WO2010017468A1 - Treatment of autoimmune and inflammatory disease - Google Patents
Treatment of autoimmune and inflammatory disease Download PDFInfo
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- WO2010017468A1 WO2010017468A1 PCT/US2009/053136 US2009053136W WO2010017468A1 WO 2010017468 A1 WO2010017468 A1 WO 2010017468A1 US 2009053136 W US2009053136 W US 2009053136W WO 2010017468 A1 WO2010017468 A1 WO 2010017468A1
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Definitions
- the present invention provides novel methods of treatment of multiple sclerosis and other autoimmune diseases, and novel isolated binding proteins for use in these methods. There is also provided a method of treating multiple sclerosis comprising the neutralization of the biological activity of IL-7 or IL-7R.
- MS Multiple Sclerosis
- oligodendrocytes which are the cells responsible for creating and maintaining a fatty layer, known as the myelin sheath.
- MS results in the thinning or complete loss of myelin. When the myelin is lost, the neurons can no longer effectively conduct their electrical signals leading to numerous neurologic dysfunctions.
- Individuals with MS produce autoreactive T cells that participate in the formation of inflammatory lesions along the myelin sheath of nerve fibres.
- the cerebrospinal fluid of patients with active MS contains activated T cells, which infiltrate the brain tissue and cause characteristic inflammatory lesions, destroying the myelin. While the multiple sclerosis symptoms and course of illness can vary from person to person, there are three forms of the disease — relapsing-remitting MS, secondary progressive MS, and primary progressive MS.
- inflammatory attacks occur over short intervals of acutely heightened disease activity. These episodes are followed by periods of recovery and remission.
- the local swelling in the nervous system lesion resolves, the immune cells become less active or inactive, and the myelin- producing cells remyelinate the axons. Nerve signalling improves, and the disability caused by the inflammation becomes less severe or goes away entirely.
- This phase of the disease is called relapsing-remitting MS (RRMS).
- RRMS relapsing-remitting MS
- the lesions do not all heal completely, though. Some remain as "chronic" lesions, which usually have a demyelinated core region which lacks immune cells.
- SPMS secondary progressive MS
- SPMS secondary progressive MS
- the disease no longer responds well to disease-modifying drugs, and patients' disabilities steadily worsen.
- the destruction of neurons from early in the natural course of MS suggests that the progressive disabilities of SPMS might be the result of an accumulated neuronal loss that eventually overwhelms the brain's compensatory abilities.
- Primary progressive MS is a type of multiple sclerosis where there are no relapses, but over a period of years, there is gradual loss of physical and cognitive functions.
- the goal of treatment in patients with relapsing-remitting multiple sclerosis is to reduce the frequency and severity of relapses (and thereby prevent exacerbations) as well as to prevent or postpone the onset of the progressive phase of the disease.
- immunomodulatory or immunosuppressive drugs have been used, but they have never found widespread acceptance owing to limited efficacy and considerable toxicity.
- large randomized controlled trials have been performed successfully with interferon beta-1 a, interferon beta-1 b, and glatiramer acetate.
- T H 1 and T H 17 cells are thought to play an important role.
- T H 1 and T H 17 cells are thought to play an important role.
- CNS central nervous system
- T reg regulatory T cells that normally keep pathogenic T H 1 and T H 17 cells in check are deficient in patients with MS, further tilting the immune system toward an pro-inflammatory state.
- SNPs genome wide single nucleotide polymorphisms
- IL-7 and IL-7 receptor are known to play an important role in T cell and B cell development and homeostasis mainly in a thymic environment. Indeed, thymic stromal cells, fetal thymus, and bone marrow are sites of IL-7 of production.
- the IL-7 receptor consists of two subunits, CD127 and a common chain (gamma chain or vc) which is shared by receptors of IL-2, IL-4, IL-9, IL-15, and IL-21.
- CD127 is also known as IL-7 receptor alpha (IL-7R ⁇ ) and p90 IL-7R.
- Human CD127 (Swiss Prot accession number P16871 ) has a total of 459 amino acids (20 signal sequence). It comprises a 219 amino acid extra cellular region, a 25 amino acid transmembrane region and a 195 amino acid intracellular region. The numbering of residues within CD127, as used herein (e.g. for the description of antibody epitopes) is based on the full length protein, including signal sequence residues.
- CD127 may exist in four isoforms, the isoform H20 (Swissprot accession number P16871-1 ) has the following amino acid sequence (including signal sequence):
- CD127 is also found in the receptor of thymic stromal derived lymphopoietin (TSLP).
- TSLP receptor is a heterodimer of CD127 and cytokine receptor-like factor 2 (CRLF2).
- Binding of IL-7 to the IL-7R activates multiple signaling pathways including the activation of JAK kinases 1 and 3 leading to the phosphorylation and activation of Stat ⁇ .
- This pathway is crucial to the survival of thymic developing T cell precursors because Stat ⁇ activation is required in the induction of the anti-apoptotic protein Bcl-2 and the prevention of the pro-apoptotic protein Bax entry into the mitochondrion.
- Another IL-7R mediated pathway is the activation of PI3 kinase, resulting in the phosphorylation of the pro-apoptotic protein Bad and its cytoplasm retention.
- CD127 is expressed in peripheral resting and memory T cells. The mechanism of IL-7 regulation of T cell survival and homeostasis and the source of IL-7 in the periphery are not completely understood.
- IL-7 may contribute to the pathogenesis of autoimmune diseases.
- CD127 has been described in WO9015870 and antagonists of IL-7 and CD127 in the treatment of multiple sclerosis have been described in WO2006052660 and US20060198822. Antagonists of TSLP have been described in, for example, US7304144 and WO2007096149.
- the present inventors have shown that IL-7/CD127 antagonism is efficacious in amelioration of Experimental Autoimmune Encephalomyelitis (EAE).
- EAE Experimental Autoimmune Encephalomyelitis
- the treatment resulted in marked reduction of T H 17 and, to a lesser degree, T H 1 cells in both spleen and spinal cord of treated mice, which was accompanied by an increased level of Foxp3+ T reg .
- the inventors have also shown that IL-7 is critically required for the expansion and survival of T H 17 cells, but that its requirement during differentiation of precursor T cells into a T H 17 cell population is minimal.
- Restoring the balance of the functional ratio of autoreactive inflammatory T H 17 and T H 1 cells and T reg with an antagonist of CD127 or IL-7 provides great potential as a therapy for multiple sclerosis and other autoimmune diseases.
- T H 17 and T H 1 cells The selective susceptibility of T H 17 and T H 1 cells was attributable to high expression of CD127 in activated pathogenic T cells and their requirement for IL-7 for expansion and survival.
- Blockade of CD127 led to altered signalling events characterized by down-regulation of phosphorylated JAK-1 and STAT-5 and BCL-2 and the increased activity of BAX, rendering CD127+ T H 17 and T H 1 cells susceptible to apoptosis.
- Foxp3+ T reg inducible T reg
- a method for the treatment of an autoimmune disease or an inflammatory disorder in a human subject comprising administering to the subject an antagonist of at least one of: IL-7 receptor mediated T H 17 expansion, and IL-7 receptor mediated T H 17 survival.
- IL-7 receptor mediated T H 17 expansion and/or survival can be observed at a cellular level by an increase or maintenance of T H 17 cell count, or by an increase in the ratio of T H 17 cell numbers compared to the numbers of other CD4+ T cells, or more specifically by an increase in the T H 17:T H 1 ratio, the T H 17:T reg ratio, the (T H 17 plus T H 1 ):T reg ratio, and/or the T H 17:(T H 1 plus T reg ) ratio.
- T H 17 expansion and/or survival can be observed by an increase in IL-17 production by a population of CD4+ T cells (or by a population of T H 17 cells).
- the antagonist of IL-7 receptor mediated T H 17 expansion and/or IL-7 receptor mediated T H 17 survival reduces IL-17 production by a population of CD4+ T cells.
- IL-7 receptor mediated T H 17 expansion and survival can also be observed by an increase in IFN- ⁇ production by a population of CD4+ T cells (or by a population of T H 17 cells).
- the antagonist of the present invention inhibits IFN- ⁇ production by a population of CD4+ T cells.
- the antagonist of IL-7 receptor mediated T H 17 expansion and/or survival may inhibit IL-7 receptor mediated STAT-5 phosphorylation.
- the invention provides a method for the treatment of an autoimmune disease or inflammatory disorder, comprising administering to a patient a antagonist of IL-7 or CD127 in an amount sufficient to reduce the T H 17 cell count in the patient.
- the invention provides a method for the treatment of an autoimmune disease in a human subject, comprising administering to the subject an antagonist of IL-7 receptor mediated STAT-5 phosphorylation.
- the present invention provides a method for treating multiple sclerosis in a patient comprising administering an antagonist of IL-7 or CD127 to said patient, wherein the patient is suffering from relapsing remitting multiple sclerosis.
- the invention provides a method of treating an autoimmune or inflammatory disease in a human subject, comprising administering to the subject an antagonist of IL-7 or IL-7R in an amount effective to reduce the ratio of T H 17 cells relative to T H 1 cells.
- the invention provides a method of treating an autoimmune or inflammatory disease in a human subject, comprising administering to the subject an antagonist of IL-7 or IL-7R in an amount effective to reduce the ratio of T H cells relative to (Foxp3+) T reg cells.
- the antagonist is selected from the group consisting of (a) a binding protein which specifically binds to CD127 (SEQ ID NO:1 ); (b) a binding protein which specifically binds to IL-7, (c) a soluble CD127 polypeptide; and (d) a combination of two or more of said antagonists.
- the binding protein which specifically binds CD127 or IL-7 is an isolated human, humanized or chimeric antibody.
- the binding protein which specifically binds to CD127 is an antibody, or an antigen-binding fragment thereof.
- the anti-CD127 binding protein inhibits the binding of IL-7 to the IL-7R receptor complex.
- anti-CD127 antibodies useful in the methods of the present invention include 9B7, 6C5, 6A3, R34.34, GR34 and 1A11 , humanised or chimeric versions thereof, analogs thereof, and antigen-binding fragments thereof.
- the binding protein which specifically binds to IL-7 is an antibody, or an antigen-binding fragment thereof.
- the invention provides a chimeric, humanised or fully human antibody or an antigen-binding fragment thereof which binds to CD127 and which is capable of inhibition of IL-7 mediated T H 17 expansion.
- anti-CD127 binding proteins are not uniformly effective at functionally neutralising the IL-7 pathway or IL-7R mediated signalling.
- regions of the human CD127 polypeptide which appear to play an important role in the signalling pathway, to the extent that an antibody which is capable of binding to one or more of these regions of human CD127 is particularly effective in neutralising the IL-7 pathway or IL-7R mediated signalling. These regions are defined by amino acid residues:
- amino acids which play a role in the interaction between the ligand IL-7 and the CD127 receptor.
- amino acids are believed to be of particular significance in the IL-7/CD127 interaction: amino acids
- the antigen-binding proteins are capable of binding to at least one amino acid within, or an amino acid flanking or structurally neighbouring, at least one or a plurality of regions (i) to (iv) as defined above. In another embodiment, the antigen- binding proteins are capable of binding to at least one amino acid within, or an amino acid, at least one of the regions (a) to (e), as defined above.
- the invention provides antigen-binding proteins which are capable of binding to at least one amino acid within a region defined by amino acid residues 202 to 219 of SEQ ID NO:1.
- the antigen-binding protein according to this embodiment may further be capable of binding to at least one amino acid within one, two, three or all four of the regions defined by amino acid residues (i) 41 to 63, (ii) 65 to 80, (iii) 84 to 105 and (iv) 148 to 169 of SEQ ID NO:1.
- the antigen-binding protein binds to at least one amino acid within a region defined by amino acids (v) 202 to 219 of SEQ ID NO:1 and at least one amino acid within a region defined by amino acids (iv) 148 to 169 of SEQ ID NO:1.
- the antigen-binding protein according to this embodiment may further be capable of binding to at least one amino acid within a region defined by amino acids (ii) 65 to 80 and/or (iii) 84 to 105 of SEQ ID NO:1.
- the antigen-binding protein binds to at least one amino acid within each of peptides (ii) 65 to 80, (iii) 84 to 105, (iv) 148 to 169, and (v) 202 to 219 of SEQ ID NO:1.
- the invention provides antigen-binding proteins which are capable of binding to at least one amino acid within a region defined by amino acid residues (e) 212 to 213 of SEQ ID NO:1 , or a flanking or structurally neighbouring amino acid.
- the antigen-binding protein according to this embodiment may further be capable of binding to at least one amino acid within, flanking or structurally neighbouring to, one, two, three or all four of the regions defined by amino acid residues (a) 51 to 53, (b) 77 to 79, (c) 97 to 103 and (d) 158 to 159 of SEQ ID NO:1.
- the binding protein binds to at least one amino acid within a region defined by amino acids (e) 212 to 213 of SEQ ID NO:1 , or a flanking or structurally neighbouring amino acid, and at least one amino acid within, flanking, or structurally neighbouring to a region defined by amino acids (d) 158 to 159 of SEQ ID NO:1.
- the binding protein according to this embodiment may further be capable of binding to at least one amino acid within, flanking or structurally neighbouring to a region defined by amino acids (b) 77 to 79 and/or (c) 97 to 103 of SEQ ID NO:1.
- the binding protein binds to at least one amino acid within each of peptides (b) 77 to 79, (c) 97 to 103, (d) 158 to 159, and (e) 212 to 213 of SEQ ID NO:1.
- Antibodies according to these aspects of the invention include 6A3, 1A11 , 6C5 and 9B7, antigen-binding fragments thereof and chimeric or humanised variants thereof. Additional antibodies of these aspects of the invention are chimeric or humanised variants of R3434 or GR34, or an antigen-binding fragment of R3434 or GR34.
- the invention provides a human, humanised or chimeric antibody, or a fragment thereof, wherein the antibody or fragment binds to an epitope of human CD127 that contains at least one amino acid residue within the region beginning at residue number 80 and ending at residue number 190.
- the invention provides an antibody or fragment thereof which binds to an epitope of human CD127 (SEQ ID NO:1 ), wherein said epitope has an amino acid residues which are present in at least one of the regions of CD127 of SEQ ID NOs:20-28, 45-50,67-70, 87-89, and 106-116.
- This binding may be measured by, inter alia, peptide ELISA, surface plasmon resonance (BIAcore) or phage display.
- the antibody or fragment thereof binds to an epitope of human CD127 (SEQ ID NO:1 ), wherein said epitope has amino acid residues which are present in: one, two, three or four of the regions of SEQ ID NOs:66-70; one, two or three of the regions of CD127 of SEQ ID NOs:87-89; or one, two or three of the regions of CD127 of SEQ ID NOs:114-116.
- the invention provides an antibody or fragment thereof which binds to an epitope of human CD127, wherein said epitope has an amino acid residue present in at least one of the following regions of CD127: 35-49 (SEQ ID NO:20), 84-105 (SEQ ID NO:21 ) 171-180 (SEQ ID NO:22), or an antibody or fragment which binds to an at least one of the following linear peptides: 35-49 (SEQ ID NO:20), 84-105 (SEQ ID NO:21 ) 171-180 (SEQ ID NO:22).
- This binding may be measured by, inter alia, peptide ELISA, suface plasmon resonance (BIAcore), or phage display.
