from the antibody once it has homed to its designated target: e. g. a tumor cell.
  • the strategies described above result in antibody-drug conjugates with either a disulfide bond or a carbonylester which are both reversible in the cell, but relatively stable in the bloodstream.
  • Candidate compounds with a directly available sulfhydryl are coupled to the antibody's' primary amines' using an SPDP linker. In the case of diethyldithiocarbamate, the resulting disulfide bond is cleaved in the reducing environment of the early endosome releasing the drug that, once it reaches the cytosol, can then be oxidized to yield Disulfiram.
  • Hydroxyls as in EGCG are targeted by a combination of Traut's reagent and PMPI to attach them to amines of the antibody. The resulting carbonylester bond will be cleaved in the late endosome by esterase.
  • Carboxyls of drugs such as Bendamustine can be used to attach the drug to an antibody via its amine.
  • EDC-activated AEDP is used.
  • the resulting linker has a disulfide and a peptide bond which will be cleaved in a two-step process ultimately releasing Bendamustine in the late endosome.
  • these drugs were effective at concentrations at least 100-fold lower than when used as unconjugated drugs.
  • the application of the peptides as described herein demonstrate that peptide-guided drug delivery to selected cell types leads to accumulation of active compounds in the cell. Such compounds would be well-tolerated and markedly less toxic than the substances already in evaluation as clinical immunotoxins.
  • the substances we have chosen are even approved for oral uptake as well as systemic treatment without the life-threatening side effects of very potent toxins such as ricin A or pseudomonas exotoxin, which, in the past, has been used to construct anti-CD22 immunotoxins.
  • the present invention relates to the medical use of the peptides, multimers and immune conjugates as described herein.
  • the peptides and immunoconjugates are particularly suited for treatment of B- cell-related medical disorders, especially B-cell- or precursor-B-cell derived lymphomas or leukaemia, B-cell proliferations or enrichments, organ- or stem cell implant/transplant rejection, autoimmune and rheumatic disorders.
  • B-cell associated cancers encompass but are not limited to B cell lymphomas, B cell leukemia, diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated Lymphatic Tissue lymphoma, small cell lymphocytic lymphoma (overlaps with Chronic lymphocytic leukemia), mantle cell lymphoma (MCL), burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular large B-cell lymphoma, Primary effusion lymphoma, or Lymphomatoid granulomatosis.
  • B cell lymphomas B cell leukemia, diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated Lymphatic Tissue lymphoma, small cell lymphocytic lymphoma (overlaps with Chronic lympho
  • the autoimmune or rheumatic disorders are selected from Takayasu Arteritis, Giant-cell arteritis, familial Mediterranean fever, Kawasaki disease, Polyarteritis nodosa, cutanous Polyarteritis nodosa, Hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, Vasculitis of connective tissue diseases, Hennoch-Schonlein purpura, Cryoglobulinemic vasculitis, Cutaneous leukocytoclastic angiitis, Tropical aortitis, Sarcoidosis, Cogan's syndrome, Wiskott-Aldrich Syndrome, Lepromatous arteritis, Primary angiitis of the CNS, Thromboangiitis obliterans, Paraneoplastic ateritis, Urticaria, Dego's disease,
  • Rheumatoid Arthritis atherosclerosis, Amyloidosis, Morbus Chron, Colitis ulcerosa, Autoimmune Myositis, Diabetes mellitus, Multiple sclerosis, Guillain-Barre Syndrome, histiocytosis, Osteoarthritis, atopic dermatitis, periodontitis, chronic rhinosinusitis, Psoriasis, psoriatic arthritis, Microscopic colitis, Pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome, Lupus erythematosus, aplastic anemia, Osteomyelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibros
  • Autoimmunlymphocytopenia Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's Thyroiditis, atropic thyroiditis, Graves disase, Autoimmune polyglandular syndrome, Autoimmune Addison Syndrome, Pemphigus vulgaris, Pemphigus foliaceus, Dermatitis herpetiformis, Autoimmune alopecia, Vitiligo, Antiphospholipid syndrome, Myasthenia gravis, Stiff- man syndrome, Goodpasture's syndrome, Sympathetic ophthalmia, Folliculitis, Sharp syndrome and/or Evans syndrome.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the inventive peptides and/or immune conjugates, optionally together with a pharmaceutically tolerable carrier.
  • the pharmaceutical composition can be used as a drug.
