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Definitions

• This invention relates to binding members, especially antibody molecules, for CXCL13.
• the binding members are useful for treatment of disorders associated with CXCL13, including arthritic disorders such as rheumatoid arthritis.
• CXCL13 is a potent B cell chemoattractant, which directs na ⁇ ve B cells into the follicles of secondary lymphoid organs and is constitutively expressed by follicular dendritic cells (FDCs) and stromal cells in the B cell rich areas of secondary lymphoid organs.
• CXCL13 is also known as B cell-attracting chemokine 1 (BCA-1).
• BCA-1 B cell-attracting chemokine 1
• CXCL13 signals through its receptor CXCR5.
• CXCR5 is a seven-transmembrane spanning G protein coupled receptor and is a member of the CXC-chemokine receptor subfamily of the class 1 GPCR family.
• CXCR5 is expressed at high levels on na ⁇ ve and activated B cells, including peripheral blood and tonsillar B cells. It is also expressed on a subset of activated peripheral blood CD4+ T cells and the majority of CD4+ cells in secondary lymphoid tissue. CXCL13 is the only known ligand for CXCR5.
• CXCL13 plays a role in the development of peripheral lymphoid organs; for example, Ansel et al [1] have shown that mice deficient in CXCL13 have severe defects in peripheral lymph node development. CXCL13 induces membrane lymphotoxin ⁇ 1 ⁇ 2 expression on na ⁇ ve B cells recruited into follicles, which promotes the maturation of FDCs and further enhances CXCL13 production [1]. CXCL13-deficient mice, immunised with a T cell-dependent antigen, form germinal centres in lymph nodes and spleen but these are small and have an irregular architecture suggesting CXCL13 is required for the recruitment and correct positioning of B cells within follicles [1].
• CXCL13 also has a role in innate immunity; CXCL13-deficient mice lack both peritoneal and pleural cavity B1 cells and are defective in the production of natural antibodies to body cavity bacterial antigens (Ansel, K M. et al. Immunity, 16: 67-76, 2002).
• MAB801 is a commercially available murine anti-human CXCL13 monoclonal antibody (R & D Systems: MAB801).
• MAB470 is a rat anti-mouse CXCL13 monoclonal.
• a binding member of the invention inhibits binding of CXCL13 to the target receptor, CXCR5. Inhibition of binding may be direct inhibition.
• binding members for CXCL13 also referred to as CXCL13-binding members, are described herein.
• the binding members bind and may neutralise human CXCL13 and non-human primate CXCL13 e.g. cynomolgus CXCL13.
• Non-human CXCL13 refers to an ortholog of CXCL13 that occurs naturally in a species other than human.
• Binding members of the invention are normally specific for CXCL13 over other members of the CXC family of chemokines, including for example, CXCL3, CXCL5, CXCL6, CXCL8, CXCL10 and/or CXCL12 and thus bind CXCL13 selectively. This may be determined or demonstrated for example in a competition assay. For example, a suitable assay is described herein in Example 2.5.
• the binding members are useful for inhibiting binding of CXCL13 to CXCR5 in vivo or in vitro, and may be used for treating disorders associated with CXCL13, such as rheumatoid arthritis, as described in detail elsewhere herein.
• binding members according to the invention have been shown to neutralise CXCL13 with high potency.
• Neutralisation means inhibition of a biological activity of CXCL13.
• Binding members of the invention may neutralise one or more activities of CXCL13.
• the inhibited biological activity is typically CXCL13 binding to a binding partner.
• the inhibited biological activity may be binding of CXCL13 to CXCR5.
• binding of human or non-human CXCL13 to CXCR5 may be inhibited, e.g. a binding member may inhibit binding of non-human primate, such as cynomolgus, CXCL13 to CXCR5.
• CXCR5 may be human or non-human, such as non-human primate, e.g. cynomolgus, or mouse CXCR5.
• the CXCR5 is of the same species as the CXCL13.
• CXCR5 is human CXCR5.
• Neutralisation of CXCL13 binding to CXCR5 may for example be measured as a function of CXCR5 signalling-mediated activity, since CXCL13 binding to CXCR5 stimulates such activity.
• CXCR5 is a G-protein coupled receptor
• the activity may be an activity associated with G-protein coupled receptors.
• a suitable assay may comprise detecting an inhibition of the decrease in cellular cAMP levels that occurs in the absence of the binding member i.e. a cAMP assay referred to herein measures the inhibition (by a binding member of the invention) of CXCL13 mediated decreases in cellular cAMP levels.
• the cells used in the cAMP assay are co-stimulated with an adenyl cyclase activator, such as NKH477. Suitable cells for use in such an assay are described herein.
• Suitable (cellular) cAMP assays include those which combine the techniques of fluorescence resonance energy transfer (FRET) and time resolved fluorescence (TRF) in an homogeneous assay format (a TRF/FRET cAMP assay as referred to herein).
• TRF/FRET cAMP assay examples include the HTRF® (Homogeneous Time Resolved Fluorescence) cAMP assay (CisBio International) (see, e.g. Gabriel D et al, (2003) Assay and Drug Development Technologies 1(2): 291-303) and the LANCE® cAMP assay (Perkin Elmer) (see, e.g. Hemmila et al (1999) J Biomol Screen 4(6): 303-308).
• FRET refers to a non-radiative energy transfer of excitation energy between a donor-fluorophore and a suitable acceptor fluorophore (a FRET donor-acceptor pair).
• the assay components include: labelled cAMP (tracer cAMP); and labelled cAMP specific antibody. Each of the tracer cAMP and the antibody are labelled with one member of a donor-acceptor pair. When the antibody binds to the tracer cAMP, FRET occurs and the signal emitted by the acceptor is determined by TRF. Free cAMP in a sample competes with the tracer cAMP for binding to antibody and reduces the FRET signal. The assay thus comprises determining the signal emitted by the acceptor.
• the assay comprises determining the specific signal of both the donor and the acceptor as an internal control to give a ratio measurement, which compensates for the presence of coloured compounds in the assay.
• the donor acceptor pair is typically such that the donor is excited by a light at about 337 to 340 nm.
• the acceptor emits a signal at about 665 nm (which can be time resolved).
• both XL665 (acceptor in the HTRF® cAMP assay) and the Alexa Fluor®-647 dye (acceptor in the LANCE® cAMP assay) emit a fluorescent signal at 665 nm.
• a donor may for example emit a signal at about 615 to 620 nm.
• An acceptor may be for example, a red-absorbing fluorescent dye, in particular a hydrophilic red-absorbing dye (Buschmann et al, Bioconjugate Chem. 2003, 14: 195-204).
• a red-absorbing fluorescent dye may be for example, Alexa Fluor®-647 (Perkin Elmer) such as is used in the LANCE® cAMP assay.
• donor-acceptor pairs include the europium cryptate (donor)/XL665 (acceptor), e.g. as used in the HTRF® cAMP assay, and the europium chelate (donor)/Alexa Fluor®-647dye (acceptor) e.g. as used in the LANCE® cAMP assay.
• Either the tracer cAMP or the antibody may be labelled with the donor or the acceptor.
• an assay may comprise use of (donor label-tracer cAMP) and (acceptor label-cAMP specific antibody), or of (acceptor label-tracer cAMP) and (donor label-cAMP specific antibody).
• an assay may comprise the use of XL665-cAMP and europium cryptate-anti-cAMP monoclonal antibody, e.g. as in the HTRF® assay.
• An assay may comprise use of europium chelate-labelled cAMP and Alexa Fluor®-647dye labelled cAMP antibody, e.g. as in the LANCE® assay.
• Fluorescent signals in a TRF/FRET assay are determined by TRF.
• a TRF/FRET cAMP assay can be used to assay inhibition of CXCL13 mediated decreases in cellular cAMP levels upon costimulation of CH0qi5 CXCR5 cells with the adenylyl cyclase activator, NKH477.
• HTRF® assay In a HTRF® assay generally, macromolecules which may bind to each other are labelled, one with europium (Eu3+) cryptate (donor) and the other with a second fluorescent label, XL665 (or XLent) (a stable cross linked allophycocyanin).
• XL665 or XLent
• FRET occurs and XL665 reemits a specific long-lived fluorescence at 665 nm.
• the specific signals of both the donor (at 620 nm) and the acceptor (at 665 nm) are measured as an internal control, giving a ratio measurement that compensates for the presence of coloured compounds in the assay.
• a HTRF® cAMP assay typically comprises: mixing XL665-cAMP (tracer cAMP), europium cryptate-anti-cAMP monoclonal antibody and a sample containing cAMP; applying light at 337 nm (thereby exciting the europium cryptate donor); determining the fluorescent signal at 620 nm (donor) and 665 nm (acceptor) by TRF; and determining the ratio of signal 665 nm/620 nm as a measure of the FRET which has occurred. Any free cAMP from a sample competes with XL665-cAMP (tracer cAMP) for binding to europium cryptate-anti-cAMP monoclonal antibody. Maximum FRET occurs when a sample does not contain any cAMP and the FRET signal decreases with increasing sample cAMP.
• the HTRF® technology thus takes advantage of the possibilities offered by fluorescence to work on both the spectral characteristics of the signal emitted (patented ratio correction) and under a time resolved detection mode (eliminating, e.g. auto fluorescence).
• the technology is homogeneous, which means that samples and detection reagents are mixed together in a plate, which can be read in a suitable plate reader, the so-called “mix and read” protocol.
• the LANCE® cAMP assay combines TRF and FRET and also the use of a red-shifted Alexa® Fluor dye (Perkin Elmer), which allows a FRET assay without the compound interference associated with blue dyes.
• a LANCE® cAMP assay typically comprises: mixing a europium-chelate of europium/streptavidin-biotin/cAMP tracer, Alexa Fluor®-647-labelled cAMP specific antibody and a sample containing cAMP; applying light at 340 nm (thereby exciting the europium donor); and determining the fluorescent signal at 665 nm (acceptor) by TRF as a measure of the FRET which has occurred. Residual energy from the chelate produces a light at 615 nm. Any free cAMP from a sample competes with europium labelled-cAMP (tracer cAMP) for binding to Alexa®-labelled-anti-cAMP monoclonal antibody. Maximum FRET occurs when a sample does not contain any cAMP and the FRET signal decreases with increasing sample cAMP.