- the invention provides an antibody or fragment thereof which binds to an epitope of human CD127 (SEQ ID NO:1 ), the epitope having an amino acid residue present within, or the epitope being present within the following regions of CD127 (SEQ ID NO:1 ): 80-94 (SEQ ID NO:23), 95-109 (SEQ ID NO:24), 170-184 (SEQ ID NO:25).
- the invention provides an antibody or fragment thereof which binds to an epitope of human CD127 (SEQ ID NO:1 ), the epitope having an amino acid residue present within, or the epitope being present within the following regions of CD127 (SEQ ID NO:1 ): 35-49 (SEQ ID NO:26), 84-105 (SEQ ID NO:27), 139-184 (SEQ ID NO:28).
- an antibody or fragment thereof which binds to a C-terminal biotinylated CD127 peptide that comprises residues 35-49, 84-105, 171-180 of CD127 as determined by surface plasmon resonance, said peptide being bound to a streptavidin sensor chip.
- the antibody or fragment thereof additionally requires at least one flanking residue or structurally neighbouring residue to said at least one residue in the 35-49, 84-105 or 171-180 regions of CD127 for binding.
- Whether or not a residue in the abovedefined regions of CD127 is required is defined by a reduction in binding affinity of the antibody to the alanine substituted CD127 compared with the wild-type CD127, wherein said reduction is more than 1 , 2, 3, 4 or 5 fold as determined by Biacore or ELISA affinity measurements.
- a structurally neighbouring residue in this context is a residue that is in close proximity in three-dimensional space to the residue in question and which is bound by the antibody.
- antigen epitopes may be either liner or non-liner peptide sequences. In the latter, non-linear case, although the residues are from different regions of the peptide chain, they may be in close proximity in the three dimensional structure of the antigen.
- Such structurally neighbouring residues can be determined through computer modelling programs or via three-dimensional structures obtained through methods known in the art, such as X-ray crystallography.
- Another aspect of the present invention relates to therapeutic antibodies and antigen-binding fragments thereof which are specific for CD127, and which are useful in the treatment of autoimmune and/or inflammatory disorders.
- the antibodies and antigen- binding fragments may inhibit T H 17 expansion and survival and/or inhibit pSTAT-5, in an assay that as that herein defined.
- These antibodies and antigen-binding fragments may represent the antagonist useful in the methods of the invention.
- an antibody or antigen-binding fragment and/or derivative thereof which binds to CD127 and which comprises at least a third heavy chain CDR (CDRH3) selected from the group consisting of: 9B7-CDRH3 (SEQ ID NO:6); 6C5-CDRH3 (SEQ ID NO:33), 6A3-CDRH3 (SEQ ID NO:55) or 1A11- CDRH3 (SEQ ID NO:75).
- CDRH3 third heavy chain CDR
- the antibody or antigen-binding fragment and/or derivative thereof comprises CDRH3 of: antibody 9B7 (SEQ ID NO:6) and one, two, three, four or all five additional CDRs of 9B7 (SEQ ID NOs:4,5,7,8,9); antibody 6C5 (SEQ ID NO:33) and one, two, three, four or all five additional CDRs of 6C5 (SEQ ID NOs: 31 ,32,34,35,36); antibody 6A3 (SEQ ID NO:55) and one, two, three, four or all five additional CDRs of 6A3 (SEQ ID NOs: 53,54,56,57,58); or antibody 1A11 (SEQ ID NO:75) and one, two, three, four or all five additional CDRs of 1 A1 1 (SEQ ID NOs:73,74,76,77,78).
- a therapeutic antibody which is an antibody or an antigen binding fragment and/or derivative thereof which binds to CD127 and which comprises the following CDR
- CDRH1 RYNVH (SEQ ID NO:4)
- CDRH2 MIWDGGSTDYNSALKS (SEQ ID NO:5)
- SEQ ID NO:5 A: CDRH1 : RYNVH (SEQ ID NO:4); CDRH2: MIWDGGSTDYNSALKS (SEQ ID NO:5);
- CDRH3 NRYESG (SEQ ID NO:6);
- CDRL1 KSSQSLLNSGNRKNYLT (SEQ ID NO:7);
- CDRL2 WASTRES (SEQ ID NO:8);
- CDRL3 QNDYTYPFTFGS (SEQ ID NO:9).
- CDRL1 RSSQSIVQSNGNTYLE (SEQ ID NO:34)
- CDRL2 KVSNRFS (SEQ ID NO:35)
- CDRL3 FQGSHVPRT (SEQ ID NO:36)
- CDRH2 YIFYSGSTTYTPSLKS (SEQ ID NO:54)
- CDRH3 GGYDVNYF (SEQ ID NO:55)
- CDRL1 LASQTIGAWLA (SEQ ID NO:56)
- CDRL2 AATRLAD (SEQ ID NO:57)
- CDRL3 QQFFSTPWT (SEQ ID NO:58)
- CDRH2 LINPYNGVTSYNQKFK (SEQ ID NO:74)
- CDRH3 GDGNYWYF (SEQ ID NO:75)
- CDRL1 SASSSVTYMHW (SEQ ID NO:76)
- CDRL2 EISKLAS (SEQ ID NO:77)
- CDRL3 QEWNYPYTF (SEQ ID NO:78).
- a therapeutic antibody which is a human, humanised or chimeric antibody or an antigen binding fragment and/or derivative thereof which binds to CD127 and which comprises the following CDRs, or analogs thereof:
- CDRH2 LINPYSGITSYNQNFK (SEQ ID NO:93)
- CDRL1 SASSSVSYMHW (SEQ ID NO:95)
- CDRL2 EISKLAS (SEQ ID NO:96)
- CDRL3 QYWNYPYTF (SEQ ID NO:97).
- CDR CDR
- CDRL1 CDR1
- CDRL2 CDRL3
- CDRH 1 CDRH2
- CDRH3 CDRH3
- a monoclonal antibody comprising:
- antibody variable domain sequences that have at least 90% identity, or at least 95% identity, or at least 98% identity, or at least 99% identity, over the whole length of the sequences of SEQ ID NOs: 2, 3, 29, 30, 51 , 52, 71 , and 72.
- Also provided by the invention is a method of treatment of an autoimmune disease or inflammatory disorder comprising administering to a patient an anti-CD127 antibody, wherein the antibody comprises: (i) the heavy chain variable region of SEQ ID NO:2 and/or the light chain variable region of SEQ ID NO:3; (ii) the heavy chain variable region of SEQ ID NO:29 and/or the light chain variable region of SEQ ID NO:30;
- the invention provides an antibody or an antigen-binding fragment thereof which binds to CD127 and which is capable of inhibition of IL-7 mediated T H 17 expansion, wherein the antibody is not R.34.34 (Dendritics lnc.,#DDX0700).
- a method for identifying antibodies or antibody fragment suitable for use in the treatment of an autoimmune disease or an inflammatory disease comprising the steps of: screening a plurality of independent antibody or antibody fragment populations to determine the ability of each antibody population to: i. inhibit the binding of IL-7 to IL-7R, ii. neutralise IL-7 induced STAT-5 phosphorylation, and/or iii. inhibit the production of IL-17 by T H 17 cells, and selecting those antibody or antibody fragment populations which are able to inhibit the binding of IL-7 to IL-7R, inhibit IL-7 induced STAT-5 phosphorylation, and/or inhibit the production of IL-17 by T H 17 cells in vivo.
- a composition or substance to act as an antagonist of IL-7 receptor mediated T H 17 expansion or IL-7 receptor mediated T H 17 survival, or to reduce T H 17 cell count, can be determined by routine methods.
- na ⁇ ve CD4 + cells can be stimulated to differentiate into T H 17 with appropriate conditions known to those of skill in the art (e.g. TGF- ⁇ 1 , IL-23, IL-6, anti-IFN- ⁇ and anti-IL-4, or IL-1 ⁇ , IL-6 and IL-23).
- TGF- ⁇ 1 , IL-23, IL-6, anti-IFN- ⁇ and anti-IL-4, or IL-1 ⁇ , IL-6 and IL-23 can then be exposed to the test agent and IL-7, following which the T H 17 cell count can be determined.
- a decrease in T H 17 cells relative to a control would indicate that the test agent is capable of inhibiting T H 17 expansion or survival.
- a method of manufacturing a medicament for the treatment of autoimmune or inflammatory disease comprising formulating an anti-CD127 or anti-IL-7 antibody or antigen-binding fragment thereof and one or more excipients into a pharmaceutically acceptable formulation.
- This method may comprise the preliminary steps of identifying an antibody, as hereinbefore defined, and/or of recombinantly producing such an antibody.
- the numbering system used refers to the full length sequence of CD127, which includes the signal sequence.
- the epitopes of human CD127 are found within the cited residues of SEQ ID NO:1.
- the binding proteins of the present invention binds to human CD127 with an affinity (KD) which is less than 2OnM, less than 15nM, less than 1OnM, less than 5nM, less than 1 nM or less than 0.5nM, as measured by surface plasmon resonance (BIAcore) .
- KD affinity
- BiAcore surface plasmon resonance
- the binding protein competitively inhibits binding of 9B7, 6C5, 3A6, 1A11 or R34.34 (Dendritics Inc. #DDX0700), or an antigen-binding fragment thereof to human CD127.
- Competitive inhibition can be determined by those skilled in the art, for example, in a competition ELISA assay, by BIAcore or Scatchard analysis.
- isolated binding proteins which compete with: i. antibody R34.34 (Dendritics lnc.,#DDX0700); ii. an antibody having a variable heavy chain region as set out in SEQ ID NO:
- the isolated binding protein of the present invention is an antibody or an antigen-binding fragment thereof which competes with: i. antibody R34.34 (Dendritics lnc.,#DDX0700); ii. an antibody having a variable heavy chain region as set out in SEQ ID NO:91;
- variable light chain region as set out in SEQ ID NO:51 and a variable light chain region as set out in SEQ ID NO:52; iii. an antibody having a variable heavy chain region as set out in SEQ ID NO:52;
- binding proteins for use in the treatment of multiple sclerosis wherein the binding proteins compete for binding to human CD127 (SEQ ID NO:1 ) with: i. antibody R34.34 (Dendritics lnc.,#DDX0700); ii. an antibody having a variable heavy chain region as set out in SEQ ID NO:2 and a variable light chain region as set out in SEQ ID NO:3; iii. an antibody having a variable heavy chain region as set out in SEQ ID
- antibody or fragment A to compete with antibody R34.34, GR34, 6A3, 1 A11 , 6C5 or 9B7 (antibody B) for a specific binding site (of human CD127), antibody A must be present in a sufficient amount to have an effect in said assay. For example, antibody A and antibody B may be present in equimolar amounts. If antibody A is a competing antibody, the presence of antibody A may reduce the binding of antibody B to human CD127 in an ELISA assay by more than 10%, 20%, 30%, 40% or 50%. A competing antibody (antibody A) may reduce the binding of antibody B to plate-bound human CD127, whereas a non-anti-CD127-specific control does not.
- human CD127 may be bound to an immunoassay plate.
- surface plasmon resonance may be used to determine competition between antibodies.
- Isolated binding proteins which are capable of competition for binding to CD127 with antibody R34.34 or the antibodies of the invention, an isolated binding protein having a V H of SEQ ID NO:2 and V L of SEQ ID NO:3, an isolated binding protein having a V H of SEQ ID NO:76 and a V L of SEQ ID NO:77, or an isolated binding protein having a V H of SEQ ID NO: 193 and a V L of SEQ ID NO: 194 may be used in the treatment of MS and other autoimmune diseases.
- the binding proteins of the present invention may comprise the CDRs of R34.34, GR34, 9B7, 6A3, 1 A11 or 6C5, or they may comprise analogs thereof.
- humanized antibodies wherein the R34.34, GR34, 9B7, 6A3, 1A1 1 or 6C5 CDRs (or analogs thereof) are grafted into a heavy chain or light chain variable domain framework.
- a polynucleotide sequence which encodes the binding proteins of the present invention.
- a polynucleotide sequence that encodes an antibody or fragment thereof which comprises one or all of the CDRs found in 9B7 (SEQ ID NOs:4-9), 6C5 (SEQ ID NOs:31- 36), 6A5 (SEQ ID NOs:53-58), 1A11 (SEQ ID NOs:73-78) or GR34 (SEQ ID NOs:92-97).
- a host cell transfected with the polynucleotides of the present invention.
- binding proteins, antibodies, antigen-binding fragments, their humanised, human or chimeric variants, and analogs, of the present invention may be used in a method of treatment of multiple sclerosis, the method comprising administering a safe and effective dose of the binding proteins of the present invention to a patient in need thereof.
- the binding protein may be an antibody which comprises one or all of the CDRs found in 9B7 (SEQ ID NOs:4-9), 6C5 (SEQ ID NOs:31-36), 6A5 (SEQ ID NOs:53-58), 1A1 1 (SEQ ID NOs:73-78) or GR34 (SEQ ID NOs:92-97).
- RRMS relapsing/remitting MS
- the invention provides a method of treating an autoimmune or inflammatory disease comprising administering to a subject in need thereof a therapeutically effective amount of an antagonist of IL-7 or IL-7R and an additional therapeutic agent.
- the additional therapeutic agent may be selected from the group consisting of: immunomodulators such as interferon beta (IFN ⁇ -1 a or IFN ⁇ -1 b) and glatiramer acetate, immunosuppresants such as cyclophosphamide, methotrexate, azathioprine, cladribine, cyclosporine and mitoxantrone, other immune therapies such as intravenous immune globulin (IVIg), plasma replacement and sulphasalazine.
- the additional therapeutic may be administered as in a manner (dosage, timing, mechanism) as prescribed by a physician.
- the additional therapeutic agent may be administered simultaneously or sequentially or separately from the antagonist of the present invention.
- the additional therapeutic agent and the antagonist are administered such that their pharmacological effects on the patient overlap; in other words, they exert their biological effects on the patient at the same time.
- the IL7/IL7R antagonist is a soluble CD127 polypeptide.
- the soluble CD127 polypeptide may comprise a polypeptide which is 90% or more identical to a polypeptide selected from the extracellular domain of CD127 (SEQ ID NO:1 ), or a polypeptide comprised of amino acids 21 to 219 of SEQ ID NO:1.
- the soluble CD127 comprises a polypeptide is amino acids 21-219 of SEQ ID NO:1.
- the soluble CD127 polypeptide may be fused to a non-CD127 moiety.
- the non-CD127 moiety may be a heterologous peptide fused to the soluble CD127 polypeptide.
- the non-CD127 moiety is selected from the group consisting of serum albumin, a targeting protein, an immunoglobulin fragment, a reporter protein or a purification-facilitating protein.
- the soluble CD127 polypeptide is fused to an Fc region of an immunoglobulin.