  • drugs in the meaning of the invention are compositions for therapeutic and prophylactic purposes, as well as pharmaceutical compositions usable as diagnostic agents.
  • drugs or pharmaceutical compositions - used in a synonymous fashion herein - are substances and formulations of substances intended to cure, alleviate or avoid diseases, illness, physical defects or pathological affection by application on or in the human body.
  • medical adjuvants are substances used as active ingredients in the production of drugs.
  • Pharmaceutical- technical adjuvants serve to suitably formulate the drug or pharmaceutical composition and, if required during the production process can even be removed thereafter, or they can be part of the pharmaceutical composition as pharmaceutically tolerable carriers.
  • a therapeutically effective amount can be determined by a skilled person, preferably by the alleviation of illness symptoms or by measurement of diagnostic markers, such as molecular markers.
  • Drug formulation or formulation of the pharmaceutical composition is optionally effected in combination with a pharmaceutically tolerable carrier and/or diluent.
  • suitable pharmaceutically tolerable carriers are well known to those skilled in the art and comprise e.g. phosphate-buffered saline, water, emulsions such as oil/water emulsions, various types of detergents, sterile solutions, and so forth.
  • Drugs or pharmaceutical compositions comprising such carriers can be formulated by means of well-known conventional methods.
  • These drugs or pharmaceutical compositions can be administered to an individual at a suitable dose, e.g. in a range of from 1 ng to 10 g of peptides per day and patient. Doses of from 1 mg to 1 g are preferred.
  • Administration can be effected on various routes, e.g. intravenous, intraperitoneal, intrarectal, intragastrointestinal, intranodal, intramuscular, local, e.g. intratumoral, but also subcutaneous, intradermal or on the skin or via mucosa.
  • Administration of nucleic acids encoding the peptide according to the invention can also be effected in the form of a gene therapy, e.g. via viral vectors.
  • the kind of dosage and route of administration can be determined by the attending physician according to clinical factors.
  • the kind of dosage will depend on various factors such as size, body surface, age, sex, or general health condition of the patient, but also on the particular agent being administered, the time period and type of administration, and on other medications possibly administered in parallel.
  • Those skilled in the art will also be familiar with the fact that the type and extent of B-cell related disease can be diagnosed first, in order to determine the required concentration of drug.
  • compositions or drugs comprise a pharmacological substance which includes one or more peptides of the invention in a suitable solution or administration form.
  • Administration thereof can be effected either alone or together with appropriate adjuvants described in connection with drugs or pharmaceutical compositions, or in combination with one or more adjuvants, e.g.
  • the pharmaceutical composition or drug can also be a combination of two or more of the inventive pharmaceutical compositions or drugs, as well as a combination with other drugs, such as antibody therapies, chemotherapies or radiotherapies, suitably administered or applied at the same time or separately in time.
  • the production of the drugs or pharmaceutical compositions proceeds according to per se known methods.
  • Figure 1 Experimental flow-chart showing molecular biological methods applied in one round of the in vitro evolution.
  • FIG. 2 Translated peptide-pools from each generation were coupled to Europium-chelat and tested on plate bound CD22 and albumin for binding.
  • Generation 5 shows the first binding to CD22. Above the amount of different sequences, e.g. clusters are listed for each generation.
  • Figure 4 Binding of peptides to full-length extracellular CD22 isolated from B-cells (CD22 B-Cell), to CD22 isolated from E.Coli (CD22 E.Coli) and to a deletion variant missing the first two Ig-like repeats isolated from B-cells (CD22 delta Ig) is shown.
  • the proteins have been immobilized on CM5-chips and stability values after peptide flow-through are shown.
  • FIG. 6 Peptide-binding to Burkitt cells in blood PBMCs. Peptide26-Cy5 binding to CSFE-labeled Namalwa cells diluted with PBMCs from a normal donor is shown
  • FIG. 8 MACS sorting by peptides. Biotinylated Peptides have been used in MACS sorting of
  • B-cells from PBMCs of a normal donor. Cells were stained with the peptide-Biotin- conjugates, washed and incubated with Anti-Biotin magnetic beads. Purity of B cells was assayed by flow cytometric staining with Anti-CD19 and Anti-CD20.
  • FIG. 10 Binding of peptide-Cy5 conjugates to spleen cells is shown. Counterstaining has been done with Anti-CD22-FITC
  • Figure 1 1 Binding of peptide-Cy5 conjugates to primary B-NHL cells isolated form a patient with low- grade lymphoma has been analyzed by flow cytometry. Only CD22+ and CD3- cells are shown.