• Suitable standards may be used to calibrate the assays.
• CXCR5 signalling-mediated activity which may be assayed, in order to determine neutralisation of CXCL13 binding to CXCR5 is CXCL13 induced calcium release.
• a binding member is assayed for an inhibitory effect on CXCL13 induced calcium release in cells expressing CXCR5 and the G-protein Gqi5.
• Stimulation of these cells with CXCL13 gives rise to increases in cytoplasmic Ca2+ in a concentration dependent manner by inducing release from intracellular stores and influx from extracellular media. This can be measured using calcium sensitive dyes in a Fluorescence Imaging Plate Reader (FLIPR).
• FLIPR Fluorescence Imaging Plate Reader
• one such assay uses human CXCL13 and a CHO cell line transfected with Gqi5 and human CXCR5.
• cAMP assays and Ca2+ release assays may be performed in vitro with a mammalian cell line, e.g. Chinese Hamster Ovary (CHO) cells such as CHO K1 cells, expressing CXCR5 and the G protein Gqi5 which contains the G ⁇ i subunit.
• CHO Chinese Hamster Ovary
• Suitable cell lines may be produced by transfecting the cells with nucleic acid encoding CXCR5 and Gqi5.
• CXCL13 is a potent B cell chemoattractant.
• CXCL13 signals through the CXCR5 receptor, which is expressed at high levels on na ⁇ ve and activated B cells, and on a subset of activated T cells. Therefore another activity, which may be assayed, in order to determine neutralisation of CXCL13 binding to CXCR5 is CXCL13 induced chemotaxis in B cells expressing CXCR5.
• the B cells in the assay may be non-primary B cells.
• One such assay uses a murine pre-B cell line that has been transfected with human recombinant CXCR5 (B300.19 hCXCR5 cells).
• primary B cells may be used.
• One such assay uses a mixed lymphocyte population isolated from murine spleen.
• suitable assays such as the cAMP, calcium release and B cell chemotaxis assays can be used to calculate a binding member's potency for inhibiting binding of CXCL13 to CXCR5, as described below.
• These assays can be used to calculate potency of a binding member for neutralising CXCL13 binding to CXCR5.
• the neutralising potency is calculated as a function of the CXCR5 signalling-mediated activity.
• the assay may measure potency of neutralising CXCL13 induced: decrease in cellular cAMP levels; increase in cellular calcium levels; or B cell chemotaxis.
• Binding members may inhibit CXCL13 biological activity by 100%, or at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the binding member.
• Neutralising potency of a binding member may be determined. Potency is normally expressed as an IC 50 value, in nM unless otherwise stated. In functional assays, IC 50 is the concentration of a binding member that reduces a biological response by 50% of its maximum. In ligand-binding studies, IC 50 is the concentration that reduces receptor binding by 50% of maximal specific binding level. IC 50 may be calculated by plotting % of maximal biological response as a function of the log of the binding member concentration, and using a software program, such as Prism (GraphPad) to fit a sigmoidal function to the data to generate IC 50 values. Potency may be determined or measured using one or more assays known to the skilled person and/or as described or referred to herein.
• Other methods that may be used for determining binding of a binding member to CXCL13 include ELISA, Western blotting, immunoprecipitation, affinity chromatography and biochemical assays.
• Neutralising potency of a binding member as calculated in an assay using CXCL13 from a first species may be compared with neutralising potency of the binding member in the same assay using CXCL13 from a second species (e.g. a non-human primate such as cynomolgus), in order to assess the extent of cross-reactivity of the binding member for CXCL13 of the two species.
• a second species e.g. a non-human primate such as cynomolgus
• cross-reactivity may be assessed in a competition binding assay, as described in more detail elsewhere herein.
• a binding member of the invention may have a potency for neutralising binding of human CXCL13 to CXCR5 that is within 10-fold of its potency for neutralising binding of cynomolgus CXCL13 to CXCR5.
• Potency may for example be as measured in a cAMP assay (e.g. a TRF-FRET assay such as a LANCE® cAMP assay) or in a B cell chemotaxis assay, with human and cynomolgus CXCL13 respectively.
• a cAMP assay e.g. a TRF-FRET assay such as a LANCE® cAMP assay
• B cell chemotaxis assay with human and cynomolgus CXCL13 respectively.
• Potency in the cAMP assay e.g.
• a LANCE® cAMP assay with human CXCL13 may, for example, be not more than 10-, 5-, 4-, 3- or 2-fold different than in a cAMP assay with cynomolgus CXCL13. Potency in the cAMP assay is as determined for a final concentration of human or cynomolgus CXCL13 of 2 nM. Examples of data obtained in a LANCE® cAMP assay with human CXCL13 and cynomolgus CXCL13 are shown in Tables 2 and 5.
• a binding member of the invention may bind human CXCL13 more strongly than cynomolgus CXCL13.
• the strength of binding of a binding member to human CXCL13 may for example be not more than 10, 9, 8, 7, 6, 5, 4, 3, or 2-fold greater than for cynomolgus CXCL13 as measured in a competition binding assay.
• the strength of binding may be not more than 4-fold greater for human CXCL13 than for cynomolgus CXCL13. Examples of data obtained in a competition assay with human and cynomolgus CXCL13 are shown in Table 8.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 12 nM in a human CXCL13 cAMP assay, as described herein, with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM, such as not more than 5 nM.
• the binding member may be in any form described herein, for example, an IgG or scFv or any other suitable form.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 12 nM in a cynomolgus CXCL13 cAMP assay, as described herein, with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM, such as not more than 3 nM.
• the binding member may be in any form described herein, for example, an IgG or scFv or any other suitable form.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 12 nM in a human CXCL13 HTRF® cAMP assay with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM, such as not more than 5 nM. Examples of data obtained in a human CXCL13 HTRF® cAMP assay are shown in Table 1.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 12 nM in a cynomolgus CXCL13 HTRF® cAMP assay with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM, such as not more than 3 nM. Examples of data obtained in a cynomolgus CXCL13 HTRF® cAMP assay are shown in Table 1.
• the binding member may have a neutralising potency or IC 50 of not more than 5 nM in a human CXCL13 cAMP assay as described herein with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 4.5, 4, 3.5, 3, 2.5, 2, 1.9, 1.7, 1.5, 1.3, 1.1, 1.0, 0.9, 0.7 or 0.5 nM, such as not more than 1.4 or not more than 1.6 nM.
• the binding member may have a neutralising potency or IC 50 of not more than 5 nM in a cynomolgus CXCL13 cAMP assay as described herein with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4 or 0.2 nM, such as not more than 0.3 nM.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 5 nM in a human CXCL13 LANCE® cAMP assay with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 4.5, 4, 3.5, 3, 2.5, 2, 1.9, 1.7, 1.5, 1.3, 1.1, 1.0, 0.9, 0.7 or 0.5 nM, such as not more than 1.4 or not more than 1.6 nM. Examples of data obtained in a human CXCL13 LANCE® cAMP assay are shown in Tables 2 and 5.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 5 nM in a cynomolgus CXCL13 LANCE® cAMP assay with a 2 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4 or 0.2 nM, such as not more than 0.3 nM. Examples of data obtained in a cynomolgus CXCL13 LANCE® cAMP assay are shown in Tables 2 and 5.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 40 nM in a human CXCL13 calcium release assay with a 100 nM final concentration of CXCL13.
• the IC 50 may for example be not more than 38, 36, 34, 32, 20, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, or 2 nM. Examples of data obtained in a human CXCL13 calcium release assay are shown in Tables 4 and 7.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 12 nM in a human CXCL13 B cell chemotaxis assay with a final concentration of human CXCL13 which gives an approximately ED80 response, and a non-primary B cell line that has been transfected with human recombinant CXCR5.
• the IC 50 may for example be not more than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 1.5 nM.
• the assay uses a B cell line, e.g. a murine pre-B cell line that has been transfected with human recombinant CXCR5, such as B300.19 cells expressing human recombinant CXCR5. Examples of data obtained in a human CXCL13 B cell chemotaxis assay with human CXCL13 are shown in Tables 3 and 6.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 10 nM in a cynomolgus CXCL13 B cell chemotaxis assay with a final concentration of cynomolgus CXCL13 which gives an approximately ED80 response and a non-primary B cell line that has been transfected with human recombinant CXCR5.
• the IC 50 may for example be not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM.
• the assay uses a B cell line, e.g. a murine pre-B cell line that has been transfected with human recombinant CXCR5, such as B300.19 cells expressing recombinant CXCR5. Examples of data obtained in a cynomolgus CXCL13 B cell chemotaxis assay with are shown in Table 6.
• a binding member of the invention may have a neutralising potency or IC 50 of not more than 40 nM in a human CXCL13 primary cell chemotaxis assay with a final concentration of human CXCL13 which gives an approximately ED80 response, and a mixed lymphocyte population.
• the IC 50 may for example be not more than 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4 or 2 nM.
• the assay uses a primary lymphocyte population freshly isolated from mouse spleen.
• Table 12 An example of data obtained in a human CXCL13 B cell chemotaxis assay with human CXCL13 and murine primary lymphocytes is shown in Table 12.
• Binding kinetics and affinity (expressed as the equilibrium dissociation constant K D ) of CXCL13 binding members for CXCL13 may be determined, e.g. using surface plasmon resonance e.g. BIAcore.
• Binding members of the invention normally have an affinity for human CXCL13 of less than 400 pM, for example, less than 380, 360, 340, 320, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, or 50 pM.
• Affinity for cynomolgus CXCL13 is normally similar to that for human CXCL13. Examples of data obtained for human CXCL13 and cynomolgus CXCL13 using BIAcore are shown in Tables 10 and 11.
• a binding member of the invention may have an affinity for human CXCL-13 and cyno CXCL-13 of less than 150 pM.
• Binding kinetics and affinity can be measured using surface plasmon resonance by flowing human or cyno CXCL-13 over a surface comprising the binding member.
• the neutralising potencies described for the assays above may apply to a binding member assayed in any form described herein.
• the potencies may apply to antibody molecules including any of the antibody fragments described herein, e.g. IgG binding members.