- Figure 1 shows inhibition of IL-7-mediated pSTAT5 by anti-mouse CD127 antibodies
- Figure 1 (B) shows inhibition of TSLP mediated pSTAT5 by anti-mouse CD127 antibodies
- Figure 2 shows a CD127 ELISA binding curve for 9B7
- Figure 3 shows that MAb 9B7 (solid line) is capable of recognizing CD127 expressed on the surface of CD127-transfected CHO cell line. An irrelevant, isotype control, antibody is shown as a dotted line;
- Figure 3(B) shows that antibody 9B7 (solid line) is not capable of recognizing CD127 in a mock transfected CHO cell line - an irrelevant, isotype control, antibody is shown as a dotted line;
- Figure 4 demonstrates an example of the inhibition of IL7-mediated pStat ⁇ signalling by purified murine anti-CD127 mAb 9B7;
- Figure 5(A) shows that the MOG-EAE clinical score was ameliorated by rat anti-murine CD127 antibody SB/14;
- Figure 5(B) shows inhibition of MOG peptide-induced T-cell proliferation by SB/14;
- Figure 5(C) shows inhibition of cytokine production by anti-CD127 antibody by SB/14;
- Figures 5(D) and 5(E) show the selective effect of anti-mCD127 antibody (SB/14) treatment on helper T cell subtypes;
- Figure 5(F) shows that the MOG-EAE clinical score was ameliorated by anti-mCD127 antibody (SB/14) treatment;
- Figure 6 shows CD127 expression in T reg , T H 1 and T H 17 cells derived ex vivo from spleen or spinal cord of EAE mice;
- Figure 7(A) shows that the effect of IL-7 on the promotion of T H 17 differentiation was modest compared to that of I L-6;
- Figure 7(B) shows that the induction of STAT-3 phosphorylation is largely driven by I L-6 independently from IL-7;
- Figure 7(C) shows that the effect of IL-7 on ROR ⁇ expression is also modest compared to that of I L-6;
- Figure 7(D) shows that the effect of anti-mCD127 antibody (SB/14) treatment was modest during disease onset in EAE;
- Figure 8(A) shows the percentage of T H 17 cells, ⁇ -interferon secreting T H 1 cells, and T reg cells in the CNS;
- Figure 8(B) shows the percentage of T H 17 cells, ⁇ -interferon secreting T H 1 cells, and T reg cells in splenocytes
- Figure 8 (C) shows the percentage of T H 17, T H 1 and T reg in the course of EAE in both treated and control mice
- Figure 9(A) shows the effect of an anti-CD127 antibody (SB/14) of T H 17 and T H 1 cell counts, but not T reg count, was inhibited when CD127 antibody was added in the onset of differentiation
- Figure 9(B) shows the effect of an anti-mCD127 antibody (SB/14) as in Figure 9(A), but on differentiated T H 17, but not T H 1 or T reg ;
- Figure 10 shows that addition of IL-7 promoted T H 17 expansion/survival and, to a lesser degree, T H 1 , but not Foxp3 in T reg , when day 9 EAE MOG-specific T cells were cultured;
- Figure 11 (A) shows an immunoblot analysis of CD4+ T cells derived ex vivo from treated or control EAE mice showing anti-CD127 antibody treatment changes in signaling pathways related to JAK-STAT and apoptosis as characterized by down-regulation of phosphorylated JAK-1 and phosphorylated STAT-5 and markedly decreased levels of a key pro-apoptotic molecule, BCL-2, and increased activity of an anti-apoptotic molecule, BAX;
- FIG. 11 (B) shows that anti-CD127 antibody treatment increased the percentage of
- Annexin-V+ apoptotic cells among CD4+CD127+ T cells compared to that of
- CD4+CD127- T cells derived from treated EAE mice CD4+CD127- T cells derived from treated EAE mice
- Figure 11 (C) shows that differentiated T H 17 cells derived from EAE mice undergo apoptosis which can be rescued with IL-7, but this process is slowed if the cells are pre- incubated with an anti-CD127 antibody;
- Figure 1 1 (D) shows that the effects of IL-7 are mediated through the JAK/STAT-5 pathway
- Figure 12 shows mAb 9B7 and R34.34 have minimal inhibitory effect on the differentiation of T H 17 from human total CD4+ cells.
- Figure 13 shows mAb 6C5 inhibition of CD127-ECD binding to immobilised IL-7
- Figure 14 shows that mAb 6C5 competes with IL-7 for binding to CD127;
- Figure 15 shows that mAb 6C5 and Dendritics antibody R.34.34 compete for binding to
- Figure 16(A) shows mAb 6A3 inhibition of CD127-ECD binding to immobilised IL-7;
- Figure 16(B) is an inhibition ratio curve of antibodies 6A3, 6C5 and R34.34 at different concentrations of antibody, showing the effect of these antibodies on the binding of
- Figure 17 shows that mAb 6A3 competes with IL-7 for binding to CD127 expressed on CHO cells
- Figure 18 shows mAb 6C5 and antibody R.34.34 both inhibit the production of I FNy by IL-
- Figure 19 shows the ability of antibodies BD, R34.34, 1A11 and 6C5 to block Stat5 signalling induced by IL-7 stimulated PBMCs;
- Figure 20 shows the ability of antibodies BD, R34.34, 1A11 and 6C5 to block Stat5 signalling induced by IL-7 stimulated CCF-CEM cells;
- Figure 21 shows the ability of mAb 6A3 to inhibit IL-17 and IFN- ⁇ production in a T H 17 expansion assay;
- Figure 22 shows the inhibitory effect of various anti-CD127 antibodies on the production of IL-17 by hCD4 + cells under IL-7 stimulation
- Figure 23 shows the inhibitory effect of mAb 6A3 on IFN- ⁇ production and IL-17 production by T H 17 cells.
- the invention is based on the discovery that IL-7/IL-7R signalling is critically required for survival and expansion of committed T H 17 cells in both mouse and human systems, while its role in T H 17 differentiation is not essential compared to that of IL-6.
- the in vivo effect on the immune system by IL-7R antagonism is highly selective in EAE, an animal model for multiple sclerosis, affects T H 17 cells and, to a lesser extent, T H 1 cells predominantly of the memory phenotype, and spares T reg cells. This selectivity appears to play an important role in rebalancing the ratio of pathogenic T H 17 cells and T reg cells by IL-7R antagonism in EAE and is attributable to the treatment efficacy.
- IL-7 neutralization or IL-7R antagonism is likely to have unique therapeutic advantages.
- the treatment offers the selectivity that distinguishes pathogenic T H 1 and T H 17 cells from T reg and unrelated immune cells.
- additional therapeutic advantages of IL-7R antagonism involve its selective effect on survival and expansion of differentiated T H 17 as opposed to T H 17 differentiation. It is conceivable that targeting in vivo maintenance of committed T H 17 versus T H 17 differentiation is more efficacious in a therapeutic context.
- Inhibition of IL-7 receptor mediated signalling therefore provides a promising therapeutic intervention for the treatment of autoimmune or inflammatory diseases.
- IL-7R mediated signalling means the biological effect instigated by the IL-7 receptor complex when bound by its ligand, IL-7.
- IL-7R mediated signalling therefore includes, but is not necessarily limited to, one or more, or all, of IL-7 induced phosphorylation of STAT-5, IL-7 induced expansion of T H 17 cells and IL-7 induced survival of T H 17 cells.
- An IL-7 pathway antagonist as used herein is any entity that functionally blocks the biological effects of IL-7, measurable by assays. At the molecular level, one can observe and measure the blocking effect by assays such as IL-7-induced P-STAT5 or Bcl-2. Exemplary p-STAT5 assays are described herein. At the cellular level, one can observe and measure the blocking effect by assays such as Th 17 secretion of IL-17 or I FNy. Exemplary assays are also described herein.
- the IL-7/IL-7R pathway antagonists useful in the present invention are capable of inhibiting, partially or in full, phosphorylation of STAT-5 induced by IL-7.
- STAT-5 phosphorylation can be determined by methods routine in the art, for instance, in an assay such as that described herein (Example 2.3).
- PBMCs are stimulated with IL-7 in the presence and absence of a test agent.
- Cells are subsequently assessed quantitatively for the level of pSTAT-5, e.g. by staining for pSTAT-5 (e.g. with a labelled anti-pSTAT-5 antibody) followed by fluorescence activated cell sorting.
- the levels of phosphorylated STAT-5 could also be determined by ELISA. Those agents which reduce the level of phosphorylated STAT-5 may be potential therapeutic candidates for autoimmune disease.
- the antagonist may be capable of reducing levels of phosphorylated STAT-5 by at least 20%, 50%, 75%, 80%, 85%, 90%, 95% or 100% when compared to STAT-5 levels in the absence of the antagonist, or when compared to a negative control, or untreated cells.
- the antagonist may have an IC 50 of 50 ⁇ g/ml, 25 ⁇ g/ml or less, 10 ⁇ g/ml or less, 5 ⁇ g/ml or less, or 2 ⁇ g/ml or less.
- the antagonist has an IC 50 of less than or equal to 1 ⁇ g/ml, less than or equal to 0.75 ⁇ g/ml, less than or equal to 0.5 ⁇ g/ml, less than or equal to 0.25 ⁇ g/ml, or less than or equal to 0.1 ⁇ g/ml.
- the antagonists of the invention are particularly effective in inhibiting the expansion of T H 17 cells.
- Expansion of T H 17 cells can be determined in a T H 17 expansion assay, which comprises stimulating a population of na ⁇ ve T cells to expand in the presence and absence of a test agent, followed by stimulating the cells to produce IL-17 and assessing the level of IL-17 produced by the cells in the presence and absence of the test agent.
- the antagonist is capable of from 20% or more inhibition of IL-
- the antagonist is capable of from 50%, from 75%, from 85% or from 90% or more inhibition of IL-17 secretion versus the control.
- the antagonist may, in some embodiments, exhibit an IC 50 of less than or equal to 50 ⁇ g/ml in the assay. In other embodiments, the IC 50 may be less than or equal to 20 ⁇ g/ml, 10 ⁇ g/ml or 5 ⁇ g/ml.
- human CD4+ T cells are differentiated into T H 17 by stimulation with T cell receptor activation in the presence of IL-1 , IL-6, and IL-23.
- CCR6+ cells are sorted out to produce an enriched T H 17 population. This population is then stimulated with human IL-7 and the increase in IL-17 and IFN- ⁇ in the supernatant are determined. Blocking the interaction between the IL-7 and CD127 by a functional IL-7/IL-7R pathway antagonist (e.g. an anti-CD127 antibody) in the incubation period should prevent the expansion of the T H 17 cells leading to the reduction of IL-17 and IFN- ⁇ production.
- a functional IL-7/IL-7R pathway antagonist e.g. an anti-CD127 antibody
- CD4+ T cells may be isolated from human peripheral blood mononuclear cells using a commercial kit (e.g. CD4+ T Cell Isolation Kit II, # 130-091- 155, Miltenyi Biotec). CD4+ T cells are then typically re-suspended in RPMI medium with 10% FCS at a concentration of 1.5x10E6/ml. Cells are pre-incubated with control or anti- IL-7R ⁇ antibodies, typically for 30 min. Cells were then cultured with or without 10 ng/ml of IL-7 for 72 h at 37 C. At the end of the incubation, cells are stimulated with 50ng/ml PMA and 1 ug/ml of lonomycin for 5 h. Cell culture supernatants were then collected and the IL-17 concentration is determined by ELISA (eBiosciences).
- a commercial kit e.g. CD4+ T Cell Isolation Kit II, # 130-091- 155, Miltenyi Biotec.
- the isolated binding proteins of the present invention may be in the form of an antibody or immunoglobulin, such as an intact antibody, a human, humanized or chimeric antibody, or fragments or domains of said antibodies.
- These antibodies of the present invention may comprise one or more, or all of the CDRs found in 9B7 (SEQ ID NOs:4-9), 6C5 (SEQ ID NOs:31-36), 6A5 (SEQ ID NOs:53-58), 1A1 1 (SEQ ID NOs:73-78) or GR34 (SEQ ID NOs:92-97).
- binding in this context it is essentially meant that the binding protein, such as an antibody, binds to (an epitope of) CD127 via an antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and (the epitope of) CD127.
- a binding protein therefore binds to CD127 or an epitope of CD127 more readily than it would bind to a random, unrelated polypeptide, or a random, unrelated epitope. In other words, there is specificity between the binding protein and (the epitope of) CD127.
- binding proteins of the invention may also be in the form of a soluble CD127 polypeptide.
- the binding proteins of the present invention may bind to CD127, such as a monoclonal antibody that specifically binds to CD127.
- the binding proteins may also be entities that reduce binding of TSLP to the TSLP receptor, and also reduces binding of IL- 7 to the IL-7 receptor, for the treatment of multiple sclerosis, such as a bispecific binding protein that binds to the IL-7 and TSLP ligands, or elements of the IL-7R and TSLPR that would give this effect, or combinations of ligands and receptors.
- TSLP antagonists are described in, for example, US7304144 and WO2007096149, and as noted supra, the TSLP receptor comprises CD127.
- the antagonists of the present invention may therefore be useful as antagonists of TSLP.
- isolated means that the binding proteins are removed from the environment in which they may be found in nature, for example, they may be purified away from substances with which they would normally exist in nature.
- binding proteins may be substantially pure, in that the mass of protein in a sample would by constituted of at least 50% or at least 80% binding protein.
- a binding protein is said to competitively inhibit the binding of a reference binding protein to CD127, to a fragment of CD127 or to an epitope within CD127 if it preferentially binds to that epitope, to the extent that it blocks, to some degree, binding of the reference binding protein to CD127, or to that fragment of CD127 or epitope within CD127.
- Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays, surface plasmon resonance (BIAcore), or Scatchard analysis.
- a binding protein may be said to competitively inhibit the binding of a reference binding protein to a given epitope if the binding of the reference antibody is reduced by at least 90%, at least 80%, at least 70%, at least 60% or at least 50%.
- the binding proteins of the present invention may be "intact antibodies".
- Intact antibodies are usually heteromultimeric glycoproteins comprising at least two heavy and two light chains. Aside from IgM, intact antibodies are heterotetrameric glycoproteins of approximately 150KDa, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond while the number of disulfide linkages between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant regions.
- V H variable domain
- Each light chain has a variable domain (V L ) and a constant region at its other end; the constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.
- V L variable domain
- the light chains of antibodies from most vertebrate species can be assigned to one of two types called Kappa and Lambda based on the amino acid sequence of the constant region.
- human antibodies can be assigned to five different classes, IgA, IgD, IgE, IgG and IgM.
- IgG and IgA can be further subdivided into subclasses, IgGI , lgG2, lgG3 and lgG4; and IgAI and lgA2.
- Species variants exist with mouse and rat having at least lgG2a, lgG2b.
- the variable domain of the antibody confers binding specificity upon the antibody with certain regions displaying particular variability called complementarity determining regions (CDRs).
- CDRs complementarity determining regions
- the more conserved portions of the variable region are called framework regions (FR).
- the variable domains of intact heavy and light chains each comprise four FR connected by three CDRs. The CDRs in each chain are held together in close proximity by the FR regions and with the CDRs from the other chain contribute to the formation of the antigen binding site of antibodies.
- the constant regions are not directly involved in the binding of the antibody to the antigen but exhibit various effector functions such as participation in antibody dependent cell- mediated cytotoxicity (ADCC), phagocytosis via binding to Fc ⁇ receptor, half- life/clearance rate via neonatal Fc receptor (FcRn) and complement dependent cytotoxicity via the C1q component of the complement cascade.
- ADCC antibody dependent cell- mediated cytotoxicity
- FcRn neonatal Fc receptor
- complement dependent cytotoxicity via the C1q component of the complement cascade.
- the human lgG2 constant region has been reported to essentially lack the ability to activate complement by the classical pathway or to mediate antibody-dependent cellular cytotoxicity.
- the lgG4 constant region has been reported to lack the ability to activate complement by the classical pathway and mediates antibody-dependent cellular cytotoxicity only weakly. Antibodies essentially lacking these effector functions may be termed 'non-lytic' antibodies.
- the binding proteins of the present invention may be "human antibodies".