  • Figure 12 CD22-specific killing of CD22- or MOCK-transfected Jurkat cells with Peptide26-vinblastin is shown.
  • Figure 13 Tumor growth inhibition by peptide26-vinblastin. 5x10 ⁇ 6 cells have been inoculated in matrigel at day 0 s,c. in IFNR-knockout-SCID-mice. Vinblastin or conjugates where injected i.v. on days 3 and 10.
  • Figure 14 Tumor growth inhibition by peptide26-vinblastin variants. 5x10 ⁇ 6 cells have been
  • Groups D, E and F have been treated with differentially linked vinblastine
  • Group G is a multimer of peptide 26 (4-mer) coupled with vinblastine via EDC.
  • Figure 15 Side effects by peptide26-vinblastin multimer (A) as compared to standard Immunotoxine (B). 5x10 ⁇ 6 cells have been inoculated in matrigel at day 0 s,c. in IFNR-knockout-SCID-mice. Mortality, tumor growth and body weight changes of the treatment groups are indicated in the figure.
  • RNA-peptide adducts A novel protocol to identify antigen-binding peptides with removal of peptides binding to other human proteins was developed (Fig 1 ). After optimization of PCR, transcription, ligation and purification of RNA-peptide adducts we sequenced the RNA after 2, 4, 5, 6 and 10 rounds (generations) of positive and negative selection. The diversity was reduced to below 100 clusters after generation 6, which was the first generation were we could identify binding of the polyclonal RNA-peptide-adducts to CD22 in an ELISA (Fig 2).
  • Peptide 26, Peptide 27, Peptide 31 , Peptide 24, Peptide 19, Peptide 14, Peptide 41 , Peptide 66, Peptide 2, Peptide 4, Peptide 56, Peptide 57, Peptide 77 and Peptide 68 are being examined in further detail with respect to in vitro binding via ELISA and pull-downs, binding to B-cells in vitro, internalisation and target-cell killing according to the approaches provided herein.
  • the peptides of the invention have also been linked to anti-B-cell agents as described herein using the linkers as described herein and are being validated as immune conjugates.
  • Peptide-Cy-5-conjugates on various cell lines by flow cytometry.
  • Peptide-26-Cy5 showed binding to B-cell-lines but to a very minor extent to T cell lines and no binding to myeloma derived cell lines (Fig. 5).
  • Peptide-26-Cy5 also stained Namalwa 55 mixed with heparinized full blood (Fig. 6).
  • peptide conjugated to alkaline phosphatase showed CD22 specific staining in cytochemistry.
  • Peptide 26 bound to B-non Hodgkin lymphoma cells (follicular lymphoma) isolated from the blood of a patient with extensive lymphocytosis (Fig. 7). Biotin conjugates of peptides 2, 10 and 26 could enrich B-cells from PBMCs via magnetic cell sorting to 8-33% purity (Fig. 8). Peptide 2 stained CD20+ B cells in human splenocytes (Fig 9).
  • Table 3a A summary of binding properties for various peptides is provided in Table 3a and Table 3b (The peptide numbers refer to table 1 ).
  • Table 3b A summary of binding properties for various peptides is provided in Table 3a and Table 3b (The peptide numbers refer to table 1 ).
  • Peptide 2 and 26 were tested as Cy5 conjugates in internalization experiments by confocal microscopy. Both were quickly internalized in a vesicle-congruent pattern, faster than a control anti- CD22-antibody labelled with FITC. Incubation with pHrodo-Dextran was used to label endosomes which take up particles and antigen via micropinocytosis and other routes. No overlap with labelled HD239 was identified; therefore CD22-bound antibodies were directed in other vesicles. Cells with apoptotic nucleus staining with DAPI did not show any internalization.
  • CD22-negative Jurkat cells were retrovirally transduced and used as low-expressing target cells as bulk culture.
  • Peptide-26-Taxol and peptide 26-vinblastine showed killing of CD22 transfected Jurkat cells but not CD22-delta-lg1 ,2 transfected Jurkat cells in a metabolic assay (Fig. 12).
  • Peptide26 was tested on various CD22-chips and showed a ten-fold higher affinity and some improvements in binding kinetics as an Anti-CD22-mAb. E.Coli derived CD22 was recognized to a lower degree, indicating that Peptide26 may recognize a folding or glycosylation-dependent site on CD22.