• Glycosylated and unglycosylated forms of human CXCL13 were obtained when CXCL13 was expressed recombinantly in mammalian cell lines (MEL cells). The majority of the human CXCL13 produced was unglycosylated, however a smaller fraction containing the glycosylated form could be obtained when expression was scaled-up. Cynomolgus CXCL13 expressed in MEL cells was glycosylated. Potency of antibody 1 for glycosylated and unglycosylated human CXCL13 was within 10-fold different and can be considered equipotent, as measured in the B cell chemotaxis assay described herein.
• Example potency data for unglycosylated human CXCL13, glycosylated human CXCL13 and glycosylated cynomolgus CXCL13 are shown in Table 6 in Example 2.3. Binding to glycosylated human CXCL13 may represent an advantage of binding members of the invention, since endogenous CXCL13 may be glycosylated and therefore glycosylated human CXCL13 may represent the therapeutic target antigen for human therapy.
• recombinant CXCL13 e.g. human CXCL13
• recombinant CXCL13 may be used in unglycosylated or glycosylated form.
• Our data indicate that binding members of the invention may bind an epitope in a region of CXCL13 unaffected by glycosylation (see Example 2.3 and Table 6).
• binding member potencies in assays described herein for human CXCL13 may apply both for unglycosylated human CXCL13 and for glycosylated human CXCL13.
• a binding member of the invention may have not more than 10-, 5-, 4-, 3-, 2.5-, or 2 fold greater potency for unglycosylated human CXCL13 as compared with glycosylated human CXCL13 in an assay described herein, e.g. the B cell chemotaxis assay. Potency for glycosylated human CXCL13 may of course be greater than potency for unglycosylated human CXCL13.
• Potency of binding members of the invention for glycosylated human CXCL13 may be similar or the same as for unglycosylated human CXCL13, e.g. it may be not more than 10-, 5-, 4-, 3-, 2.5- or 2-fold different as measured in a competition binding assay, a cAMP assay, calcium release assay or B cell chemotaxis assay as described herein.
• a binding member of the invention may comprise an antibody molecule, e.g. a human antibody molecule.
• the binding member normally comprises an antibody VH and/or VL domain.
• VH and VL domains of binding members are also provided as part of the invention.
• Within each of the VH and VL domains are complementarity determining regions, (“CDRs”), and framework regions, (“FRs”).
• CDRs complementarity determining regions
• FRs framework regions
• a VH domain comprises a set of HCDRs
• a VL domain comprises a set of LCDRs.
• An antibody molecule may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework.
• VH and VL domains and CDRs are as listed in the appended sequence listing that forms part of the present disclosure.
• All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention.
• a “set of CDRs” comprises CDR1, CDR2 and CDR3.
• a set of HCDRs refers to HCDR1, HCDR2 and HCDR3
• a set of LCDRs refers to LCDR1, LCDR2 and LCDR3.
• a “set of CDRs” includes HCDRs and LCDRs.
• binding members of the invention are monoclonal antibodies.
• a binding member of the invention may comprise an antigen-binding site within a non-antibody molecule, normally provided by one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.
• Antibody 1 refers to antibodies with the set of CDRs of antibody 1. Sequences for an antibody 1 are shown in the appended Sequence Listing, with the following sequences in the following order: nucleotide sequence encoding VH domain (SEQ ID NO: 1); amino acid sequence of VH domain (SEQ ID NO: 2); VH CDR1 amino acid sequence (SEQ ID NO: 3); VH CDR2 amino acid sequence (SEQ ID NO: 4); VH CDR3 amino acid sequence (SEQ ID NO: 5); nucleotide sequence encoding VL domain (SEQ ID NO: 6); amino acid sequence of VL domain (SE1 ID NO: 7); VL CDR1 amino acid sequence (SEQ ID NO: 8); VL CDR2 amino acid sequence (SEQ ID NO: 9); VL CDR3 amino acid sequence (SEQ ID NO: 10).
• a binding member of the invention may comprise one or more CDRs as described herein, e.g. a CDR3, and optionally also a CDR1 and CDR2 to form a set of CDRs.
• the CDR or set of CDRs may be a CDR or set of CDRs of antibody 1, or may be a variant thereof as described herein.
• the invention provides binding members comprising an HCDR1, HCDR2 and/or HCDR3 of antibody 1 and/or an LCDR1, LCDR2 and/or LCDR3 of antibody 1 e.g. a set of CDRs of antibody 1.
• the binding member may comprise a set of VH CDRs of antibody 1.
• it may also comprise a set of VL CDRs of antibody 1, and the VL CDRs may be from the same or a different antibody as the VH CDRs.
• a VH domain comprising a set of HCDRs of antibody 1, and/or a VL domain comprising a set of LCDRs of antibody 1, are also provided by the invention.
• a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although as discussed further below a VH or VL domain alone may be used to bind antigen.
• the antibody 1 VH domain may be paired with the antibody 1 VL domain, so that an antibody antigen-binding site is formed comprising both the antibody 1 VH and VL domains.
• the antibody 1 VH is paired with a VL domain other than the antibody 1 VL.
• Light-chain promiscuity is well established in the art.
• the VH of antibody 1 may be paired with the VL of antibody 1 or of another antibody.
• a binding member may comprise a set of H and/or L CDRs of antibody 1 with one or more amino acid mutations within the disclosed set of H and/or L CDRs.
• the mutation may be an amino acid substitution, insertion or deletion.
• there may be up to 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations, e.g. substitutions, within the set of H and/or L CDRs.
• there may be one or up to 5, 4, 3 or 2 mutations, e.g. substitutions in HCDR3 and/or there may be one or up to 5, 4, 3 or 2 mutations, e.g. substitutions, in LCDR3.
• a binding member may comprise an antibody molecule having one or more CDRs, e.g. a set of CDRs, within an antibody framework.
• CDRs e.g. a set of CDRs
• one or more CDRs or a set of CDRs of an antibody may be grafted into a framework (e.g. human framework) to provide an antibody molecule.
• the framework regions may be of human germline gene segment sequences.
• the framework may be germlined, whereby one or more residues within the framework are changed to match the residues at the equivalent position in the most similar human germline framework.
• a binding member of the invention may be an isolated human antibody molecule having a VH domain comprising a set of HCDRs in a human germline framework, e.g. human germline framework, e.g. human VH3-23.
• the binding member also has a VL domain comprising a set of LCDRs, e.g. in a human germline framework, e.g. human VK1 L12.
• a germlined VH or VL domain may or may not be germlined at one or more Vernier residues.
• a non-germlined antibody molecule has the same CDRs, but different frameworks, compared to a germlined antibody molecule.
• the antibody sequences for antibody 1 shown herein in the appended sequence listing, are germlined. As described herein in the Examples, germlining from antibody 1 VH and VL domains as isolated involved making the following mutations in the VH domain: Q1E, V5L, R16G, V23A, G24A, H39Q, G83R and R105Q; and the following mutations in the VL domain: I15V, A58V and D70E.
• a VH domain according to the invention may comprise the amino acid sequence in SEQ ID NO:2 but with 1 or more, e.g. 2, 3, 4, 5, 6, 7 or all 8, of the above VH mutations reversed. In one embodiment, the VH domain is not substituted at positions 30, 97 and 98 of SEQ ID NO. 2.
• a VL domain according to the invention may comprise the amino acid sequence in SEQ ID NO: 7 but with 1, 2, or all 3 of the above VL mutations reversed. In one embodiment, the VL domain is not substituted at position 2 of SEQ ID NO. 7.
• a binding member of the invention may comprise (i) a VH domain comprising the amino acid sequence SEQ ID NO:2 or the amino acid sequence SEQ ID NO:2 with one or more amino acid alterations (e.g. substitutions) in one or more of the framework regions (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 substitutions); and (ii) a VL domain comprising the amino acid sequence in SEQ ID NO:7 or the amino acid sequence SEQ ID NO: 7 but with one or more amino acid alterations (e.g. substitutions) in one or more of the framework regions (e.g. 1, 2 or 3 substitutions).
• the VH domain of the binding member e.g.
• the VH domain may comprise the amino acid sequence in SEQ ID NO:2 or may comprise the amino acid sequence in SEQ ID NO:2 with one or more (e.g. 1, 2 or all) of the following amino acid substitutions: G30S; T97A; R98K.
• the VL domain may comprise the amino acid sequence in SEQ ID NO:7 or may comprise the amino acid sequence in SEQ ID NO:2 with the following amino acid substitution: T2Ile.
• the 3′ cgt codon, and corresponding Arginine residue, shown in the nucleotide and amino acid sequence for the kappa VL domain of Antibody 1 was included in the expressed scFv and IgG sequences of this antibody.
• the C terminal Arginine residue of the sequence corresponds to Kabat residue 108. The origin of this residue and its encoding triplet cgt is explained below.
• a nucleotide sequence encoding the antibody light chain comprising a first exon encoding the VL domain, a second exon encoding the CL domain, and an intron separating the first exon and the second exon.
• the intron is spliced out by cellular mRNA processing machinery, joining the 3′ end of the first exon to the 5′ end of the second exon.
• DNA having the said nucleotide sequence was expressed as RNA
• the first and second exons were spliced together.
• Translation of the spliced RNA produces a polypeptide comprising the VL domain and CL domain.
• the Arg at Kabat residue 108 is encoded by the last base (c) of the VL domain framework 4 sequence and the first two bases (gt) of the CL domain.
• the Arginine residue at Kabat residue 108 may be considered to be the C terminal residue of the VL domain of the antibody molecule.
• a binding member of the invention may be one which competes for binding to CXCL13 with any binding member which
• the invention provides a binding member which competes for binding with an scFv antibody molecule having SEQ ID NO: 11.
• the sequence in SEQ ID NO: 11 corresponds to the VH domain of SEQ ID NO:2 linked to the VL domain of SEQ ID NO:7 by a linker sequence (Gly120 to Ser134).
• the binding member may comprise a CDR or set of CDRs of antibody 1, or a variant thereof as described elsewhere herein.
• the binding member may be an antibody molecule e.g. an IgG (e.g. IgG1) or scFv, as described elsewhere herein.
• a binding member may show complete or partial competition in a competition assay.