- Human antibodies may be produced by a number of methods known to those of skill in the art. Human antibodies can be made by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines see Kozbor J.Immunol 133, 3001 , (1984) and Brodeur, Monoclonal Antibody Production Techniques and Applications, pp51-63 (Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice both of which utilize human V region repertories (see Winter G, (1994), Annu.Rev.lmmunol 12,433-455, Green LL (1999), J. Immunol. methods 231 , 1 1-23).
- mice Several strains of transgenic mice are now available wherein their mouse immunoglobulin loci has been replaced with human immunoglobulin gene segments (see Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature Biotechnol. 14,845- 851 , Mendez MJ, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing a repertoire of human antibodies from which antibodies of interest can be selected.
- Phage display technology can be used to produce human antibodies (and fragments thereof), see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD et al (1994) EMBO 13:3245-3260.
- the binding proteins of the present invention may be "chimeric" or "humanized” antibodies.
- the use of intact non-human antibodies in the treatment of human diseases or disorders carries with it the now well established problems of potential immunogenicity especially upon repeated administration of the antibody: that is the immune system of the patient may recognise the non-human intact antibody as non-self and mount a neutralising response.
- various techniques have been developed over the years to overcome these problems and generally involve reducing the composition of non-human amino acid sequences in the intact therapeutic antibody whilst retaining the relative ease in obtaining non-human antibodies from an immunised animal e.g. mouse, rat or rabbit. Broadly two approaches have been used to achieve this.
- Chimaeric antibodies are typically produced using recombinant DNA methods. DNA encoding the antibodies (e.g. cDNA) is isolated and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the H and L chain variable regions of the antibody of the invention, e.g.
- a chimaeric antibody comprising a V H domain having the sequence: SEQ ID NO:2 and a V L domain having the sequence: SEQ ID N0:3 fused to a human constant region (which maybe of a IgG isotype e.g. IgGI ) .
- the second approach involves the generation of humanised antibodies wherein the non-human content of the antibody is reduced by humanizing the variable regions.
- Two techniques for humanisation have gained popularity.
- the first is humanisation by CDR grafting.
- CDRs build loops close to the antibody's N-terminus where they form a surface mounted in a scaffold provided by the framework regions.
- Antigen-binding specificity of the antibody is mainly defined by the topography and by the chemical characteristics of its CDR surface. These features are in turn determined by the conformation of the individual CDRs, by the relative disposition of the CDRs, and by the nature and disposition of the side chains of the residues comprising the CDRs.
- a large decrease in immunogenicity can be achieved by grafting only the CDRs of a non-human (e.g.
- human V regions showing the greatest sequence homology (typically 60% or greater) to the non-human donor antibody maybe chosen from a database in order to provide the human framework (FR).
- the selection of human FRs can be made either from human consensus or individual human antibodies. Where necessary key residues from the donor antibody are substituted into the human acceptor framework to preserve CDR conformations.
- Computer modelling of the antibody maybe used to help identify such structurally important residues, see WO99/48523.
- humanisation maybe achieved by a process of "veneering".
- a statistical analysis of unique human and murine immunoglobulin heavy and light chain variable regions revealed that the precise patterns of exposed residues are different in human and murine antibodies, and most individual surface positions have a strong preference for a small number of different residues (see Padlan E.A. et al; (1991 ) Mol.lmmunol.28, 489-498 and Pedersen JT. et al (1994) J. MoI. Biol. 235; 959-973). Therefore it is possible to reduce the immunogenicity of a non-human Fv by replacing exposed residues in its framework regions that differ from those usually found in human antibodies.
- the binding proteins of the present invention may be "multi-specific” or "bispecific” antibodies.
- a multispecific or bispecific antibody is an antibody derivative which prevents or reduces binding of both IL-7 and TSLP to their receptors, the antibody having binding specificities for at least two proteins selected from IL-7, TSLP, CD127, IL7R gamma chain or CRLF2, also forms part of the invention.
- the binding protein of the invention may also have binding specificity for IL-23, which is expressed on the cell surface of T H 17 cells, for example, the binding protein may have specificity for both IL-23R (or IL-23) and CD127, or lL-2R (or lL-23) and IL-7.
- the CH 1 region containing the site necessary for light chain binding present in at least one of the fusions.
- DNA encoding these fusions, and if desired the L chain are inserted into separate expression vectors and are then cotransfected into a suitable host organism. It is possible though to insert the coding sequences for two or all three chains into one expression vector.
- the bispecific antibody is composed of an H chain with a first binding specificity in one arm and an H-L chain pair, providing a second binding specificity in the other arm, see WO94/04690. See also Suresh et al Methods in Enzymology 121 , 210, 1986.
- bispecific antibody or bispecific fragment such as described supra wherein a first specificity is towards an epitope of IL-7 and a second specificity towards TSLP.
- Another potential approach is a is to produce a bispecific antibody or bispecific fragment such as described supra wherein a first specificity is towards an epitope of IL-7 and a second specificity towards IL-6.
- the binding proteins of the present invention may be "antibody fragments".
- a therapeutic antibody which is an antigen binding fragment.
- Such fragments may be functional antigen binding fragments of intact and/or humanised and/or chimaeric antibodies such as Fab, Fd, Fab', F(ab') 2 , Fv, ScFv fragments of the antibodies described supra.
- the fragments may also be human, camellid or shark or other species, single variable domain antibodies or larger constructs comprising them. Fragments lacking the constant region lack the ability to activate complement by the classical pathway or to mediate antibody-dependent cellular cytotoxicity.
- fragments are produced by the proteolytic digestion of intact antibodies by, e.g., papain digestion (see for example, WO 94/29348) but may be produced directly from recombinantly transformed host cells.
- papain digestion see for example, WO 94/29348
- ScFv see Bird et al ; (1988) Science, 242, 423-426.
- antibody fragments may be produced using a variety of engineering techniques as described below.
- Fv fragments appear to have lower interaction energy of their two chains than Fab fragments.
- V H and V L domains have been linked with peptides (Bird et al, (1988) Science 242, 423-426, Huston et al, PNAS, 85, 5879- 5883), disulphide bridges (Glockshuber et al, (1990) Biochemistry, 29, 1362-1367) and "knob in hole” mutations (Zhu et a/ (1997), Protein ScL, 6, 781-788).
- ScFv fragments can be produced by methods well known to those skilled in the art see Whitlow et al (1991 ) Methods companion Methods Enzymol, 2, 97-105 and Huston et al (1993)
- ScFv may be produced in bacterial cells such as E.Coli but are more typically produced in eukaryotic cells.
- One disadvantage of ScFv is the monovalency of the product, which precludes an increased avidity due to polyvalent binding, and their short half-life.
- Attempts to overcome these problems include bivalent (ScFv') 2 produced from ScFV containing an additional C terminal cysteine by chemical coupling ( Adams et al (1993) Can. Res 53, 4026-4034 and McCartney et al (1995) Protein Eng. 8, 301-314) or by spontaneous site-specific dimerization of ScFv containing an unpaired C terminal cysteine residue (see Kipriyanov et al (1995) Cell.
- ScFv can be forced to form multimers by shortening the peptide linker to between 3 to 12 residues to form "diabodies", see Holliger et al PNAS (1993), 90, 6444-6448. Reducing the linker still further can result in ScFV trimers ("triabodies”, see Kortt et al (1997) Protein Eng, 10, 423-433) and tetramers ("tetrabodies", see Le Gall et al (1999) FEBS Lett, 453, 164-168).
- ScFv-Sc-Fv tandems (ScFV) 2 ) may also be produced by linking two ScFv units by a third peptide linker, see Kurucz et al (1995) J.lmmol.154, 4576-4582.
- Bispecific diabodies can be produced through the noncovalent association of two single chain fusion products consisting of V H domain from one antibody connected by a short linker to the V L domain of another antibody, see Kipriyanov et al (1998), Int. J. Can 77,763-772.
- the stability of such bispecific diabodies can be enhanced by the introduction of disulphide bridges or "knob in hole” mutations as described supra or by the formation of single chain diabodies (ScDb) wherein two hybrid ScFv fragments are connected through a peptide linker see Kontermann et al (1999) J. Immunol. Methods 226 179-188.
- Tetravalent bispecific molecules are available by e.g.
- Smaller tetravalent bispecific molecules can also be formed by the dimerization of either ScFv-ScFv tandems with a linker containing a helix- loop-helix motif (DiBi miniantibodies, see Muller et al (1998) FEBS Lett 432, 45-49) or a single chain molecule comprising four antibody variable domains (V H and V L ) in an orientation preventing intramolecular pairing (tandem diabody, see Kipriyanov et al, (1999) J. MoI. Biol. 293, 41-56).
- Bispecific F(ab')2 fragments can be created by chemical coupling of Fab' fragments or by heterodimerization through leucine zippers (see Shalaby et al, (1992) J.Exp.Med. 175, 217-225 and Kostelny et al (1992), J.Immunol. 148, 1547- 1553). Also available are isolated V H and V L domains, see US 6, 248,516; US 6,291 ,158; US 6, 172,197.
- the binding proteins of the present invention may comprise other modifications to enhance or change their effector functions.
- the interaction between the Fc region of an antibody and various Fc receptors (Fc ⁇ R) is believed to mediate the effector functions of the antibody which include antibody-dependent cellular cytotoxicity (ADCC), fixation of complement, phagocytosis and half-life/clearance of the antibody.
- ADCC antibody-dependent cellular cytotoxicity
- phagocytosis phagocytosis and half-life/clearance of the antibody.
- Various modifications to the Fc region of antibodies of the invention may be carried out depending on the desired effector property.
- human constant regions which essentially lack the functions of a) activation of complement by the classical pathway; and b) mediating antibody-dependent cellular cytotoxicity include the lgG4 constant region, the lgG2 constant region and IgGI constant regions containing specific mutations as for example mutations at positions 234, 235, 236, 237, 297, 318, 320 and/or 322 disclosed in EP0307434 (WO8807089), EP 0629 240 (WO9317105) and WO 2004/014953.
- Human Fc ⁇ receptors include Fc ⁇ R (I), Fc ⁇ Rlla, Fc ⁇ Rllb, Fc ⁇ Rllla and neonatal FcRn. Shields et al, (2001 ) J.Biol.Chem 276, 6591-6604 demonstrated that a common set of IgGI residues is involved in binding all Fc ⁇ Rs, while Fc ⁇ RII and Fc ⁇ RIII utilize distinct sites outside of this common set.
- One group of IgGI residues reduced binding to all Fc ⁇ Rs when altered to alanine: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain and clustered near the hinge joining CH1 and CH2.
- Fc ⁇ RI utilizes only the common set of IgGI residues for binding
- Fc ⁇ RII and Fc ⁇ RIII interact with distinct residues in addition to the common set.
- Alteration of some residues reduced binding only to Fc ⁇ RII (e.g. Arg-292) or Fc ⁇ RIII (e.g. Glu-293).
- Some variants showed improved binding to Fc ⁇ RII or Fc ⁇ RIII but did not affect binding to the other receptor (e.g., Ser-267Ala improved binding to Fc ⁇ RII but binding to Fc ⁇ RIII was unaffected).
- Other variants exhibited improved binding to Fc ⁇ RII or Fc ⁇ RIII with reduction in binding to the other receptor (e.g.
- Ser-298Ala improved binding to Fc ⁇ RIII and reduced binding to Fc ⁇ RII).
- Fc ⁇ Rllla the best binding IgGI variants had combined alanine substitutions at Ser-298, Glu-333 and Lys-334.
- the neonatal FcRn receptor is believed to be involved in protecting IgG molecules from degradation and thus enhancing serum half life and the transcytosis across tissues (see Junghans R. P (1997) Immunol. Res 16. 29-57 and Ghetie et al (2000) Annu.Rev.lmmunol. 18, 739-766).
- Human IgGI residues determined to interact directly with human FcRn include Ne253, Ser254, Lys288, Thr307, Gln311 , Asn434 and His435.
- the therapeutic antibody of the invention may incorporate any of the above constant region modifications.
- the therapeutic antibody essentially lacks the functions of a) activation of complement by the classical pathway; and b) mediating antibody-dependent cellular cytotoxicity.
- the present invention provides therapeutic antibodies of the invention having any one (or more) of the residue changes detailed above to modify half-life/clearance and/or effector functions such as ADCC and/or complement dependent cytotoxicity and/or complement lysis.
- the therapeutic antibody has a constant region of isotype human IgGI with alanine (or other disrupting) substitutions at positions 235 (e.g., L235A) and 237 (e.g., G237A) (numbering according to the EU scheme outlined in Kabat).
- Other derivatives of the invention include glycosylation variants of the antibodies of the invention. Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly effector functioning, such as those described above, see for example, Boyd et al (1996), MoI. Immunol. 32, 131 1-1318. Glycosylation variants of the therapeutic antibodies of the present invention wherein one or more carbohydrate moiety is added, substituted, deleted or modified are contemplated.
- Analogs In this context of the present invention, also provided are analogs of the antibodies described.
- the invention provides analogs of the R34.34, GR34, 9B7, 6A3, 1A11 or 6C5 CDRs (R34.34 analogs, GR34 analogs, 9B7 analogs, 6A3 analogs, 1A11 analogs or 6C5 analogs).
- Analogs of a parental antibody e.g. 6A3 or 9B7 will have the same or similar functional properties to those containing the CDRs of the parental antibody, respectively, in that the 9B7 analog antibodies or 6A3 analog antibodies bind to the same target protein or epitope with the same or similar binding affinity.
- the analogs may comprise one or more amino acid substitutions within each or all of its CDRs, and in one embodiment at least 75% or 80% of the amino acid residues in the CDRs of the parental antibody are unaltered, in another embodiment at least 90% of the CDRs are unaltered, and in another embodiment at least 95% of the amino acid residues in the CDRs are unaltered.
- the CDR H3 of the parental antibody is unaltered in its entirety, whilst the other CDRs may be the same as the corresponding parental antibody CDRs or may be analogs thereof.
- the binding proteins of the present invention may be produced by methods known to the man skilled in the art.
- Antibodies of the present invention may be produced in transgenic organisms such as goats (see Pollock et al (1999), J. Immunol. Methods 231 :147-157), chickens (see Morrow KJJ (2000) Genet.Eng.News 20:1-55), mice (see Pollock et al ibid) or plants (see Doran PM, (2000) Curr.Opinion Biotechnol. 1 1 , 199-204, Ma JK-C (1998), Nat.Med. 4; 601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000) Plant MoI. Biol.
- Antibodies may also be produced by chemical synthesis. However, antibodies of the invention are typically produced using recombinant cell culturing technology well known to those skilled in the art.
- a polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for further propagation or expression in a host cell.
- a replicable vector such as a plasmid for further propagation or expression in a host cell.
- One useful expression system is a glutamate synthetase system (such as sold by Lonza Biologies), particularly where the host cell is CHO or NSO (see below).
- Polynucleotide encoding the antibody is readily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes).
- Vectors that may be used include plasmid, virus, phage, transposons, minichromsomes of which plasmids are a typical embodiment.
- such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription termination sequences operably linked to the light and/or heavy chain polynucleotide so as to facilitate expression.
- Polynucleotide encoding the light and heavy chains may be inserted into separate vectors and introduced (e.g. by transformation, transfection, electroporation or transduction) into the same host cell concurrently or sequentially or, if desired both the heavy chain and light chain can be inserted into the same vector prior to such introduction.
- Antibodies of the present invention maybe produced as a fusion protein with a heterologous signal sequence having a specific cleavage site at the N terminus of the mature protein.