  • CD22-positive Namalwa cells were titrated in SCID-IFNR-knock/out mice. Treatment after day 3 with peptide-26-vinblastin led to a dose-dependent reduction in tumour growth. The same tumour growth inhibiting activity as unconjugated vinblastin was found with 10% of the dose of peptide-26-vinblastin (Fig. 13). Comparing the same concentration of vinblastine, the pure peptide and various conjugates the Peptide 26-vinblastine conjugates showed a dramatic growth delay of about 10 days (Fig 14). A multimer (four repeats) of Peptide 26 conjugated to vinblastine with introduced lysine-containing linkers was produced in a recombinant insect cell system to improve binding, conjugation and plasma half-life.
  • CD22 is an ideal target antigen to design immunoconjugates as it is expressed by B-NHL-cells, and some human acute and chronic leukemias.
  • Specific antibodies clones HD239, HD6 and RFB4 are available and taken up by the endosome. Binding of these agents to other tissues leads however to side-effects.
  • To overcome this limitation we developed a new technology to negatively select peptide populations after mRNA display by in vitro evolution.
  • RNA library covering all possible 12-mer peptides was created consisting of 10 ⁇ 16 different molecules. Only covalent peptide-RNA-adducts would be stable enough to allow stringent selection procedures which are crucial to remove non-specific binding peptides.
  • Puromycin is the best studied linker and was used in this study. In marked contrast to previous approaches using mRNA display we used a 1 : 1 ratio of RNA to ribosomes during in vitro translation as the linked puromycin will inevitably block the ribosome.
  • Polyclonal peptide populations labelled with Europium-chelat show binding to CD22 after six generations of selection.
  • individual Peptides were synthesized after their sequences were identified by deep sequencing.
  • the peptides resemble all functions of monoclonal antibodies except the binding to immune- receptors.
  • Conjugated to Vinblastin, peptide 26 leads to dramatic growth delay in a xeno-model which is even more pronounced after multimerization of the peptide. Surprisingly no toxicity could be noted in the animal model in marked contrast to the clinically used Anti-CD22-ozogamicin. Functional activity of the peptides was shown and conjugation with various peptide-drug conjugates

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Abstract

L'invention concerne des peptides synthétiques de liaison à CD22, non dérivés d'anticorps, utilisables comme mimétiques d'anticorps, comme agents actifs ou comme agents de ciblage de conjugués immunitaires.
PCT/EP2013/069569 2012-09-20 2013-09-20 Peptides de liaison à cd22 WO2014044793A2 (fr)

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WO2017176651A1 (fr) * 2016-04-04 2017-10-12 True North Therapeutics, Inc. Anticorps anti-facteur bb du complément et utilisations de ceux-ci
CN109432087A (zh) * 2018-12-21 2019-03-08 陕西师范大学 鞣花酸在制备预防和治疗多发性硬化症药物中的应用
US11192944B2 (en) 2017-10-11 2021-12-07 Bioverativ Usa Inc. Methods of inducing complement activity
JP2022510964A (ja) * 2018-12-19 2022-01-28 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 二重特異性抗ｃｄ２８ｘ抗ｃｄ２２抗体及びその使用
US11242382B2 (en) 2020-04-20 2022-02-08 Genzyme Corporation Humanized anti-complement factor Bb antibodies

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017176651A1 (fr) * 2016-04-04 2017-10-12 True North Therapeutics, Inc. Anticorps anti-facteur bb du complément et utilisations de ceux-ci
US10934347B2 (en) 2016-04-04 2021-03-02 Genzyme Corporation Anti-complement factor BB antibodies and uses thereof
US11851482B2 (en) 2016-04-04 2023-12-26 Genzyme Corporation Anti-complement factor Bb antibodies and uses thereof
US11192944B2 (en) 2017-10-11 2021-12-07 Bioverativ Usa Inc. Methods of inducing complement activity
JP2022510964A (ja) * 2018-12-19 2022-01-28 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 二重特異性抗ｃｄ２８ｘ抗ｃｄ２２抗体及びその使用
CN109432087A (zh) * 2018-12-21 2019-03-08 陕西师范大学 鞣花酸在制备预防和治疗多发性硬化症药物中的应用
US11242382B2 (en) 2020-04-20 2022-02-08 Genzyme Corporation Humanized anti-complement factor Bb antibodies
US11999780B2 (en) 2020-04-20 2024-06-04 Genzyme Corporation Humanized anti-complement factor Bb antibodies and uses thereof

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