• a binding member may show at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% competition for binding to CXCL13 in a competition assay, for example, with an antibody scFv molecule having the sequence of SEQ ID NO: 11.
• Competition between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one binding member which can be detected in the presence of one or more other untagged binding members, to enable identification of binding members which bind the same epitope or an overlapping epitope.
• a TRF-FRET epitope competition assay using the TRF-FRET technology described herein in relation to cAMP assays, may be used (a TRF-FRET epitope competition assay), e.g. the HTRF® epitope competition assay.
• a HTRF® epitope competition assay typically comprises: mixing XL665-labelled CXCL13, europium cryptate-labelled reference antibody (e.g. an scFv antibody molecule having SEQ ID NO: 11) and a test binding member; applying light at 337 nm (thereby exciting the europium cryptate donor); determining the fluorescent signal at 620 nm (donor) and 665 nm (acceptor) by TRF; and determining the ratio of signal 665 nm/620 nm as a measure of the FRET which has occurred.
• a test binding member e.g. IgG or ScFv antibody molecule, which competes with the reference antibody for binding to CXCL13 reduces the FRET which occurs.
• a detailed method for this assay is provided in the Examples.
• a further aspect of the present invention provides a binding member comprising a human antibody antigen-binding site that competes with an antibody molecule, for example especially an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3, or set of CDRs of antibody 1 for binding to CXCL13.
• an antibody molecule for example especially an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3, or set of CDRs of antibody 1 for binding to CXCL13.
• the present invention provides a binding member comprising a human antibody antigen-binding site which competes with an antibody antigen-binding site for binding to CXCL13, wherein the antibody antigen-binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise a set of CDRs of antibody 1 disclosed herein.
• a binding member of the invention may bind an epitope of human CXCL-13 wherein said epitope includes at least one residue of the sequence Ile-Leu-Pro-Arg-Gly-Asn-Gly-Cys-Pro-Arg-Lys-Glu (SEQ ID NO: 20) at positions 31-42 of mature human IL-17A. It may for example bind one, two, three, four, five or more than five residues of SEQ ID NO: 20.
• a binding member of the invention may bind other regions of human CXCL-13 in addition to SEQ ID NO: 20.
• Any suitable method may be used to determine the sequence of residues bound by a binding member, e.g. hydrogen-deuterium exchange, site-directed mutagenesis, mass spectrometry, NMR and X-ray crystallography.
• Peptide amide hydrogen exchange is a very well described methodology used to study proteins (Englander, S. W. et al. Methods Enzymol. 232:26-42, 1994). More recently this has been further developed to use deuterium labelled proteins that can exchange for protons and coupling this to mass spectrometry to measure the rates of exchange across a whole protein (Pantazatos, D. et al. Proc. Natl. Acad. Sci. 101(3):751-756, 2004). This rate of hydrogen/deuterium exchange (H/D exchange) can be modified significantly by accessibility to solvent such that when a part of the protein is involved in binding to another molecule the rate of exchange will slow significantly.
• the invention provides an isolated nucleic acid which comprises a sequence encoding a binding member, VH domain and/or VL domain according to the present invention, and methods of preparing a binding member, a VH domain and/or a VL domain of the invention, which comprise expressing said nucleic acid under conditions to bring about production of said binding member, VH domain and/or VL domain, and recovering it.
• Another aspect of the present invention provides nucleic acid, generally isolated, encoding a VH CDR or VL CDR sequence disclosed herein.
• a further aspect provides a host cell containing or transformed with nucleic acid of the invention.
• compositions containing binding members of the invention and their use in methods of inhibiting and/or neutralising CXCL13, including methods of treatment of the human or animal body by therapy.
• Binding members according to the invention may be used in a method of treatment or diagnosis, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in the human or animal body (e.g. in a human patient), which comprises administering to said patient an effective amount of a binding member of the invention.
• a method of treatment which may include prophylactic treatment
• Conditions treatable in accordance with the present invention include any in which CXCL13 plays a role, as discussed in detail elsewhere herein.
• CXCL13 is chemokine (C—X—C motif) ligand 13.
• a sequence of full length human CXCL13 is deposited under Accession number NP — 006410 SEQ ID NO: 12.
• the full length amino acid sequence includes a 22 residue N-terminal peptide which is cleaved in vivo to generate the mature form.
• Mature CXCL13 has amino acid sequence SEQ ID NO: 13.
• Mature CXCL13 is the in vivo target antigen for therapeutic and diagnostic applications, and references herein to human CXCL13 are to mature CXCL13 unless otherwise specified.
• human CXCL13 was expressed in the MEL cell line.
• Human CXCL13 expressed from MEL cells was truncated at the C-terminus, and its amino acid sequence is SEQ ID NO: 14.
• SEQ ID NO: 14 is the MEL-expressed human CXCL13 as used in the assays described herein. Mature, non-truncated CXCL13 (SEQ ID NO: 13) would also be suitable for use in assays and the truncation is not believed to affect the results obtained.
• Biotinylated human CXCL13 was used in some of the assays described herein. This CXCL13 was synthetically produced and has the mature CXCL13 sequence SEQ ID NO: 13, carrying biotin at the most C terminal lysine residue.
• CXCL13 may be cynomolgus CXCL13.
• the cynomolgus CXCL13 When expressed from MEL cells for the experimental work herein, the cynomolgus CXCL13 was C-terminally truncated by 5 amino acids and has amino acid sequence SEQ ID NO: 16.
• a proposed sequence of mature untruncated cynomolgus CXCL13 is SEQ ID NO 15. As shown the full untruncated cyno sequence is proposed to include KRKIP at the C-terminus as for human and rhesus CXCL13. This is assumed because the rhesus primer used for cloning was designed to anneal to the region that is removed from the mature human CXCL13 when it is truncated.
• CXCL13 may be recombinant, and/or may be either glycosylated or unglycosylated. Glycosylated and/or unglycosylated CXCL13 may be expressed in recombinant systems, e.g. in mammalian cells such as human cell lines, murine cell lines e.g. MEL cells, or in Chinese hamster ovary (CHO) cells.
• mammalian cells such as human cell lines, murine cell lines e.g. MEL cells, or in Chinese hamster ovary (CHO) cells.
• CXCR5 is the receptor for CXCL13 as described elsewhere herein.
• Human CXCR5 exists in two isoforms.
• An amino acid sequence of CXCR5 isoform 1 is deposited under Accession number NP — 001707 and is shown herein as SEQ ID NO: 17.
• An amino acid sequence of CXCR5 isoform 2 is deposited under Accession number NP — 116743 and is shown herein as SEQ ID NO: 18.
• Isoform 2 is truncated by 45 amino acids at the N-terminus. Isoform 2 therefore lacks the translation initiation codon and extracellular domain of variant 1 and is not likely to bind ligand and be functional Isoform 1 of human CXCR5, having SEQ ID NO: 17, was used in the experiments herein. Accordingly, references herein to CXCR5 are to human isoform 1 unless otherwise indicated.
• CXCR5 referred to herein may be human or non-human as indicated by the context.
• CXCR5 from an appropriate species may be selected.
• An appropriate species may be human or non-human CXCR5 where the binding partner is human or non-human CXCL13 from the same species.
• human or non-human CXCL13 may be used in assays with human or non-human CXCR5 from a different species where human or non-human CXCL13 is known to cross-react.
• binding pair This describes one member of a pair of molecules that bind one another.
• the members of a binding pair may be naturally derived or wholly or partially synthetically produced.
• One member of the pair of molecules has an area on its surface, or a cavity, which binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules.
• types of binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, and enzyme-substrate.
• the present invention is concerned with antigen-antibody type reactions.
• a binding member normally comprises a molecule having an antigen-binding site.
• a binding member may be an antibody molecule or a non-antibody protein that comprises an antigen-binding site.
• An antigen-binding site may be provided by means of arrangement of CDRs on non-antibody protein scaffolds, such as fibronectin or cytochrome B etc. [2, 3, 4], or by randomising or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et al. [4]. Protein scaffolds for antibody mimics are disclosed in WO/0034784, which is herein incorporated by reference in its entirety, in which the inventors describe proteins (antibody mimics) that include a fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g.
• a set of HCDRs may be provided by any domain member of the immunoglobulin gene superfamily.
• the scaffold may be a human or non-human protein.
• An advantage of a non-antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties, such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, or to bind within protein cavities of the target antigen.
• Use of antigen binding sites in non-antibody protein scaffolds is reviewed in Wess, 2004 [5].
• Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen-binding site that binds the target antigen.
• proteins include the IgG-binding domains of protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain), lipocalins as well as gamma-crystalline and other AffilinTM scaffolds (Scil Proteins).
• Examples of other approaches include synthetic “Microbodies” based on cyclotides—small proteins having intra-molecular disulphide bonds, Microproteins (VersabodiesTM, Amunix) and ankyrin repeat proteins (DARPins, Molecular Partners).
• a binding member according to the present invention may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen.
• Binding members of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e.g. via a peptidyl bond or linker).
• a binding member may comprise a catalytic site (e.g. in an enzyme domain) as well as an antigen binding site, wherein the antigen-binding site binds to the antigen and thus targets the catalytic site to the antigen.
• the catalytic site may inhibit biological function of the antigen, e.g. by cleavage.
• CDRs can be carried by non-antibody scaffolds
• the structure for carrying a CDR or a set of CDRs of the invention will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes.
• the structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, et al., 1987 [6] and updates thereof.
• a number of academic and commercial on-line resources are available to query this database. For example, see ref. [7] and the associated on-line resource, currently at the web address of http://www.bioinf.org.uk/abs/simkab.html.
• CDR region or CDR it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulin as defined by Kabat et al. 1991 [8], and later editions.
• An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.
• the term CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.
• the third CDR of the heavy chain (HCDR3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It may be as short as 2 amino acids although the longest size known is 26. CDR length may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody [9, 10, 11, 12, 13, 14, 15, 16].
• antibody antigen-binding site any polypeptide or protein comprising an antibody antigen-binding site. It must be understood here that the invention does not relate to the antibodies in natural form, that is to say they are not in their natural environment but that they have been able to be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they can then contain unnatural amino acids as will be described later.
• Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab′, Fab′-SH, scFv, Fv, dAb and Fd.
• antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab 2 , Fab 3 , diabodies, triabodies, tetrabodies and minibodies.
• Antibody molecules and methods for their construction and use are described in [17].
• antibody molecule should be construed as covering any binding member or substance having an antibody antigen-binding site with the required specificity and/or binding to antigen.
• this term covers antibody fragments and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic.
• Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide (e.g. derived from another species or belonging to another antibody class or subclass) are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, and a large body of subsequent literature.
• mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies [19].
• Humanised antibodies can be produced using techniques known in the art such as those disclosed in for example WO91/09967, U.S. Pat. No. 5,585,089, EP592106, U.S. Pat. No. 565,332 and WO93/17105.
• WO2004/006955 describes methods for humanising antibodies, based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, e.g. germline antibody gene segments.
• Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences.
• the subset members may be further ranked by amino acid similarity between the human and the non-human CDR sequences.
• top ranking human sequences are selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies.
• Chimeric antibodies made according to the method are also disclosed.
• Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et al. [20] or Krebs et al. [21].
• binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH 1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment [22, 23, 24], which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen-binding site [25, 26]; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent
• Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains [28].
• Minibodies comprising a scFv joined to a CH3 domain may also be made [29].
• Other examples of binding fragments are Fab′, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab′-SH, which is a Fab′ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
• Antibody fragments of the invention can be obtained starting from antibody molecule 1, by methods such as digestion by enzymes e.g. pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction.
• the antibody fragments comprised in the present invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers, such as those supplied by the company Applied Biosystems, etc., or by nucleic acid synthesis and expression.
• Functional antibody fragments according to the present invention include any functional fragment whose half-life is increased by a chemical modification, especially by PEGylation, or by incorporation in a liposome.
• a dAb domain antibody is a small monomeric antigen-binding fragment of an antibody, namely the variable region of an antibody heavy or light chain [24].
• VH dAbs occur naturally in camelids (e.g. camel, llama) and may be produced by immunizing a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and suitable for selection and affinity maturation.
• Camelid VH dAbs are being developed for therapeutic use under the name “nanobodiesTM”.
• a binding member of the present invention may be a dAb comprising a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.
• bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule [30]. Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumour cells.
• bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways [31], e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
• These antibodies can be obtained by chemical methods [32,33] or somatic methods [34, 35] but likewise and preferentially by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought [36].
• bispecific antibodies include those of the BiTETM technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.
• Bispecific antibodies can be constructed as entire IgG, as bispecific Fab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against CXCL13, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al., 1996 [37].
• the antibodies may be monoclonal antibodies, especially of human, murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art.
• Monoclonal antibodies can be obtained, for example, from an animal cell immunized against CXCL13, or one of its fragments containing the epitope recognized by said monoclonal antibodies. Suitable fragments and peptides or polypeptides comprising them are described herein, and may be used to immunise animals to generate antibodies against CXCL13. Said CXCL13, or one of its fragments, can especially be produced according to the usual working methods, by genetic recombination starting with a nucleic acid sequence contained in the cDNA sequence coding for CXCL13 or fragment thereof, by peptide synthesis starting from a sequence of amino acids comprised in the peptide sequence of the CXCL13 and/or fragment thereof.
• the monoclonal antibodies can, for example, be purified on an affinity column on which CXCL13 or one of its fragments containing the epitope recognized by said monoclonal antibodies, has previously been immobilized. More particularly, the monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion-exchange chromatography aimed at eliminating the residual protein contaminants as well as the DNA and the LPS, in itself, followed or not followed by exclusion chromatography on Sepharose gel in order to eliminate the potential aggregates due to the presence of dimers or of other multimers. In one embodiment, the whole of these techniques can be used simultaneously or successively.
• an antibody antigen-binding site comprises the part of the antibody that binds to and is complementary to all or part of the target antigen.
• an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
• An antibody antigen-binding site may be provided by one or more antibody variable domains.
• An antibody antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
• binding members of the invention or nucleic acid encoding such binding members, will generally be in accordance with the present invention.
• binding members, VH and/or VL domains, and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.
• Isolated members and isolated nucleic acid will be free or substantially free of material with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.
• Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
• Binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
• heterologous eukaryotic cells e.g. CHO or NS0 (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
• Heterogeneous preparations comprising anti-CXCL13 antibody molecules also form part of the invention.
• such preparations may be mixtures of antibodies with full-length heavy chains and heavy chains lacking the C-terminal lysine, with various degrees of glycosylation and/or with derivatized amino acids, such as cyclization of an N-terminal glutamic acid to form a pyroglutamic acid residue.
• the phrase “substantially as set out” refers to the characteristic(s) of the relevant CDRs of the VH or VL domain of binding members described herein will be either identical or highly similar to the specified regions of which the sequence is set out herein.
• the phrase “highly similar” with respect to specified region(s) of one or more variable domains it is contemplated that from 1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4, amino acid substitutions may be made in the CDR and/or VH or VL domain.
• a binding member in accordance with the present invention binds CXCL13 and may neutralise a biological activity of CXCL13.
• a binding member of the present invention may be subjected to potency optimisation, to further improve its neutralizing potency.
• potency optimisation involves mutating the sequence of a selected binding member (normally the variable domain sequence of an antibody) to generate a library of binding members, which are then assayed for potency and the more potent binding members are selected.
• selected “potency-optimised” binding members tend to have a higher potency than the binding member from which the library was generated.
• high potency binding members may be obtained without optimisation.
• high potency binding members may be obtained directly from an initial screen e.g. a biochemical neutralization assay.
• a “potency optimized” binding member refers to a binding member with an optimized potency of neutralization of a particular activity or downstream function of CXCL13. Assays and potencies are described in more detail elsewhere herein.
• Potency-optimized and non-optimized binding members are aspects of the invention, as well as methods for potency optimization from a selected binding member.
• the present invention thus allows the skilled person to generate binding members having high potency.
• the present invention provides a method of obtaining one or more binding members able to bind the antigen, the method including bringing into contact a library of binding members according to the invention and said antigen, and selecting one or more binding members of the library able to bind said antigen.
• the library may be displayed on particles or molecular complexes, e.g. replicable genetic packages, such as yeast, bacterial or bacteriophage (e.g. T7) particles, viruses, cells or covalent, ribosomal or other in vitro display systems, each particle or molecular complex containing nucleic acid encoding the antibody VH variable domain displayed on it, and optionally also a displayed VL domain if present.
• particles or molecular complexes e.g. replicable genetic packages, such as yeast, bacterial or bacteriophage (e.g. T7) particles, viruses, cells or covalent, ribosomal or other in vitro display systems, each particle or molecular complex containing nucleic acid encoding the antibody VH variable domain displayed on it, and optionally also a displayed VL domain if present.
• Phage display is described in WO92/01047 and e.g. U.S. Pat. No. 5,969,108, U.S. Pat. No. 5,565,33
• nucleic acid may be taken from a bacteriophage or other particle or molecular complex displaying a said selected binding member.
• nucleic acid may be used in subsequent production of a binding member or an antibody VH or VL variable domain by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage or other particle or molecular complex displaying a said selected binding member.
• An antibody VH variable domain with the amino acid sequence of an antibody VH variable domain of a said selected binding member may be provided in isolated form, as may a binding member comprising such a VH domain.
• Ability to bind CXCL13 may be further tested, also ability to compete with a binding member e.g. an antibody 1, (e.g. in scFv format and/or IgG format, e.g. IgG1) for binding to CXCL13 may be determined.
• a binding member e.g. an antibody 1, (e.g. in scFv format and/or IgG format, e.g. IgG1) for binding to CXCL13 may be determined.
• Ability to neutralize CXCL13 may be tested, as discussed further elsewhere herein.
• a binding member according to the present invention may bind CXCL13 with the affinity of antibody 1, e.g. scFv or IgG1, or with an affinity that is better.
• a binding member according to the present invention may neutralise a biological activity of CXCL13 with the potency of antibody 1, e.g. scFv, or IgG1, or with a potency that is better.
• Binding affinity and neutralization potency of different binding members can be compared under appropriate conditions.
• Variants of the VH and VL domains and CDRs of the present invention including those for which amino acid sequences are set out herein, and which can be employed in binding members for CXCL13 can be obtained by means of methods of sequence alteration or mutation and screening for antigen-binding members with desired characteristics.
• desired characteristics include but are not limited to:
• Variants of antibody molecules disclosed herein may be produced and used in the present invention.
• quantitative activity-property relationships of antibodies can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [41, 42, 43, 44, 45, 46].
• the properties of antibodies can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of antibody sequence, functional and three-dimensional structures and these properties can be considered singly and in combination.
• An antibody antigen-binding site composed of a VH domain and a VL domain is typically formed by six loops of polypeptide: three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH).
• VL light chain variable domain
• VH heavy chain variable domain
• Analysis of antibodies of known atomic structure has elucidated relationships between the sequence and three-dimensional structure of antibody combining sites [47,48]. These relationships imply that, except for the third region (loop) in VH domains, binding site loops have one of a small number of main-chain conformations: canonical structures.
• the canonical structure formed in a particular loop has been shown to be determined by its size and the presence of certain residues at key sites in both the loop and in framework regions [47, 48].
• sequence-structure relationship can be used for prediction of those residues in an antibody of known sequence, but of an unknown three-dimensional structure, which are important in maintaining the three-dimensional structure of its CDR loops and hence maintain binding specificity. These predictions can be backed up by comparison of the predictions to the output from lead optimization experiments.
• a model can be created of the antibody molecule [49] using any freely available or commercial package, such as WAM [50].
• a protein visualisation and analysis software package, such as Insight II (Accelrys, Inc.) or Deep View [51] may then be used to evaluate possible substitutions at each position in the CDR. This information may then be used to make substitutions likely to have a minimal or beneficial effect on activity.
• Variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind and/or neutralize CXCL13 and/or for any other desired property.
• VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed.
• a further aspect of the invention is an antibody molecule comprising a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain of antibody 1 shown in the appended sequence listing, and/or comprising a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of antibody 1 shown in the appended sequence listing.
• Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST [52], FASTA [53], or the Smith-Waterman algorithm [54], e.g. employing default parameters.
• VH domains, VL domains and/or CDRs or sets of CDRs may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue).
• Variants may include less than about 20 alterations, such as less than about 15, less than about 10 or less than about 5 alterations, e.g. 5, 4, 3, 2 or 1.
• Alterations may be made in one or more framework regions and/or one or more CDRs.
• the alterations normally do not result in loss of function, so a binding member comprising a thus-altered amino acid sequence may retain an ability to bind and/or neutralize CXCL13. It may retain the same quantitative binding and/or neutralizing ability as a binding member in which the alteration is not made, e.g. as measured in an assay described herein.
• the binding member comprising a thus-altered amino acid sequence may have an improved ability to bind and/or neutralize CXCL13.
• Alteration may comprise replacing one or more amino acid residue with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein.
• Naturally occurring amino acids include the 20 “standard” L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes.
• Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue.
• Non-standard amino acids may be naturally occurring or non-naturally occurring.
• Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-acetylserine, etc. [55].
• Those amino acid residues that are derivatised at their N-alpha position will only be located at the N-terminus of an amino-acid sequence.
• an amino acid is an L -amino acid, but it may be a D -amino acid.
• Alteration may therefore comprise modifying an L -amino acid into, or replacing it with, a D -amino acid.
• Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and amino acids in the present invention may be subject to such modification.
• Amino acid sequences in antibody domains and binding members of the invention may comprise non-natural or non-standard amino acids described above.
• Non-standard amino acids e.g. D -amino acids
• D -amino acids may be incorporated into an amino acid sequence during synthesis, or by modification or replacement of the “original” standard amino acids after synthesis of the amino acid sequence.
• non-standard and/or non-naturally occurring amino acids increases structural and functional diversity, and can thus increase the potential for achieving desired CXCL13-binding and neutralizing properties in a binding member of the invention.
• D -amino acids and analogues have been shown to have different pharmacokinetic profiles compared with standard L -amino acids, owing to in vivo degradation of polypeptides having L -amino acids after administration to an animal e.g. a human, meaning that D -amino acids are advantageous for some in vivo applications.
• Novel VH or VL regions carrying CDR-derived sequences of the invention may be generated using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain.
• Such a technique is described by Gram et al. [56], who used error-prone PCR.
• one or two amino acid substitutions are made within an entire variable domain or set of CDRs.
• Another method that may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al. [57] and Schier et al. [58].
• a further aspect of the invention provides a method for obtaining an antibody antigen-binding site for CXCL13, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein, a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a binding member or an antibody antigen-binding site for CXCL13 and optionally with one or more desired properties, e.g. ability to neutralize CXCL13 activity.
• Said VL domain may have an amino acid sequence which is substantially as set out herein.
• An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.
• a CDR amino acid sequence substantially as set out herein may be carried as a CDR in a human antibody variable domain or a substantial portion thereof.
• the HCDR3 sequences substantially as set out herein represent embodiments of the present invention and each of these may be carried as a HCDR3 in a human heavy chain variable domain or a substantial portion thereof.
• Variable domains employed in the invention may be obtained or derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus or actual sequences of known human variable domains.
• a variable domain can be derived from a non-human antibody.
• a CDR sequence of the invention e.g. CDR3
• CDR3 may be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology.
• Marks et al. [59] describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5′ end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3.
• the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide binding members of the invention.
• the repertoire may then be displayed in a suitable host system, such as the phage display system of WO92/01047, which is herein incorporated by reference in its entirety, or any of a subsequent large body of literature, including Kay, Winter & McCafferty [60], so that suitable binding members may be selected.
• a repertoire may consist of from anything from 10 4 individual members upwards, for example at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 or at least 10 10 members or more.
• Other suitable host systems include, but are not limited to yeast display, bacterial display, T7 display, viral display, cell display, ribosome display and covalent display.
• a method of preparing a binding member for CXCL13 antigen comprises:
• VL CDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain that either include a CDR3 to be replaced or lack a CDR3 encoding region.
• one or more, or all three CDRs may be grafted into a repertoire of VH or VL domains that are then screened for a binding member or binding members for CXCL13.
• one or more of the antibody 1 HCDR1, HCDR2 and HCDR3 or the antibody 1 set of HCDRs may be employed, and/or one or more of the antibody 1 LCDR1, LCDR2 and LCDR3 or the antibody 1 set of LCDRs may be employed.
• VH and VL domains sets of CDRs and sets of HCDRs and/or sets of LCDRs disclosed herein may be employed.
• a substantial portion of an immunoglobulin variable domain may comprise at least the three CDR regions, together with their intervening framework regions.
• the portion may also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region.
• Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions.
• construction of binding members of the present invention made by recombinant DNA techniques may result in the introduction of N— or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps.
• Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including antibody constant regions, other variable domains (for example in the production of diabodies) or detectable/functional labels as discussed in more detail elsewhere herein.
• binding members comprise a pair of VH and VL domains
• single binding domains based on either VH or VL domain sequences form further aspects of the invention. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner. For example, see the discussion of dAbs above.
• these domains may be used to screen for complementary domains capable of forming a two-domain binding member able to bind CXCL13.
• This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in WO92/01047, herein incorporated by reference in its entirety, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain binding member is selected in accordance with phage display techniques, such as those described in that reference. This technique is also disclosed in Marks et al, ibid.
• Binding members of the present invention may further comprise antibody constant regions or parts thereof, e.g. human antibody constant regions or parts thereof.
• a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human C ⁇ or C ⁇ chains.
• a binding member based on a VH domain may be attached at its C-terminal end to all or part (e.g. a CH1 domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1 and IgG4.
• IgG1 is advantageous, due to its effector function and ease of manufacture. Any synthetic or other constant region variant that has these properties and stabilizes variable regions may also be useful in the present invention.
• Binding members of the invention may be labelled with a detectable or functional label.
• a binding member or antibody molecule can be present in the form of an immunoconjugate so as to obtain a detectable and/or quantifiable signal.
• An immunoconjugate may comprise an antibody molecule of the invention conjugated with detectable or functional label.
• a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers.
• binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
• Suitable labels include, by way of illustration and not limitation,
• Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S. Pat. No. 4,275,149, and Boguslaski, et al., U.S. Pat. No. 4,318,980, each of which are herein incorporated by reference in their entireties.
• Suitable fluorescers and chemiluminescers are disclosed in Litman, et al., U.S. Pat. No. 4,275,149, which is incorporated herein by reference in its entirety.
• Labels further include chemical moieties, such as biotin that may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin or streptavidin. Detectable labels may be attached to antibodies of the invention using conventional chemistry known in the art.
• Immunoconjugates or their functional fragments can be prepared by methods known to the person skilled in the art. They can be coupled to enzymes or to fluorescent labels directly or by the intermediary of a spacer group or of a linking group, such as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or in the presence of coupling agents, such as those mentioned above for the therapeutic conjugates.
• Conjugates containing labels of fluorescein type can be prepared by reaction with an isothiocyanate.
• the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents.
• the label can also be bound to another binding member that binds the antibody of the invention, or to a support.
• the label can directly produce a signal, and therefore, additional components are not required to produce a signal.
• Numerous organic molecules for example fluorescers, are able to absorb ultraviolet and visible light, where the light absorption transfers energy to these molecules and elevates them to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength. This second wavelength emission may also transfer energy to a labelled acceptor molecule, and the resultant energy dissipated from the acceptor molecule by emission of light for example fluorescence resonance energy transfer (FRET).
• FRET fluorescence resonance energy transfer
• Other labels that directly produce a signal include radioactive isotopes and dyes.
• the label may need other components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal, which may include substrates, coenzymes, enhancers, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances.
• suitable signal producing systems can be found in Ullman, et al. U.S. Pat. No. 5,185,243, which is herein incorporated herein by reference in its entirety.
• the present invention provides a method comprising causing or allowing binding of a binding member as provided herein to CXCL13.
• binding may take place in vivo, e.g. following administration of a binding member, or nucleic acid encoding a binding member, or it may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immunoprecipitation, affinity chromatography, and biochemical or cell-based assays, such as a cAMP, calcium release or B cell chemotaxis assay.
• the present invention also provides for measuring levels of antigen directly, by employing a binding member according to the invention for example in a biosensor system.
• the present invention comprises a method of detecting and/or measuring binding to CXCL13, comprising, (i) exposing said binding member to CXCL13 and (ii) detecting binding of said binding member to CXCL13, wherein binding is detected using any method or detectable label described herein.
• This, and any other binding detection method described herein may be interpreted directly by the person performing the method, for instance, by visually observing a detectable label.
• this method may produce a report in the form of an autoradiograph, a photograph, a computer printout, a flow cytometry report, a graph, a chart, a test tube or container or well containing the result, or any other visual or physical representation of a result of the method.
• the amount of binding of binding member to CXCL13 may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest. Screening for CXCL13 binding and/or the quantitation thereof may be useful, for instance, in screening patients for diseases or disorders referred to herein and/or any other disease or disorder involving aberrant CXCL13 expression and/or activity.
• CXCL13 levels are a useful diagnostic and/or prognostic indicator for various disorders, for example, inflammatory and/or autoimmune diseases, as described elsewhere herein.
• elevated levels of CXCL13 in e.g. serum or synovial fluid samples may be used as a predictor of rheumatoid arthritis.
• a diagnostic method of the invention may comprise (i) obtaining a tissue or fluid sample from a subject, (ii) exposing said tissue or fluid sample to one or more binding members of the present invention; and (iii) detecting bound CXCL13 as compared with a control sample, wherein an increase in the amount of CXCL13 binding as compared with the control may indicate an aberrant level of CXCL13 expression or activity.
• Tissue or fluid samples to be tested include blood, serum, urine, biopsy material, tumours, or any tissue suspected of containing aberrant CXCL13 levels. Subjects testing positive for aberrant CXCL13 levels or activity may also benefit from the treatment methods disclosed later herein.
• Radioimmunoassay is one possibility. Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the binding member. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the binding member determined. The more antigen there is in the test sample the less radioactive antigen will bind to the binding member.
• a competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule.