- the signal sequence should be recognised and processed by the host cell.
- the signal sequence may be an alkaline phosphatase, penicillinase, or heat stable enterotoxin Il leaders.
- yeast secretion the signal sequences may be a yeast invertase leader, ⁇ factor leader or acid phosphatase leaders see e.g. WO90/13646.
- viral secretory leaders such as herpes simplex gD signal and native immunoglobulin signal sequences (such as human Ig heavy chain) are available.
- the signal sequence is ligated in reading frame to polynucleotide encoding the antibody of the invention.
- Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins e.g. ampicillin, neomycin, methotrexate or tetracycline or (b) complement auxotrophic deficiencies or supply nutrients not available in the complex media or (c) combinations of both.
- the selection scheme may involve arresting growth of the host cells that contain no vector or vectors. Cells, which have been successfully transformed with the genes encoding the therapeutic antibody of the present invention, survive due to e.g. drug resistance conferred by the co-delivered selection marker.
- One example is the DHFR-selection system wherein transformants are generated in DHFR negative host strains (eg see Page and Sydenham 1991 Biotechnology 9: 64-68).
- the DHFR gene is co-delivered with antibody polynucleotide sequences of the invention and DHFR positive cells then selected by nucleoside withdrawal.
- the DHFR inhibitor methotrexate is also employed to select for transformants with DHFR gene amplification.
- DHFR gene amplification results in concomitant amplification of the desired antibody sequences of interest.
- CHO cells are a particularly useful cell line for this DHFR/methotrexate selection and methods of amplifying and selecting host cells using the DHFR system are well established in the art see Kaufman RJ. et al J. MoI. Biol.
- Suitable promoters for expressing antibodies of the invention are operably linked to DNA/polynucleotide encoding the antibody.
- Promoters for prokaryotic hosts include phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan and hybrid promoters such as Tac.
- Promoters suitable for expression in yeast cells include 3-phosphoglycerate kinase or other glycolytic enzymes e.g.
- Inducible yeast promoters include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein and enzymes responsible for nitrogen metabolism or maltose/galactose utilization.
- Promoters for expression in mammalian cell systems include RNA polymerase Il promoters including viral promoters such as polyoma, fowlpox and adenoviruses (e.g. adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular the immediate early gene promoter), retrovirus, hepatitis B virus, actin, rous sarcoma virus (RSV) promoter and the early or late Simian virus 40 and non-viral promoters such as EF-1 alpha (Mizushima and Nagata Nucleic Acids Res 1990 18(17):5322.
- the choice of promoter may be based upon suitable compatibility with the host cell used for expression.
- enhancer elements can be included instead of or as well as those found located in the promoters described above.
- suitable mammalian enhancer sequences include enhancer elements from globin, elastase, albumin, fetoprotein, metallothionine and insulin.
- an enhancer element from a eukaroytic cell virus such as SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculoviral enhancer or murine lgG2a locus (see WO04/009823).
- enhancers are typically located on the vector at a site upstream to the promoter, they can also be located elsewhere e.g. within the untranslated region or downstream of the polyadenylation signal.
- the choice and positioning of enhancer may be based upon suitable compatibility with the host cell used for expression.
- polyadenylation signals are operably linked to polynucleotide encoding the antibody of this invention. Such signals are typically placed 3' of the open reading frame.
- signals include those derived from growth hormones, elongation factor-1 alpha and viral (eg SV40) genes or retroviral long terminal repeats.
- yeast systems non-limiting examples of polydenylation/termination signals include those derived from the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH) genes.
- PGK phosphoglycerate kinase
- ADH alcohol dehydrogenase 1
- polyadenylation signals are typically not required and it is instead usual to employ shorter and more defined terminator sequences. The choice of polyadenylation/termination sequences may be based upon suitable compatibility with the host cell used for expression ⁇
- codon usage of the antibody of this invention thereof can be modified to accommodate codon bias of the host cell such to augment transcript and/or product yield (eg Hoekema A et al MoI Cell Biol 1987 7(8):2914-24).
- the choice of codons may be based upon suitable compatibility with the host cell used for expression.
- Suitable host cells for cloning or expressing vectors encoding antibodies of the invention are prokaroytic, yeast or higher eukaryotic cells.
- Suitable prokaryotic cells include eubacteria e.g. enterobacteriaceae such as Escherichia e.g. E.Coli (for example ATCC 31 ,446; 31 ,537; 27,325), Enterobacter, Erwinia, Klebsiella Proteus, Salmonella e.g. Salmonella typhimurium, Serratia e.g.
- Serratia marcescans and Shigella as well as Bacilli such as B.subtilis and B.licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa and Streptomyces.
- yeast host cells Saccharomyces cerevisiae, schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424; 24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris (EP183, 070, see also Peng et al J.Biotechnol.
- host cells of the present invention are vertebrate cells. Suitable vertebrate host cells include mammalian cells such as COS-1 (ATCC No. CRL 1650) COS-7 (ATCC CRL 1651 ), human embryonic kidney line 293, , PerC6 (Crucell), baby hamster kidney cells (BHK) (ATCC CRL.1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO.
- CRL 1573 Chinese hamster ovary cells CHO (e.g. CHO-K1 , ATCC NO: CCL 61 , DHFR minus CHO cell line such as DG44 (Urlaub et al, Somat Cell MoI Genet (1986) VoI 12 pp555-566), particularly those CHO cell lines adapted for suspension culture, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells (ATCC CRL-1587), HELA cells, canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells e.g. NSO (see US 5,807,715), Sp2/0, YO.
- CHO-K1 ATCC NO: CCL 61
- DHFR minus CHO cell line such as DG44 (Urlaub et al, Somat Cell MoI Genet (1986) VoI 12 pp555-566)
- DG44 Urlaub et al, So
- a stably transformed host cell comprising a vector encoding a heavy chain and/or light chain of the therapeutic antibody as described herein.
- host cells comprise a first vector encoding the light chain and a second vector encoding said heavy chain.
- Such host cells may also be further engineered or adapted to modify quality, function and/or yield of the antibody of this invention.
- Non-limiting examples include expression of specific modifying (eg glycosylation) enzymes and protein folding chaperones.
- Host cells transformed with vectors encoding the therapeutic antibodies of the invention may be cultured by any method known to those skilled in the art.
- Host cells may be cultured in spinner flasks, shake flasks, roller bottles, wave reactors (eg System 1000 from wavebiotech.com) or hollow fibre systems but it is preferred for large scale production that stirred tank reactors or bag reactors (eg Wave Biotech, Somerset, New Jersey USA) are used particularly for suspension cultures.
- the stirred tankers are adapted for aeration using e.g. spargers, baffles or low shear impellers.
- For bubble columns and airlift reactors direct aeration with air or oxygen bubbles maybe used.
- the host cells are cultured in a serum free culture media this can be supplemented with a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration process.
- a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration process.
- either microcarriers maybe used as growth substrates for anchorage dependent cell lines or the cells maybe adapted to suspension culture (which is typical).
- the culturing of host cells, particularly vertebrate host cells may utilise a variety of operational modes such as batch, fed-batch, repeated batch processing (see Drapeau et al (1994) cytotechnology 15: 103-109), extended batch process or perfusion culture.
- recombinantly transformed mammalian host cells may be cultured in serum-containing media such media comprising fetal calf serum (FCS), it is preferred that such host cells are cultured in serum -free media such as disclosed in Keen et al (1995) Cytotechnology 17:153-163, or commercially available media such as ProCHO-CDM or UltraCHOTM (Cambrex NJ, USA), supplemented where necessary with an energy source such as glucose and synthetic growth factors such as recombinant insulin.
- FCS fetal calf serum
- serum -free media such as disclosed in Keen et al (1995) Cytotechnology 17:153-163, or commercially available media such as ProCHO-CDM or UltraCHOTM (Cambrex NJ, USA), supplemented where necessary with an energy source such as glucose and synthetic growth factors such as recombinant insulin.
- the serum-free culturing of host cells may require that those cells are adapted to grow in serum free conditions.
- One adaptation approach is to culture such host cells in serum containing media and repeatedly exchange 80% of the culture medium for the serum-free media so that the host cells learn to adapt in serum free conditions (see e.g. Scharfenberg K et al (1995) in Animal Cell technology: Developments towards the 21st century (Beuvery E. C. et al eds), pp619-623, Kluwer Academic publishers).
- Antibodies of the invention secreted into the media may be recovered and purified from the media using a variety of techniques to provide a degree of purification suitable for the intended use.
- the use of therapeutic antibodies of the invention for the treatment of human patients typically mandates at least 95% purity as determined by reducing SDS-PAGE, more typically 98% or 99% purity, when compared to the culture media comprising the therapeutic antibodies.
- cell debris from the culture media is typically removed using centrifugation followed by a clarification step of the supernatant using e.g. microfiltration, ultrafiltration and/or depth filtration.
- the antibody can be harvested by microfiltration, ultrafiltration or depth filtration without prior centrifugation.
- HA hydroxyapatite
- affinity chromatography optionally involving an affinity tagging system such as polyhistidine
- hydrophobic interaction chromatography HIC, see US 5, 429,746
- the antibodies of the invention following various clarification steps, are captured using Protein A or G affinity chromatography followed by further chromatography steps such as ion exchange and/or HA chromatography, anion or cation exchange, size exclusion chromatography and ammonium sulphate precipitation.
- virus removal steps are also employed (e.g. nanofiltration using e.g. a DV-20 filter).
- a purified (typically monoclonal) preparation comprising at least 10mg/ml or greater e.g. 100mg/ml or greater of the antibody of the invention is provided and therefore forms an embodiment of the invention. Concentration to 100mg/ml or greater can be generated by ultracentrifugation. Suitably such preparations are substantially free of aggregated forms of antibodies of the invention.
- Bacterial systems are particularly suited for the expression of antibody fragments.
- Insoluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art, see Sanchez et al (1999) J.Biotechnol. 72, 13- 20 and Cupit PM et al (1999) Lett Appl Microbiol, 29, 273-277.
- compositions for use in the treatment of human diseases and disorders may be incorporated into pharmaceutical compositions for use in the treatment of human diseases and disorders such as those outlined above.
- compositions further comprise a pharmaceutically acceptable (i.e. inert) carrier as known and called for by acceptable pharmaceutical practice, see e.g. Remingtons Pharmaceutical Sciences, 16th ed, (1980), Mack Publishing Co.
- pharmaceutically acceptable carriers include sterilised carrier such as saline, Ringers solution or dextrose solution, buffered with suitable buffers such as sodium acetate trihydrate to a pharmaceutically acceptable pH, such as a pH within a range of 5 to 8.
- Pharmaceutical compositions for injection e.g.
- intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal) or continuous infusion are suitably free of visible particulate matter and may comprise from 1 mg to 10g of therapeutic antibody, typically 5mg to 1g, more specifically 5mg to 25mg or 50mg of antibody.
- Methods for the preparation of such pharmaceutical compositions are well known to those skilled in the art.
- pharmaceutical compositions comprise from 1 mg to 10g of therapeutic antibodies of the invention in unit dosage form, optionally together with instructions for use.
- Pharmaceutical compositions of the invention may be lyophilised (freeze dried) for reconstitution prior to administration according to methods well known or apparent to those skilled in the art.
- a chelator of metal ions including copper such as citrate (e.g. sodium citrate) or EDTA or histidine, may be added to the pharmaceutical composition to reduce the degree of metal-mediated degradation of antibodies of this isotype, see EP0612251.
- Pharmaceutical compositions may also comprise a solubiliser such as arginine base, a detergent/anti-aggregation agent such as polysorbate 80, and an inert gas such as nitrogen to replace vial headspace oxygen.
- Effective doses and treatment regimes for administering the antibody of the invention are generally determined empirically and are dependent on factors such as the age, weight and health status of the patient and disease or disorder to be treated. Such factors are within the purview of the attending physican. Guidance in selecting appropriate doses may be found in e.g. Smith et al (1977) Antibodies in human diagnosis and therapy, Raven Press, New York.
- the antagonists of the present invention may be used in the therapy of multiple sclerosis and in other autoimmune or inflammatory diseases, particularly those in which pathogenic T H 17 cells are implicated. Such diseases are associated with high levels of IL-17 expression. Elevated levels of IL-17 have been reported in serum and CSF of MS patients (Matusevicius, D. et al.; Mult. Scler. 5, 101-104; 1999) and in the synovial fluid obtained from rheumatoid arthritis patients. IL-17 has also been implicated in psoriasis (Homey et al.; J. Immunol.
- Inhibition of IL-7 receptor mediated signalling may also be useful in the treatment of inflammatory (non-autoimmune) diseases in which elevated IL-17 has been implicated, such as asthma.
- inflammatory and/or autoimmune diseases of the invention include inflammatory skin diseases including psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma, hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute-lung injury; Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheuma
- the antagonists of the present invention may be useful in the therapy of multiple sclerosis, in all its forms, including neuromyelitis optica.
- Treatment with an antagonist of the present invention is predicted to be most efficacious when administered in the context of active inflammatory disease, i.e. when used in the treatment of clinically isolated syndrome or relapsing forms of MS.
- These stages of disease can be defined clinically and/or by imaging criteria such as gadolinium enhancement or other more sensitive techniques, and/or other as yet undefined biomarkers of active disease.
- the antagonists of the invention can be used to treat RRMS (via intravenous, sub-cutaneous, oral or intramuscular delivery) when the patients are entering or are in relapse.
- the antagonist of the invention is administered to the patient at the onset of relapse, or within 1 hr, 2hrs, 3hrs, 6hrs, 12hrs, 24hrs, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days from the onset of relapse.
- biomarkers such as CD127 expression and intracellular cytokine staining (e.g. IL-17 staining) provides criteria for applying the therapeutic anti-CD127 binding protein.
- a subgroup of MS patients with increased T H 17 in their CD4+ T cells are primary candidates for the treatment.
- the methods of treatment are methods of the present invention are methods to treat those patients that express high level of CD127 on their T cells, making them susceptible to anti-CD127 treatment.
- Treatment with anti-CD127 may likely shorten the time of relapse and quicken the attenuation of the clinical activities measurable by EDSS or MRI. Once the patients enter remission, the treatment may be stopped to avoid complications such as the inhibition of normal T cell development and homeostasis.
- the use of the anti-CD127 antibody may also prolong the period between relapses and improve patients' quality of life. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used and understood by those of ordinary skill in the art.
- Example 1 Characterization of monoclonal antibodies that bind to mouse CD127
- splenocytes were prepared from C57B/6 mouse spleens by a standard protocol; CD4 + T cells were then purified from the splenocytes using a Miltenyi magnetic isolation kit (Cat# 130-049- 201 ); one million CD4 + T cells per ml were first incubated with the indicated antibodies and concentrations as shown in the figure below for 30 min.
- the antibodies used were BD Biosciences control rat lgG2a (#553926), BD Biosciences anti-CD127 (Clone SB/14, #550426), eBiosciences anti-CD127 (Clone:A7R34, #16-1271 ), Abeam anti- CD127 (Clone SB199, #ab36428), R&D anti-CD127 (MAB7471 and 7472) ; cells were then either untreated or treated with 1 ng/ml mouse IL-7 for 60 min. at 37°C; cells were collected and immediately put on ice after the IL-7 treatment; cells were then washed with ice-cold PBS once and fixed in 1 % paraformaldehyde for 10 min.
- SB/14 was also tested for its inhibition of IL-7-driven expansion of differentiated T H 17 in vitro.
- Experimental autoimmune encephalomyelitis EAE was induced in mice by immunization of myelin oligodendrocyte glycoprotein (MOG) as described in Example 3 below.
- CD4+ T cells were harvested from the spleens or lymph nodes of EAE mice and were cultured in vitro in the absence or presence of IL-7 for 3 days. As shown in Figure 11 C, IL-7 promoted the expansion of T H 17 cells, detectable by IL-17 intracellular staining.
- Antibody SB/14 against mouse IL-7Ra but not a control IgG inhibited the IL-7-driven expansion of Th17 cells.
- the antibodies were also tested for the inhibition of TSLP-mediated pStat ⁇ in mouse thymocytes.
- CD4- cells in thymocytes expressed functional TSLP receptors and were gated in the FACS analysis.
- IL-7-induced and TSLP- induced pStat ⁇ was inhibited by SB/14 (BD) and A7R34 (eBio). Therefore, antibodies against mouse CD127 (SB/14 and A7R34) inhibited both IL-7- mediated and TSLP- mediated signalling.
- Random peptide libraries displayed on filamentous bacteriophage M13 has been used as a tool to map the epitopes of monoclonal antibodies (Scott and Smith, 1990, searching for peptide ligands with an epitope library, Science, 249:386-390).
- Enriched phage displayed peptide consensus sequences or mimotopes from identified phage peptides were employed to predict possible epitopes of mouse antibody (phage peptide mimotopes: antibody interaction site on the surface of the antigen mimicked by phage peptide or a mimic of an epitope) (Geysen et al., 1986, a priori delineation of a peptide which mimics a discontinuous antigenic determinant. MoI. Immunol., 23:709-715;
- Random peptide libraries 1. Ph.D-12 phage displayed random peptide library (from New England Biolabs Inc., #E8110S) 2. fGWXIO phage displayed random peptide library (GSK in house library)
- the biopanning procedure using fGWXIO phage displayed random peptide library The in house phage library, fGWXIO, displaying 10-mer random peptide sequences was constructed as described previously (Deng et al., 2004, Identification of peptides that inhibit the DNA binding, frans-activator, and DNA replication functions of the human papillomavirus type 1 1 E2 protein, J. Virol., 78: 2637-2641 ). Briefly,
- anti-mouse CD127 antibodies were assessed using a Biacore T100 system (GE Healthcare). Briefly, anti-mouse CD127 antibodies was immobilized on a CM5 biosensor chip with a final level of -100 RU (response units) using the standard amine coupling kit and procedure. HBS-EP buffer pH 7.4 (consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20) was used as running buffer. Sensograms were run against a reference cell that was activated/deactivated using EDC/NHS/ethanol amine.
- Table 1 shows epitope regions of mouse CD127 (NP_032398) for two mouse antibodies, BD Biosciences Clone SB/14 and eBiosciences Clone A7R34 that identified by one or more of the methods phage peptide library, peptide ELISA and Biacore Table 1 : Summary of epitope studies for anti-murine CD127 antibodies SB/14 and A7R34
- Monoclonal antibodies were produced by hybridoma cells generally in accordance with the method set forth in E Harlow and D Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
- Antigen used to generate hybridomas including 9B7 and 6C5 was a dimeric recombinant human CD127 extracellular domain (ECD)-Fc (R&D Systems #306-IR), comprising amino acid 21-262 of human CD127 (SEQ ID No:1 ).
- Antigen used to generate hybridomas including 6A3 and 1 A11 was a construct containing the full ECD of CD127 (amino acids 21-219 of SEQ ID NO:1 ).
- Balb/c mice were primed and boosted by intraperitoneal injection with Antigen in
- FCA or FIA Sigma-Aldrich, #F5881 , #F5506) (1 :1 ; vohvol).
- Spleens from responder animals were harvested and fused to SP/0 myeloma cells to generate hybridomas.
- Hybridomas of interest were monocloned using semi-solid media (methyl cellulose solution) and manually picked up into 96-well plate. The hybridoma supernatant material was screened for binding to CD127ECD using ELISA, CHO-CD127 transfected cell FACS, pStat ⁇ FACS and BIAcore T100 (results shown below).
- Selected purified mAbs (isolated from hybridoma supernatants 9B7, 6C5, 6A3 and 1A11 ) were tested for inhibition of IL-7 induced IFN- ⁇ and IL-17 in a T H 17 expansion assay.
- commercially available anti-hCD127 R34.34 was shown to inhibit IL-7 induced IFN- ⁇ and IL-17 in a T H 17 expansion assay and was also selected for further analysis.
- Mock transfected CHO or CHO-CD127 cells (2x10 6 cells/ml) were stained with hybridoma supernatants or purified antibodies at 1 ⁇ g/ml for 1 hour with 4% FCS in PBS (FACS buffer). Cells were also stained in a suitable negative control mouse antibody and anti-human CD127 positive control (R34.34 Dendritics Inc. #DDX0700). Cells were washed in FACS buffer and then stained with an anti-mouse IgG ALEXA488 secondary antibody 1 :2000 (Invitrogen Inc. #13-A1 1017). After washing in FACS buffer, cells were analysed in LSR Il (BD Biosciences Inc.). Results for the 9B7 antibody are shown in Figure 3.
- CD127 hybridoma culture medium, positive control antibody (R34.34, Dendritics Inc) at
- T H 17 cells in a population of normal human CD4+ T cells are stimulated to expand for three days. These T H 17 cells are then activated by PMA and ionomycin to stimulate the production of IL-17. Blocking the interaction between the IL-7 and CD127 by a functional anti-CD127 antibody in the three day incubation period should prevent the expansion of the T H 17 cells leading to the reduction of IL-17 production.
- CD4+ T cells were isolated from human peripheral blood mononuclear cells using a commercial kit (CD4+ T Cell Isolation Kit II, # 130-091-155, Miltenyi Biotec). CD4+ T cells were resuspended in RPMI medium with 10% FCS at a concentration of 1.5x10E6/ml. Cells were pre-incubated with control or anti-IL-7R ⁇ antibodies for 30 min. Cells were then cultured with or without 10 ng/ml of IL-7 for 72 h at 37 C. At the end of the incubation, cells were stimulated with 50ng/ml PMA and 1 ug/ml of Ionomycin for 5 h.
- Approximately 1x10 6 VmI of the CD4+ cells in 100 ⁇ l were mixed with equal volume of 2x Th17 differentiation medium (2 ⁇ g/ml anti-CD28 + 10 ⁇ g/ml anti-IFN- ⁇ + 10 ⁇ g/ml anti-IL-4 + 12.5ng/ml IL-1 ⁇ + 20ng/ml IL-23 + 50ng/ml IL-6) and cultured in 37°C with 5% CCv for 5 days.
- Treatment by the various cytokines and growth factors in the T H 17 medium preferentially differentiated the CD4+ cells into T H 17 cells.
- CCR6+ cells from the differentiated cultured cells at day 5 were sorted using BD FACS SORP Aria II. The CCR6+ cells were then adjusted to 2 x10 6 VmI for the IL-17 production assay.
- IL-17 and IFN- ⁇ level 10O ⁇ l of CCR6+ cells were pre-incubated with testing antibody for 1 h at 37°C, and then mixed with 10O ⁇ l of 10ng/ml IL-7. The cells were cultured for 24-40 hours in 37°C with supplement of 5% CO . IFN- ⁇ and IL-17 levels in 100 ul of culture supernatant were measured by FlowCytomix (Bender MedSystems) at 24 h and 40 h, respectively.
- the binding kinetics of anti-CD127 antibodies for human CD127 was assessed using a Biacore T100 system (GE Healthcare). Briefly, recombinant human CD127 ECD was immobilized on a CM5 biosensor chip with a final level of -100 RU (response units) using the standard amine coupling kit and procedure. HBS-EP buffer pH 7.4 (consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20) was used as running buffer. Sensograms were run against a reference cell that was activated/deactivated using EDC/NHS/ethanol amine.
- Analytes (anti-CD127 antibodies) were injected at various concentrations for 120s at a flow rate of 30uL/min.
- the antigen surfaces were regenerated with 1OmM Glycine-HCI, pH2.5. Kd values were calculated using the Biacore evaluation software package. The runs were carried out at 25°C.
- the isotype of 9B7 was determined to be IgGI with a kappa light chain constant region.
- the following assay was used to evaluate the binding kinetics of anti-CD127 antibodies 6C5, 6A3, 1A11 and GR34.
- Antibody kinetics were assessed using a Biacore T100 system (GE Healthcare) with a reaction temperature of 25 0 C.
- Rabbit anti-mouse IgG antibody was immobilized on a CM5 biosensor chip with a final level of -10000 RU (response units) using the standard amine coupling kit and procedure.
- HBS-EP buffer pH 7.4 Consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20 was used as running buffer. Sensograms were run against a reference cell that was blank immobilized using EDC/NHS/ethanol amine. For ligand capturing, 25nM of 6C5 was injected over the chip surface for 30 s at 10 ⁇ l_/min. Analytes (recombinant human CD127 ECD) were then injected at various concentrations for 500 s at 30 ⁇ L/min. The sensor chip surfaces were regenerated with 10 mM Glycine-HCI, pH 1.7. Kd values were calculated using the Biacore evaluation software package.
- Antibody 9B7 was found to bind tightly to CD127 with a dissociation constant of 556 pM. It was also capable of partially blocking the binding of IL-7 to CD127, correlating to the partial blocking of IL-7-induced STAT-5 phosphorylation in human CD4 cells ( Figure 4).
- Antibody 6C5 (mouse IgGI ) was determined to inhibit pSTAT5 signalling with an IC 50 Of 50 ⁇ g/ml.
- Antibody 6A3 (mouse IgGI ) was determined to inhibit pSTAT5 signalling with an IC 50 Of 50 ⁇ g/ml.
- Antibody 1A1 1 was determined to inhibit pSTAT5 signalling with an IC 50 of 0.088 ⁇ g/ml in the assay described herein. It had an affinity for the IL-7R ⁇ EDC of 3.44nM (KD) and a Kd of 2.5IxI O "4 . It was capable of binding to IL-7R ⁇ expressed on CHO with an EC 50 of 0.16 ⁇ g/ml, and blocked IL-7/IL-7R ⁇ with an IC 50 of 1.79 ⁇ g/ml. 1A11 was determined to bind to amino acids within CD127 epitope regions 2, 3, 4 and 5 (SEQ ID NOs:118-121 ).
- Antibody GR34 (mouse IgGI ) was determined to inhibit pSTAT5 signalling with an IC 50 of 0.22 ⁇ g/ml in the assay described herein. It had an affinity for the IL-7R ⁇ EDC of 15.3nM (KD) and a Kd of 8.75X10 "4 . It was capable of binding to IL-7R ⁇ expressed on CHO with an EC 50 of 0.27 ⁇ g/ml, and blocked IL-7/IL-7R ⁇ with an IC 50 of 2.29 ⁇ g/ml. GR34 was determined to bind to amino acids within CD127 epitope regions 2, 3, 4 and 5 (SEQ ID NOs:118-121 ).
- R.3434 (Dendritics) was determined to inhibit pSTAT5 signalling with an IC 50 of 0.67 ⁇ g/ml in the assay described herein. It had an affinity for the IL-7R ⁇ EDC of 7.74nM (KD) and a Kd of 1.46x1 ⁇ It was capable of binding to IL-7R ⁇ expressed on CHO with an EC 50 of 0.01 ⁇ g/ml, and blocked IL-7/IL-7R ⁇ with an IC 50 of 1.38 ⁇ g/ml. R.3434 was determined to bind to amino acids within CD127 epitope regions 2, 3, 4 and 5 (SEQ ID NOs:118-121 ).
- the purified RT-PCR fragments were cloned into pMD18-T vector (Takara) and a consensus sequence was obtained for each hybridoma by sequence alignment, database searching and alignment with known immunoglobulin variable sequences listed in KABAT (Kabat, E.A., Wu, T.T., Perry, H. H., Gottesman, K.S., Foeller, C, 1991. Sequences of proteins of Immunological Interest, 5 th edition, US Department of Health and Human Services, Public Health Service, NIH).
- Rearranged VH of mAb 9B7 used a V segment of the Igh-VQ52 VH2 family.
- CDR1 sequence RYNVH (SEQ ID NO:4)
- FR2 sequence WVRQPPGKGLEWLG (SEQ ID NO:13)
- CDR2 sequence MIWDGGSTDYNSALKS (SEQ ID NO:5)
- FR3 sequence RLSITKDNSKSQVFLKMNSLQTDDTAMYYCAR (SEQ ID NO:14)
- CDR3 sequence NRYESG (SEQ ID NO:6)
- FR4 sequence MDYWGQGTTVTVSS (SEQ ID NO: 15)
- Rearranged Vk of mAb 9B7 used a V segment of the IGKV8 family DIVMTQTPSSLTVTAGEKVTMSCKSSQSLLNSGNRKNYLTWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFTLIISSVQAEDLAVYYCQNDYTYPFTFGSGTKLEIKR (SEQ ID NO:3)
- FR1 sequence DIVMTQTPSSLTVTAGEKVTMSC (SEQ ID NO:16)
- CDR1 sequence KSSQSLLNSGNRKNYLT (SEQ ID NO:7)
- CDR2 sequence WASTRES (SEQ ID NO:8)
- FR3 sequence GVPDRFTGSGSGTDFTLIISSVQAEDLAVYYC (SEQ ID NO:18)
- CDR3 sequence QNDYTYPFTFGS (SEQ ID NO:9)
- FR 4 sequence GTKLEIKR (SEQ ID NO:19) (CDR regions are in bold.
- Ig gene Immunoglobulin gene.
- VH Antibody heavy chain variable region.
- VL Antibody light chain variable region.
- FR Framework region.