• the reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red, and lanthanide chelates or cryptates. Suitable chromogenic dyes include diaminobenzidine.
• Other reporters include macromolecular colloidal particles or particulate material, such as latex beads that are colored, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
• These molecules may be enzymes, which catalyze reactions that develop, or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
• the signals generated by individual binding member-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant binding member binding in samples (normal and test).
• kits comprising a binding member according to any aspect or embodiment of the present invention is also provided as an aspect of the present invention.
• the binding member may be labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Further the binding member may or may not be attached to a solid support.
• Components of a kit are generally sterile and in sealed vials or other containers. Kits may be employed in diagnostic analysis or other methods for which binding members are useful.
• a kit may contain instructions for use of the components in a method, e.g. a method in accordance with the present invention. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention.
• the ancillary materials include a second, different binding member which binds to the first binding member and is conjugated to a detectable label (e.g., a fluorescent label, radioactive isotope or enzyme).
• a detectable label e.g., a fluorescent label, radioactive isotope or enzyme.
• Antibody-based kits may also comprise beads for conducting an immunoprecipitation. Each component of the kits is generally in its own suitable container. Thus, these kits generally comprise distinct containers suitable for each binding member. Further, the kits may comprise instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay.
• the present invention also provides the use of a binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a binding member as provided by the present invention in a competition assay. This may be where the physical separation of bound from unbound antigen is not required.
• Linking a reporter molecule to the binding member so that a physical or optical change occurs on binding is one possibility.
• the reporter molecule may directly or indirectly generate detectable signals, which may be quantifiable.
• the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
• the present invention extends to a binding member that competes for binding to CXCL13, e.g. human CXCL13, with any binding member defined herein, e.g. antibody 1, e.g. in IgG1 format.
• Competition between binding members may be assayed easily in vitro, for example by tagging a specific reporter molecule to one binding member which can be detected in the presence of other untagged binding member(s), to enable identification of binding members which bind the same epitope or an overlapping epitope.
• Competition may be determined for example using ELISA in which CXCL13 is immobilized to a plate and a first tagged or labelled binding member along with one or more other untagged or unlabelled binding members is added to the plate. Presence of an untagged binding member that competes with the tagged binding member is observed by a decrease in the signal emitted by the tagged binding member.
• a HTRF® epitope competition assay may be used.
• the present invention includes a method of identifying a CXCL13 binding compound, comprising (i) immobilizing CXCL13 to a support, (ii) contacting said immobilized CXCL13 simultaneously or in a step-wise manner with at least one tagged or labelled binding member according to the invention and one or more untagged or unlabelled test binding compounds, and (iii) identifying a new CXCL13 binding compound by observing a decrease in the amount of bound tag from the tagged binding member.
• Such methods can be performed in a high-throughput manner using a multiwell or array format.
• Such assays may be also be performed in solution. See, for instance, U.S. Pat. No. 5,814,468, which is herein incorporated by reference in its entirety.
• binding methods of the invention may produce a report in the form of an autoradiograph, a photograph, a computer printout, a flow cytometry report, a graph, a chart, a test tube or container or well containing the result, or any other visual or physical representation of a result of the method.
• epitope mapping may be used to identify the epitope bound by a CXCL13 binding member which optionally may have optimized neutralizing and/or modulating characteristics.
• Such an epitope can be linear or conformational.
• a conformational epitope can comprise at least two different fragments of CXCL13, wherein said fragments are positioned in proximity to each other when CXCL13 is folded in its tertiary or quaternary structure to form a conformational epitope which is recognized by an inhibitor of CXCL13, such as a CXCL13-binding member.
• a peptide fragment of the antigen may be employed, especially a peptide including or consisting essentially of an epitope of interest.
• a peptide having the epitope sequence plus one or more amino acids at either end may be used.
• Binding members according to the present invention may be such that their binding for antigen is inhibited by a peptide with or including the sequence given.
• the present invention further provides an isolated nucleic acid encoding a binding member of the present invention.
• Nucleic acid may include DNA and/or RNA.
• the present invention provides a nucleic acid that codes for a CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody molecule, e.g. scFv or IgG1, of the invention as defined above.
• the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.
• the present invention also provides a recombinant host cell that comprises one or more constructs as above.
• Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
• Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
• a yet further aspect provides a method of production of an antibody VH variable domain, the method including causing expression from encoding nucleic acid.
• Such a method may comprise culturing host cells under conditions for production of said antibody VH variable domain.
• a method of production may comprise a step of isolation and/or purification of the product.
• a method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.
• Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.
• the expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see for example Plückthun [62].
• a common bacterial host is E. coli.
• Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.
• Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
• Vectors may be plasmids e.g. phagemid, or viral e.g. ‘phage, as appropriate [66].
• phagemid e.g. phagemid
• viral e.g. ‘phage e.g. ‘phage, as appropriate [66].
• Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. [67].
• a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein.
• a host cell may be in vitro and may be in culture.
• Such a host cell may be in vivo. In vivo presence of the host cell may allow intra-cellular expression of the binding members of the present invention as “intrabodies” or intra-cellular antibodies. Intrabodies may be used for gene therapy.
• a still further aspect provides a method comprising introducing nucleic acid of the invention into a host cell.
• the introduction may employ any available technique.
• suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
• Introducing nucleic acid in the host cell in particular a eukaryotic cell may use a viral or a plasmid based system.
• the plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci.
• suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
• the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
• the purification of the expressed product may be achieved by methods known to one of skill in the art.
• Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.
• the present invention also provides a method that comprises using a construct as stated above in an expression system in order to express a binding member or polypeptide as above.
• Binding members of the present invention may be used in methods of diagnosis or treatment in human or animal subjects, e.g. humans. Binding members for CXCL13 may be used to treat disorders associated with CXCL13, e.g. associated with aberrant CXCL13 expression and/or activity. There is evidence for involvement of CXCL13 in a variety of disorders, as discussed elsewhere herein. Binding members of the invention may be used to inhibit binding of CXCL13 to CXCR5 and thereby to treat a disease or disorder mediated by CXCL13 binding to CXCR5. Disorders associated with CXCL13 include disorders that are caused and/or exacerbated by CXCL13 binding to its receptor CXCR5. Aspects of the invention relate to treatment of such disorders by alleviating or ameliorating one or more symptoms and/or causes of the disorder.
• Binding members for CXCL13 may be used to inhibit or reduce aberrant formation and/or development of lymphoid follicles, e.g. ectopic lymphoid follicles, such as those found in arthritic synovium. Binding members may inhibit or reduce: CXCL13-CXCR5 signalling mediated recruitment of B cells and/or dendritic cells and/or follicular B helper T cells, to follicles; and/or reduce immunoglobulin production by follicular B cells; and/or inhibit or reduce follicular B cell production of cytokines and/or activation of T cells. Binding members may also be used to inhibit or reduce aberrant bone and/or cartilage destruction.
• lymphoid follicles e.g. ectopic lymphoid follicles, such as those found in arthritic synovium. Binding members may inhibit or reduce: CXCL13-CXCR5 signalling mediated recruitment of B
• Binding members may thus be used for the treatment of diseases or disorders associated with aberrant or ectopic lymphoid follicles.
• Disorders associated with ectopic lymphoid follicles include rheumatoid arthritis, Sjogrens syndrome, multiple sclerosis, myasthenia gravis, systemic lupus erythmatosis (SLE), autoimmune thyroid diseases (e.g. Grave's disease, Hashimoto's thyroiditis), chronic infection e.g. Lyme Neuroborreliosis and acute cardiac rejection featuring Quilty effect.
• inflamed arthritic synovium is generally characterised by: ectopic lymphoid follicles containing germinal centres; inflammatory cell infiltrate; B cell production of auto-antibodies and cytokines; B cell activation of T cells; and/or bone and cartilage destruction. Binding members may disrupt formation of the ectopic follicles as described herein and thus inhibit B cell autoantibody production and/or synovial inflammation. Thus binding members may be used in the treatment of arthritic disorders such as rheumatoid arthritis (RA), and/or osteoarthritis.
• RA rheumatoid arthritis
• Binding members of the invention may be used for treatment of bone and/or joint diseases or disorders, especially diseases or disorders associated with destruction and/or remodelling of bone and/or cartilage, e.g. aberrant turnover of bone or cartilage.
• binding members may be used to treat rheumatoid arthritis and/or osteoarthritis.
• Binding members of the invention may be used to inhibit lymphocyte (e.g. B and/or T cell) chemotaxis, and may thus be used to treat disorders associated with lymphocyte chemotaxis such as viral infection (e.g. HIV infection) and leukaemia (e.g. T-cell lineage acute lymphocytic leukaemia and B-cell lineage acute and chronic lymphocytic leukaemia).
• lymphocyte e.g. B and/or T cell
• T cell e.g. B and/or T cell chemotaxis
• disorders associated with lymphocyte chemotaxis such as viral infection (e.g. HIV infection) and leukaemia (e.g. T-cell lineage acute lymphocytic leukaemia and B-cell lineage acute and chronic lymphocytic leukaemia).
• Binding members of the invention may be used to inhibit proliferation of lymphomas and may thus be used to treat disorders associated with lymphoma proliferation, such as leukaemia (e.g. T-cell lineage acute lymphocytic leukaemia and B-cell lineage acute and chronic lymphocytic leukaemia).
• leukaemia e.g. T-cell lineage acute lymphocytic leukaemia and B-cell lineage acute and chronic lymphocytic leukaemia.
• Binding members for CXCL13 may be used to inhibit aberrant formation and/or development of lymphoid follicles, e.g. ectopic lymphoid follicles, inhibit aberrant bone and/or cartilage destruction and/or remodelling, and/or to inhibit chemotaxis of lymphocytes and/or proliferation of lymphomas as described herein. Accordingly, the invention provides a method of inhibiting aberrant formation and/or development of lymphoid follicles, e.g.
• ectopic lymphoid follicles inhibiting aberrant bone and/or cartilage destruction and/or remodelling, and/or inhibiting chemotaxis of lymphocytes and/or proliferationof lymphomas, comprising administering to a patient in need thereof an effective amount of one or more binding members of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the aberrant formation and/or development of lymphoid follicles, e.g. ectopic lymphoid follicles, aberrant bone and/or cartilage destruction and/or remodelling, and/or chemotaxis of lymphocytes and/or proliferation of lymphomas is or are inhibited.