- CDR Complementarity determining region
- the 6C5 antibody was determined to have the following heavy and light chain variable regions (the CDRs of 6C5, according to Kabat, are shown in bold):
- FR1 sequence EVKLLESGGGLVQPGGSLKLSCAASGFAFS (SEQ ID NO:37)
- CRDH1 sequence AYWMS (SEQ ID NO:31)
- CDRH2 sequence EINPDSSTINCTPSLKD (SEQ ID NO:32)
- FR3 sequence KFIISRDNAKNTLSLQMNKVRSEDTALYYCAR (SEQ ID NO:39)
- CDRH3 sequence RLRPFWYFDVW (SEQ ID NO:33)
- FR4 sequence GAGTTVTVSS (SEQ ID NO:40)
- CDRL1 sequence RSSQSIVQSNGNTYLE (SEQ ID NO:34)
- FR3 sequence GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO:43)
- CDRL3 sequence FQGSHVPRT (SEQ ID NO:36)
- the 6A3 antibody was determined to have the following heavy and light chain variable regions (the CDRs of 6A3, according to Kabat, are shown in bold):
- CRDH 1 sequence TDYAWN (SEQ ID NO:53)
- FR2 sequence WIRQFPGNKLEWMG (SEQ ID NO:60)
- CDRH2 sequence YIFYSGSTTYTPSLKS (SEQ ID NO:54)
- CDRL1 sequence LASQTIGAWLA (SEQ ID NO:56)
- FR2 sequence WYQQKPGKSPQLLIY (SEQ ID NO:64)
- CDRL2 sequence AATRLAD (SEQ ID NO:57)
- CDRL3 sequence QQFFSTPWT (SEQ ID NO:58)
- FR4 sequence FGGGTKLEIK (SEQ ID NO:66)
- the 1A1 1 antibody was determined to have the following heavy and light chain variable regions (the CDRs of 1 A11 , according to Kabat, are shown in bold):
- VSS (SEQ ID NO:71 )
- FR1 sequence EVQLQQSGPELLKPGASMKISCKASGYSFT (SEQ ID NO:79)
- CDRH1 sequence GYTMN (SEQ ID NO:73)
- FR2 sequence WVKQSHGKNLEWIG (SEQ ID NO:80)
- CDRH2 sequence LINPYNGVTSYNQKFK (SEQ ID NO:74)
- FR3 sequence GKATLTVAKSSSTAYMELLSLTSEDSAVYYCAR (SEQ ID NO:81 )
- CDRH3 sequence GDGNYWYF (SEQ ID NO:75)
- CDRL1 sequence SASSSVTYMHW (SEQ ID NO:76)
- FR2 sequence YQQKSGTSPKPWIY (SEQ ID NO:84)
- CDRL2 sequence EISKLAS (SEQ ID NO:77)
- CDRL3 sequence QEWNYPYTF (SEQ ID NO:78)
- FR4 sequence GGGTKLEIK (SEQ ID NO:86)
- R3434 is commercially available from Dendritics, Inc. In-house sequence analysis of in-gel digested protein involved N-terminal sequence analysis using Edman degradation on an ABI Procise 494 automated protein sequencer (Applied Biosystems, Foster City, Ca., USA), peptide mass fingerprinting and MALDI-LI FT-MS/MS sequencing on a Bruker Ultraflex III Maldi-TOF mass spectrometer and additional LC-ESI-MS/MS sequencing on a Bruker HCT+ ion-trap mass spectrometer (both from Bruker Daltonics, Bremen, Germany).
- the reverse engineered clone was named GR34, the sequence of which is below. Rearranged VH of mAb GR34
- CDR1 sequence GYTMN (SEQ ID NO:92)
- FR2 sequence WVKQSHGKNLEWIG (SEQ ID NO:99)
- CDR2 sequence LINPYSGITSYNQNFK (SEQ ID NO:93)
- CDR3 sequence GDGNYWYF (SEQ ID NO:94)
- FR4 sequence DVWGAGTTVTVSS (SEQ ID NO:101 )
- CDR2 sequence EISKLAS (SEQ ID NO:96)
- FR3 sequence GVPARFSGSGSGTSYSLTISSMEAEDAAIYYC (SEQ ID NO:104)
- CDR3 sequence QYWNYPYTF (SEQ ID NO:97)
- FR4 sequence GGGTKLEIK (SEQ ID NO:105)
- the epitope of the anti-hCD127 antibody 9B7 was determined by peptide ELISA as before (1.2). The results of this mapping are shown in Table 3. Table 4. shows three positive regions of hCD127 identified by peptide ELISA using clone 9B7
- anti-CD127 antibodies to 15 mers synthesized peptide of human CD127 was assessed using a Biacore T100 system (GE Healthcare). Briefly, anti-CD127 antibody was immobilized on a CM5 biosensor chip with a final level of -1000 RU (response units) using the standard amine coupling kit and procedure. HBS-EP buffer pH 7.4 (consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20) was used as running buffer. Sensograms were run against a reference cell that was activated/deactivated using EDC/NHS/ethanol amine. 1 ⁇ M peptides were injected for 120s at a flow rate of 10uL/min. Data were analyzed using the Biacore evaluation software package. The runs were carried out at 25°C.
- Table 5 shows positive region for 6C5 and R34.34 antibody identified by BIAcore
- phage display was carried out as before (Section 1.3) on antibodies 9B7, 6C5, R3434, 6A3 and 1 A1 1.
- Table 6 shows three positive regions of hCD127 identified by phage peptide library using clone 9B7
- Table 7 shows 6C5 epitope regions identified by phage peptide library
- Table 8 shows R34.34 epitope regions identified by phage peptide library
- Table 9 shows 6A3 epitope regions identified by phage peptide libraries
- these regions may be closely neighbouring regions within an important effector site of CD127, potentially involved in the site of IL-7 binding.
- Table 10 shows 1A11 epitope regions identified by phage peptide libraries
- these regions may be closely neighbouring regions within an important effector site of CD127, potentially involved in the site of IL-7 binding.
- the assay was carried out using a BIAcore T100 system (GE Healthcare) with a reaction temperature of 25 0 C.
- Recombinant human IL-7 was immobilized on a CM5 biosensor chip with a final level of -500 RU (response units) using the standard amine coupling kit and procedure.
- HBS-EP buffer pH 7.4 Consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20 was used as running buffer. Sensograms were run against a reference cell that was blank immobilized using EDC/NHS/ethanol amine.
- 10 ⁇ g/mL recombinant human CD127 ECD was mixed with various concentrations of anti-CD127 antibodies in separate vials and allowed to incubate for 30 min at 4 0 C. These mixtures, as well as 10 ⁇ g/mL recombinant human CD127 ECD alone, were then injected over the chip surface for 30s at 10 ⁇ L/min. After each injection the sensor chip surfaces were regenerated with 10 mM Glycine-HCI, pH 2.0. At 100 ug/ml antibody 6C5 completely inhibited CD127-ECD binding to IL-7 on the sensor chip. The results for 6C5 are shown in Figure 13.
- the assay was repeated for 6A3, using a Biacore T100 system (GE Healthcare) with a reaction temperature of 25 0 C.
- Recombinant human IL-7 was immobilized on a CM5 biosensor chip with a final level of -1000 RU (response units) using the standard amine coupling kit and procedure.
- HBS-EP buffer pH 7.4 Consisting of 10 mM HEPES, 0.15 M sodium chloride, 3 mM EDTA and 0.005% v/v surfactant P20 was used as running buffer. Sensograms were run against a reference cell that was blank immobilized using EDC/NHS/ethanol amine.
- CHO-CD127 cells were prepared and washed by cold Dulbecco's Phosphate- Buffered Saline (DPBS) for 3 times and 2 X 10 5 cells were then incubated with 2 ⁇ g/ml_ recombinant IL-7 in separated vials at 4 0 C for 30 min. After the incubation, anti-CD127 antibodies were added and the incubation continued for additional 30 min in 4% FCS in DPBS (FACS buffer). Afterward cells were washed in FACS buffer 3 times and stained with anti-mouse IgG ALEXA488 secondary antibody at 1 :2000 dilution (Invitrogen Inc. #13-A1 1017). The cells were then washed 3 times in in FACS buffer and were analyzed in LSR Il (BD Biosciences Inc.).
- DPBS cold Dulbecco's Phosphate- Buffered Saline
- CHO-CD127 cells were prepared and washed by cold DPBS for 3 times. Fluorescence labelled anti-CD127 antibody (BD Biosciences lnc #552853) was diluted in FACS buffer and mixed with various concentrations of non-labelled same antibody, or mixed with testing anti-CD127 antibodies, R34.34 and 6C5. The antibody mixtures were then incubated with the CHO-CD127 cells for 30 min at 4 0 C. After washing in FACS buffer for 3 times, binding of fluorescent labelled BD antibody is measured in LSR Il (BD Biosciences Inc.).
- Example 1 The potential for the murine antibodies described in Example 1 , to treat MS, was assessed in a mouse EAE model. This experiment has been repeated on multiple occasions; a single representative example is described below.
- mice Male C57BL/6 mice (6-8 wk; Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China) were immunized s.c. with a synthetic peptide (300 ⁇ g) of myelin oligodendrocyte glycoprotein (MOG residues 35-55). Immunization was performed by mixing MOG peptide in complete Freunds adjuvant (CFA, containing 5 mg/ml heat-killed H37Ra strain of Mycobacterium tuberculosis (Difco Laboratories)). Two hundred nanograms of pertussis toxin (List Biological Laboratories) in PBS was administered i.v. on the day of immunization and 48 h later.
- CFA complete Freunds adjuvant
- H37Ra strain of Mycobacterium tuberculosis Difco Laboratories
- test antibodies or control IgG was administered at 200 ⁇ g per mouse i.p. every other day from day 10 onwards till a total of 5 injections. In some experiments, control IgG was replaced by PBS as for the control group. Mice were weighed and examined daily for disease symptoms.
- Tissues for histological analysis were removed from mice 21 days after immunization and immediately fixed in 4% paraformaldehyde. Paraffin-embedded 5- to 10- ⁇ m sections of spinal cord were stained with Luxol fast blue or H&E and then examined by light microscopy. For immunofluorescence staining of CD4+ T cells and CD11 b+ monocytes/macrophages, spinal cords were removed from mice, perfused with PBS, and incubated in 30% sucrose at 4°C overnight. The tissue was subsequently dissected and embedded in optimal cutting temperature (OCT) compound. Frozen specimens were sectioned at 7 ⁇ m with a cryostat, and the sections mounted upon slides, air dried, and fixed for 10 min with 100% acetone. After blocking with 3% BSA, the sections were incubated overnight with primary rat anti-mouse CD4 or CD11 b Abs (BD
- splenocytes (5 ⁇ 10 5 per well) derived from EAE mice were cultured in triplicate in RPMI 1640 in 96-well plates. Cells were cultured in the presence or absence of the MOG peptide (20 ⁇ g/ml) or Con A (2 ⁇ g/ml) at 37°C in 5% CO2 for 72 h. Cells were pulsed with 1 ⁇ Ci of [3H] thymidine during the last 16 -18 h of culture before harvest. [3H] thymidine incorporation in cpm was measured by a MicroBeta counter (PerkinElmer).
- Protein extracts were loaded onto 10% or 12% SDS-polyacrylamide gels and subjected to electrophoresis, lmmunoblot analysis was performed by initial transfer of proteins onto Immobilon-P membrane (Millipore) using a Mini Trans-Blot apparatus (Bio- Rad).
- the membranes were incubated overnight at 4°C with specific primary Abs against P-JAK1 , JAK1 , P-AKT, AKT, P-Stat3, Stat3, P-Stat5, Stat5, Bcl-2, Bcl-xL, Bim, Bad, P-Bad (All the aforementioned antibodies are from Cell Signal), MCL-1 (Bio-legend), Bax (BD Bioscience), ROR ⁇ t (Abeam), Foxp3 (Santa Cruz Biotechnology), Actin (Santa Cruz Biotechnology) respectively.
- RNA Three micrograms of total RNA were reverse transcribed into biotin-16- deoxy-UTP-labeled single-strand cDNA using an AmpLabeling-LPR Kit (SuperArray). After prehybridization, membranes were hybridized with biotin-labeled sample cDNA and incubated with alkaline phosphatase-conjugated streptavidin (Chemiluminescent Detection kit; SuperArray) to visualize the signal. The results were analyzed using the GEArray Expression Analysis Suite (SuperArray). The results are representative of three experiments using independent splenocyte preparations.
- apoptosis Analysis for apoptosis was performed using an annexin V-FITC apoptosis detection kit (BD Biosciences), splenocytes derived from EAE mice were washed and incubated with 5 ⁇ l of annexin V-FITC and 5 ⁇ l of 7-AAD for 15 min at room temperature. Stained cells were analyzed subsequently using a FACS LSRII instrument (BD) within 1 h.
- BD Biosciences annexin V-FITC apoptosis detection kit
- Mononuclear cells were prepared from brain and spinal cord using gradient centrifugation. In brief, mice were perfused with 30 ml PBS to remove blood from internal organs. The dissociated brain and spinal cord tissue were grinded and filtered through a
- CD4 + T cells were first purified using CD4 microbeads (Miltenyi) from spleen and lymph nodes of na ⁇ ve mice. The resulting cells were labeled subsequently with CD44, CD62L and CD25 antibodies and further purified for the CD44 l0 CD62L hl CD25 population by FACS sorting (FACSAria II, Becton Dickinson).
- CD4 + CD25 hl and CD4 + CD25 " T cells single cell suspensions were incubated with FITC-labelled anti-CD4 antibody and PE-labeled anti-CD25 antibody (BD Biosciences) on ice for 30 min.
- CD4 + CD25 hl and CD4 + CD25 " T cells were sorted by a FACSAria instrument (Becton Dickinson). Similar approaches were employed to isolate human CD4 + CD25 + and CD4 + CD25 " T cells.
- CD4 + T cells were first purified from PBMCs using a CD4 + No-touch T cell isolation kit (Miltenyi Biotec), and CD4 + CD25 " T cells were isolated by negative selection using anti-CD25 microbeads (Miltenyi Biotec). The purity of CD4 + , CD4 + CD25 + , and CD4 + CD25 " T cell fractions was always greater than 95%.
- Naive mouse CD4+ T cells were plated in 96-well flat-bottomed plates (Costar) at a density of 1 * 10 6 cells/ ml. Cells were stimulated with plate-bound anti-CD3 Ab (5 ⁇ g/ml; BD Bioscience) and anti-CD28 Ab (5 ⁇ g/ml; BD Bioscience) in complete medium.
- T cells were cultured in T H 1 conditions ⁇ recombinant IL-12 (10 ng/ml; eBioscience) lus anti-IL-4 (10 ⁇ g/ml; BD Bioscience) ⁇ , or T H 17 conditions ⁇ TGF- ⁇ 1 (1 ng/ml; R&D Systems), IL-23 (10 ng/ml; R&D Systems) and IL-6 (10 ng/ml; eBioscience) plus anti-IFN ⁇ (10 ⁇ g/ml; BD Bioscience) and anti-IL-4(10 ⁇ g/ml) ⁇ for 4 days.
- CD4 + CD25 + T reg from CD4 + CD25 " T cells purified human or mouse CD4 + CD25 " T cells were cultured at 2 x 10 6 cells/ml with TGF- ⁇ 1 (10 ng/ml) and IL-2 (50 Ill/ml, R&D Systems) in the presence of coated anti-CD3 antibody (5 ⁇ g/ml) and 5 ⁇ g/ml anti-CD28 antibody for 4 days. In some cases, medium was washed out from the aforementioned culture systems and cells were then cultured in fresh medium for 1 h or 48 h in the presence or absence of IL-7 (10 ng/ml).
- CD4 For surface staining of CD4, CD25, CD8, B220 and CD127, cells were resuspended in PBS containing 1% BSA (Sigma-Aldrich) and 0.1% sodium azide and incubated with fluorochrome-conjugated antibodies to the indicated cell surface markers (BD Bioscience or eBioscience) for 30 minutes on ice.
- BSA Sigma-Aldrich
- fluorochrome-conjugated antibodies to the indicated cell surface markers (BD Bioscience or eBioscience) for 30 minutes on ice.
- intracellular cytokine staining freshly isolated mononuclear cells from lymph nodes, spleens and CNS of EAE mice or in vitro cultured cells were re-stimulated for 5 h with PMA (20 ng/ml) and lonomycin (1 ⁇ M) in the presence of GolgiPlug (1 :1000 diluted; BD Bioscience).
- the cells were surface stained with fluorescently labeled antibodies, resuspended in Fixation/Permeabilization solution (BD Bioscience), and stained for intracellular cytokines according to the manufacturer's instructions. Particularly, for IL-7 intracellular staining, cells were firstly incubated with antibodies against mouse CD16/CD32 (BD Bioscience) for 30 min at 4 0 C and followed by fixation/permeabilization using BD Bioscience solution, cells were then stained with goat anti-mouse IL-7 IgG (R&D Systems) or goat IgG (R&D Systems) as primary antibodies and Alexa Fluor® 488 donkey anti-goat IgG (Jackson Immunol) as a secondary antibody.
- BD Bioscience Fixation/Permeabilization solution
- Bcl-2 intracellular staining was performed with the same protocol but without the PMA and lonomycin stimulation.
- intracellular staining of Foxp3 cells were fixed and permeabilized with Foxp3 staining buffer (eBioscience). Permeabilized cells were stained with PE or FITC-conjugated anti-human or anti-mouse Foxp3 mAbs (0.5 ⁇ g/10 6 cells; eBioscience).
- intracellular staining of phosphorylated yes, cells were fixed for 10 min at 37 0 C with 2% (wt/vol) paraformaldehyde, made permeable for 30 min on ice with 90% (vol/vol) methanol, and stained for anti- phosphorylated Stat5 (BD Bioscience) staining.
- Flow cytometric analysis was performed on BD LSR Il (Becton Dickinson) instruments and results were analyzed using FlowJo software (Tree Star Inc.).
- the treatment effect correlated with a selective reduction in the production of IL-17 among other inflammation-related cytokines in MOG-reactive T cells (Figure 5C), and in percentage of T H 17 cells and to a lesser extent, T H 1 cells in both spleen and spinal cord of treated EAE mice ( Figure 5D).
- the absolute numbers of CNS-infiltrating T H 17 cells were decreased by 10-fold in treated mice compared to those of control mice.
- T reg cells increased reciprocally over the course of EAE ( Figure 5D).
- IL-7R There was differential expression of IL-7R in the three subsets (Figure 5E).
- T H 17 and T H 1 cells seen after onset of EAE were exclusively the CD44 + CD62L " memory phenotype and susceptible to IL-7R antibody treatment (data not shown).
- CD4 + T cell infiltration in spinal cord was markedly reduced, the absolute number and overall composition of peripheral CD4 + and CD8 + T cells and B220 + B cells were not significantly altered (data not shown).
- the results indicate that CD4 + T cells of the memory phenotype in EAE were highly enriched for pathogenic T H 17 and T H 1 subsets and susceptible to IL-7R antagonism, which tilts the T H 17/T H 1 to T reg ratio towards a new balance in treated EAE mice.
- T H 17 The in vivo development and function of pathogenic T H 17 is a dichotomic process comprised of differentiation and survival and expansion.
- Pro-inflammatory cytokines such as IL-6, IL-1 ⁇ and IL-21 are critical to T H 17 differentiation and the initiation of autoimmune inflammation in EAE, while survival and expansion of T H 17 cells is poorly understood and may involve I L-23.
- the inventors investigated whether IL-7/IL-7R signalling was associated with T H 17 differentiation using purified na ⁇ ve CD4 + T cells of the CD44 l0 CD62L hl CD25 " phenotype. The effect of IL-7 was examined by stimulating the resulting cells with CD3/CD28 antibodies in the presence and absence of TGF- ⁇ .
- T H 17 cells derived from EAE mice underwent self- initiated or programmed apoptosis that could be reverted by the addition of IL-7.
- the process was abolished by pre-incubation of susceptible cells with an anti-IL-7R antibody but not a control antibody.
- IL-7 significantly altered the expression levels of BCL-2, which correlated reciprocally with the levels of Annexin-V + apoptotic cells ( Figure 11 C).
- T H 17 development is a two-step process; "Step 1 " being T H precursor cell differentiation, and “Step 2" being T H 17 survival/expansion.
- Step 1 being T H precursor cell differentiation
- Step 2 being T H 17 survival/expansion.
- These two processes are controlled by different cytokines, the expression of which are further regulated by various transcription factors. Both processes contribute critically to the clinical outcome of autoimmune disease.
- T H 17 differentiation is mainly induced by IL-6 through JAK/STAT-3 pathway.
- T H 17 differentiation was further validated in a human experimental system.
- T H 17 differentiation was minimal affected as shown in Figure 12, indicating that IL-7 plays a minor role in this process.
- our results showed that the major role of IL-7/IL-7R signalling in this two-step cell development process is in Step 2 - pathogenic T H 17 cell survival and expansion.
- the role of IL- 7 is superior to IL-23 through the JAK/STAT-5 pathway.
- anti-human IL-7R mAb was given after cells had already committed to T H 17 cells, the cells are susceptible to apoptosis as shown in Figure 22.
- the study provides compelling evidence for a novel role of IL-7/IL-7R signalling in pathogenic T H 17 cell development and functions in EAE and lends strong rationale for IL-7R antagonism as a potential treatment for MS and other autoimmune conditions.
- PBMCs were initially screened and selected on the basis of a positive result with antibody R34.34 (Dendritics Inc). Fresh or thawed PBMCs were plated at 2x10 5 cells/well in 96 well in RPMI 1640 containing 10% FBS. Purified testing antibody 6C5, positive control antibody R34.34 (Dendritics Inc) and anti-human IL-7 (R&D), plus isotype control antibody mouse IgGI (R&D) were incubated at 10 ⁇ g/ml and 100 ⁇ g/ml with cells at 37 0 C for 30 minutes before 10ng/ml IL-7 was supplemented. Cells briefly treated with IL-7 served as negative control while non-treated cells as background.
- test sample antibodies and positive control antibody were prepared in 3 fold serial dilution starting from a top concentration of 120ug/ml, and added to 2x10 5 PBMC cells for 30mins at 37 0 C before stimulation with IL-7 at 1 ng/ml for 15mins at 37 0 C.
- the cells without antibody and IL7 treatment were used as background control.
- Relative activity (%) (RFU(sample) - RFU(background control)) /
- CCF-CEM cells were cultured in growth medium (RPM11640, 10% FBS, 100U/ml Penicillin, 100ug/ml Streptomycin, 1 mM Sodium Butyrate) and treated with 1 uM Dexamethasone (Sigma #D4902) overnight for IL7 receptor induction before the experiment.
- growth medium RPM11640, 10% FBS, 100U/ml Penicillin, 100ug/ml Streptomycin, 1 mM Sodium Butyrate
- Dexamethasone Sigma #D4902
- Test sample antibodies and positive control antibody were prepared in 3 fold serial dilution starting from a top concentration of 120ug/ml, and added to 2x10 5 CCF-CEM cells for 30mins at 37 0 C before stimulation with IL-7 at 1 ng/ml for 15mins at 37 0 C.
- the cells without antibody and IL7 treatment were used as background control.
- the cells treated with IL7 but not with antibody samples were used as full activity control.
- Cells after treatment were lysed by lysis buffer (PerkinElmer #TGRS5S500) for 5mins at 37 0 C and the lysates were incubated with Reaction Buffer plus Activation Buffer mix (PerkinElmer
- Luminescence (RFU) from AlphaScreen beads were analyzed on Envision with its default alphascreen mode (top read; Ex 680nm; Em 570nm). Results for testing samples were converted to relative activity based on the following formula:
- Relative activity (%) (RFU(sample) - RFU(background control)) /
- Fresh PBMCs were suspended in serum free RPMI 1640 medium.
- Test sample antibodies and positive control antibody (6A3 and R34.34, Dendritics lnc #DDX0700) were diluted to achieve final concentrations from 20 ug/ml to 0.01 ug/ml in the culture and were added to 1x10 6 PBMC cells per sample.
- PBMCs were incubated with antibodies for 50mins at 37 0 C before stimulation with IL-7 at 1 ng/ml for 15mins.
- Figure 21 shows the inhibition of IL-7-induced P-STAT5 relative to no antibody control at increasing concentrations of R34.34 and 6A3.
- CD4+ cells from six donors were isolated according to the manual (#130-091 -155, Miltenyi). Approximately 1x106/ml of the CD4+ cells in 100 ul were mixed with equal volume of 2x T H 17 medium (2 ⁇ g/ml anti-CD28 + 10 ⁇ g/ml anti-IFN ⁇ + 10 ⁇ g/ml anti-IL-4 + 12.5ng/ml IL-1 ⁇ + 20ng/ml IL-23 + 50ng/ml IL-6) and cultured in 37 0 C with 5% CO 2 for 5 days. Treatment by the various cytokines and growth factors in the T H 17 medium preferentially differentiated the CD4+ cells into T H 17 cells. CCR6+ cells from the differentiated cultured cells at day 5 were sorted using BD FACS SORP Aria II. The
- CCR6+ cells were then adjust to 2 x10 6 VmI for the IL-17 production assay.
- 10O ⁇ l of CCR6+ cells were pre-incubated with testing antibody for 1 h at 37°C, and then mixed with 10O ⁇ l of 20ng/ml IL-7.
- the cells were cultured for 3 days in 37°C with supplement of 5% CO2.
- IL-17 level in "l OO ⁇ l of the culture supernatant were measured by FlowCytomix (Bender MedSystems).
- Table 1 1 1 shows the IL-7 and testing antibody (R34.34 and 6C5) concentrations used in generation of the results in Figure 22 (results from a single donor).
- R34.34 inhibited IL-17 production in IL-7 induced differentiated T cells in 6/6 donors; 6C5 inhibited IL-17 production in IL-7 induced differentiated T cells in 4/6 donors.
- CD4+ cells were isolated according to the manual (#130-091-155, Miltenyi) . Approximately 7x10 5 /ml of the CD4+ cells in 100 ul were mixed with equal volume of 2x T H 17 medium (2 ⁇ g/ml anti- CD28 + 10 ⁇ g/ml anti-IFN- ⁇ + 10 ⁇ g/ml anti-IL-4 + 12.5ng/ml IL-1 ⁇ + 20ng/ml IL-23 + 50ng/ml IL-6) and cultured in 37°C with 5% CO2 for 5 days. Treatment by the various cytokines and growth factors in the T H 17 medium preferentially differentiated the CD4+ cells into Th 17 cells. CCR6+ cells from the differentiated cultured cells at day 5 were sorted using BD FACS SORP Aria II. The CCR6+ cells were then adjusted to 2 x10 6 /ml for the IL-17 production assay.
- IL-17 and IFN- ⁇ level 10O ⁇ l of CCR6+ cells from individual donors were pre-incubated with testing antibody for 1 h at 37°C, and then mixed with 100 ⁇ l of 20ng/ml IL-7. The cells were cultured for 3 days in 37°C with supplement of 5% CO2. IFN- ⁇ and IL-17 levels in 100 ul of the culture supernatant were measured by FlowCytomix (Bender MedSystems) at 24 h and 40 h, respectively. Table 12 shows the IL-7 and testing antibody concentrations used in generation of the results in Figure 23. The results are representative of 5/6 donors.
- IL-7/IL-7R signalling is critically required for survival and expansion of committed T H 17 cells in both mouse and human systems, while its role in T H 17 differentiation is not essential compared to that of IL-6.
- IL-7 or IL-7R antagonism administered after EAE onset significantly affect the clinical course of disease.
- the inventors have therefore shown that IL-7 or IL-7R antagonism provides real therapeutic potential in the treatment of autoimmune diseases and inflammatory disorders in which pathogenic T H 17 cells are implicated, particularly MS, and more particularly still the relapsing/remitting course of MS (RRMS).
- T H 17 development and function is controlled chiefly by IL-6 through JAK/STAT-3 for T H 17 differentiation and IL-7 through JAK/STAT-5 for T H 17 maintenance.
- IL-7 not only provides a survival signal for pathogenic T H 17 cells but directly induces in vivo T H 17 cell expansion, critically contributing to sustained autoimmune pathology in EAE.
- T H 17 cells of the memory phenotype represent an in vivo pathogenic T cell subset and are susceptible to self-initiated or programmed apoptosis.
- This process appears to be dependent on IL-7/IL-7R signalling through regulation of pro- and anti-apoptotic proteins, such as Bcl-2 and Bax, in susceptible T H 17 cells.
- IL-7 serves as a critical survival signal that prevents differentiated T H 17 cells from programmed apoptosis.
- increased IL-7 production and highly expressed IL-7R in pathogenic T cells as seen in the acute phase of autoimmune diseases provide the milieu required for sustained T cell survival and expansion.
- T H 17 cells are altered in the in vitro but not in vivo system.
- the discrepancies may be explained by different cytokine milieu between the in vitro setting where exogenous IL-7 is added and in vivo micro-environment involving interplay of multiple cytokines.
- T H 17 over T reg is readily explained by differential expression of IL-7R, rendering T H 17 cells susceptible and T reg cells resistant to IL-7R antagonism.
- This selectivity appears to play an important role in rebalancing the ratio of pathogenic T H 17 cells and T reg cells by IL-7R antagonism in EAE and is attributable to the treatment efficacy.
- the discrepancies in the magnitude of IL-7-induced responsiveness and susceptibility to IL-7R antagonism between T H 17 and T H 1 could not be simply explained by the expression of IL-7R as both subsets highly express IL-7R.
- the intrinsic expression and activity of SOCS-1 is responsible for the discrepancies.
- IL-7 neutralization or IL-7R antagonism is likely to have unique therapeutic advantages.
- the treatment offers the selectivity that distinguishes pathogenic T H 1 and T H 17 cells from T reg and unrelated immune cells.
- additional therapeutic advantages of IL-7R antagonism involve its selective effect on survival and expansion of differentiated T H 17 as opposed to T H 17 differentiation. Targeting, with an inhibitor of the IL-7/IL-7R pathway, the in vivo maintenance of committed T H 17 versus T H 17 differentiation may be more efficacious in a therapeutic context.
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BRPI0916945A BRPI0916945A2 (en) | 2008-08-08 | 2009-08-07 | methods for treating an autoimmune disease or inflammatory disorder in a human subject, for treating multiple sclerosis in an individual, and for identifying antibodies, isolated human, humanized or chimeric antibody or antigen binding fragment thereof, and, isolated connection. |
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-
2011
- 2011-02-02 DO DO2011000041A patent/DOP2011000041A/en unknown
- 2011-02-03 IL IL211034A patent/IL211034A0/en unknown
- 2011-02-07 ZA ZA2011/00974A patent/ZA201100974B/en unknown
- 2011-02-08 CL CL2011000269A patent/CL2011000269A1/en unknown
- 2011-02-11 CO CO11016609A patent/CO6341640A2/en unknown
- 2011-03-03 CR CR20110118A patent/CR20110118A/en not_active Application Discontinuation
- 2011-03-08 MA MA33682A patent/MA32621B1/en unknown
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Also Published As
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AU2009279471A1 (en) | 2010-02-11 |
MA32621B1 (en) | 2011-09-01 |
NZ590994A (en) | 2012-09-28 |
KR20110044777A (en) | 2011-04-29 |
CA2733432A1 (en) | 2010-02-11 |
EP2318442A1 (en) | 2011-05-11 |
MX2011001477A (en) | 2011-03-25 |
AR072985A1 (en) | 2010-10-06 |
PE20110382A1 (en) | 2011-06-27 |
DOP2011000041A (en) | 2011-02-28 |
US20100040616A1 (en) | 2010-02-18 |
CR20110118A (en) | 2011-07-28 |
TW201018482A (en) | 2010-05-16 |
UY32038A (en) | 2010-03-26 |
CL2011000269A1 (en) | 2012-07-20 |
ZA201100974B (en) | 2012-10-31 |
CN102177179A (en) | 2011-09-07 |
CO6341640A2 (en) | 2011-11-21 |
EA201100150A1 (en) | 2011-10-31 |
US20110287000A1 (en) | 2011-11-24 |
IL211034A0 (en) | 2011-04-28 |
JP2011530533A (en) | 2011-12-22 |
BRPI0916945A2 (en) | 2015-11-24 |
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