• lymphoid follicles e.g. ectopic lymphoid follicles, aberrant bone and/or cartilage destruction and/or remodelling, and/or chemotaxis of lymphocytes and/or proliferation of lymphomas is or are inhibited.
• Binding members of the invention may be used in the diagnosis of diseases or disorders associated with CXCL13 e.g. in which levels of CXCL13 are altered e.g. elevated relevant to normal levels. Binding members may be used in diagnosis of one or more diseases or disorders described herein, including for example rheumatoid arthritis, Sjogren's syndrome, HIV, myasthenia gravis, angioimmunoblastic T-cell lymphoma and transmissible spongiform encephalopathy (TSE).
• diseases or disorders associated with CXCL13 e.g. in which levels of CXCL13 are altered e.g. elevated relevant to normal levels. Binding members may be used in diagnosis of one or more diseases or disorders described herein, including for example rheumatoid arthritis, Sjogren's syndrome, HIV, myasthenia gravis, angioimmunoblastic T-cell lymphoma and transmissible spongiform encephalopathy (TSE).
• the invention provides a method of treating or reducing the severity of at least one symptom of any of the disorders mentioned herein, comprising administering to a patient in need thereof an effective amount of one or more binding members of the present invention alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein such that the severity of at least one symptom of any of the above disorders is reduced.
• Inflamed synovium is characterised by an inflammatory cell infiltrate and the presence of organised lymphocyte aggregates that resemble lymphoid tissue. These ectopic lymphoid structures contain features of germinal centres including the presence of FDCs, and high endothelial venules (HEVs). Germinal centres provide a microenvironment that supports B cell differentiation into plasma cells and the generation of high affinity antibodies such as rheumatoid factor [70].
• CXCL13 protein in RA synovium has been immunolocalised to FDCs within germinal centres [69] and macrophages in the cellular infiltrate [71]. In RA lesions, CXCL13 is strongly predictive of ectopic lymphoid follicle formation and the development of germinal centre reactions (Takemura, S. et al. J. Immunol. 167: 1072-1080, 2001).
• CXCL13 The role of CXCL13 in RA is further supported by in vivo evidence from animal models of disease. Prophylactic dosing of a CXCL13 neutralising antibody reduces disease severity in collagen induced arthritis (CIA) in the mouse, with a concomitant reduction in inflammatory cell infiltration into the arthritic joint and a reduction in the degree of cartilage and bone erosion [72].
• CIA collagen induced arthritis
• follicles in both the spleen and joints of mice treated with an anti-CXCL13 antibody contain fewer germinal centres and the germinal centres are smaller in size compared with control mice [72].
• mice deficient in CXCR5 develop less severe symptoms in an adjuvant-induced arthritis (AIA) model with a reduction in synovial inflammation and joint destruction (Hartmann, S. et al, European Congress on Immunology, abstract 2672, 2006).
• AIA adjuvant-induced arthritis
• CXCL13 A role for CXCL13 in bone and cartilage turnover has also been demonstrated by in vitro evidence from cultured chondrocytes and osteoblasts.
• CXCR5 is expressed on chondrocytes isolated from articular cartilage from osteoarthritis (OA) patients.
• MMPs matrix metalloproteinases
• CXCL13 also induces the proliferation of cultured osteoblasts derived from OA patients and the expression of interleukin-1 (IL-1) mRNA [74].
• IL-1 interleukin-1
• CXCL13 protein is immunolocalised to mononuclear cell aggregates in bone marrow containing features of lymphoid neogenesis [76].
• the CXCR5 receptor is expressed on a subset of memory T cells with B cell helper function, which are designated follicular B helper T cells (T FH ).
• CXCL13 induces the chemotaxis of CXCR5-expressing T FH cells isolated from human tonsil [77], while in secondary lymphoid organs, CXCR5-positive T FH cells localise to B cell follicles and germinal centres where they support immunoglobulin production [78].
• T FH cells freshly isolated from germinal centres secrete low levels of CXCL13 but this is significantly elevated following stimulation through the TCR and CD28 (Kim, C H. et al. Blood, 104: 1952-1960, 2004).
• a subset of dendritic cells has been identified that express CXCR5 and which localise to lymphoid follicles in response to CXCL13 [79]. This response is dependent on B cell-derived membrane bound lymphotoxin which stimulates the expression of CXCL13 by follicular stromal cells and establishes the chemotactic gradient necessary to recruit CXCR5-positive cells into the follicles.
• B cell depletion therapy is unprecedented in the clinic, there is clinical precedent for efficacy of B cell depletion therapy in RA patients [80].
• B cell depletion with a chimeric anti-CD20 monoclonal antibody is effective and well tolerated with patients experiencing sustained periods of disease remission ranging from months to years following a single course of treatment.
• Reduction of autoantibody levels is a key mechanism of the success of B cell depletion as clinical relapse correlates with the return of serum autoantibodies to pre-treatment levels [81].
• B cells are thought to contribute to RA progression through cytokine production and activation of T cells through antigen presentation.
• CXCL13 neutralisation The inhibition of B cell trafficking by CXCL13 neutralisation represents a novel approach for the treatment of RA.
• CXCL13 neutralisation has the potential both to inhibit autoantibody production and synovial inflammation through disruption of ectopic follicles and germinal centre reactions in RA synovium.
• CXCL13 neutralisation may also provide additional benefit by preventing bone and cartilage destruction by direct and indirect mechanisms.
• CXCL13 Elevated levels of CXCL13 have been described in HIV (Widney, D P. et al. J. Int. Cyt. Res. 25: 702-706, 2005).
• TSE transmissible spongiform encephalopathy
• a binding member of the invention may be used in the treatment of any of the following:
• Binding members of the invention may be used in animals or in animal models of disease, including mice, rats, rabbits, guinea pigs, monkeys, fish, dogs, cows, goats, horses, etc.
• Animal models involving the CXCL13/CXCR5 signalling system are known in the art.
• animal models of rheumatoid arthritis are known, e.g. CIA, AIA and Streptococcal cell wall (SCW) models.
• An animal model of MS is the experimental autoimmune encephalomyelitis (EAE) mouse model.
• Mouse models of SLE include the MRL/lpr mouse model and the NZB ⁇ NZW F 1 (BWF 1 ) mouse model.
• the binding members of the present invention are useful as therapeutic agents in the treatment of diseases or disorders involving CXCL13, e.g. CXCL13 expression and/or activity, especially aberrant expression/activity.
• a method of treatment may comprise administering an effective amount of a binding member of the invention to a patient in need thereof, wherein aberrant expression and/or activity of CXCL13 is decreased.
• a method of treatment may comprise (i) identifying a patient demonstrating aberrant CXCL13 levels or activity, for instance using the diagnostic methods described above, and (ii) administering an effective amount of a binding member of the invention to the patient, wherein aberrant expression and/or activity of CXCL13 is decreased.
• An effective amount according to the invention is an amount that decreases the aberrant expression and/or activity of CXCL13 so as to decrease or lessen the severity of at least one symptom of the particular disease or disorder being treated, but not necessarily cure the disease or disorder.
• the invention also provides a method of antagonising at least one effect of CXCL13 comprising contacting with or administering an effective amount of one or more binding members of the present invention such that said at least one effect of CXCL13 is antagonised.
• Effects of CXCL13 that may be antagonised by the methods of the invention include binding to CXCR5, and any downstream effects that arise as a consequence of these binding reactions.
• the binding members of the present invention are useful as therapeutic agents in the treatment of diseases or disorders involving CXCL13-CXCR5 signalling, especially aberrant CXCL13-CXCR5 signalling.
• aspects of the invention provide methods of treatment comprising administration of a binding member as provided, pharmaceutical compositions comprising such a binding member, and use of such a binding member in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the binding member with a pharmaceutically acceptable excipient.
• a pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation.
• These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.
• Binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the binding member.
• pharmaceutical compositions according to the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
• a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
• carrier or other material will depend on the route of administration, which may be oral, inhaled, intra-tracheal, topical, intra-vesicular or by injection, as discussed below.
• compositions for oral administration such as for example single domain antibody molecules (e.g. “nanobodiesTM”) etc are also envisaged in the present invention.
• Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form.
• a tablet may comprise a solid carrier, such as gelatin or an adjuvant.
• Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
• Physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
• the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
• buffers such as phosphate, citrate and other organic acids
• antioxidants such as ascorbic acid and methionine
• preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or ly
• Binding members of the present invention may be formulated in liquid, semi-solid or solid forms depending on the physicochemical properties of the molecule and the route of delivery.
• Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants.
• Liquid formulations may include a wide range of antibody concentrations and pH.
• Solid formulations may be produced by lyophilisation, spray drying, or drying by supercritical fluid technology, for example.
• Formulations of anti-CXCL13 will depend upon the intended route of delivery: for example, formulations for pulmonary delivery may consist of particles with physical properties that ensure penetration into the deep lung upon inhalation; topical formulations (e.g. for treatment of scarring, e.g.
• a binding member may be prepared with a carrier that will protect the binding member against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art [82].
• Anti-CXCL13 treatment may be given orally (such as for example single domain antibody molecules (e.g. “nanobodiesTM”)) by injection (for example, subcutaneously, intra-articular, intra-venously, intra-peritoneal, intra-arterial or intra-muscularly), by inhalation, intra-tracheal, by the intra-vesicular route (instillation into the urinary bladder), or topically (for example intra-ocular, intra-nasal, rectal, into wounds, on skin).
• the treatment may be administered by pulse infusion, particularly with declining doses of the binding member.
• the route of administration can be determined by the physicochemical characteristics of the treatment, by special considerations for the disease or by the requirement to optimize efficacy or to minimize side-effects.
• One particular route of administration is intra-venous.
• Another route of administering pharmaceutical compositions of the present invention is subcutaneously. It is envisaged that anti-CXCL13 treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous.
• a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• a binding member for CXCL13 may be used as part of a combination therapy in conjunction with an additional medicinal component. Combination treatments may be used to provide significant synergistic effects, particularly the combination of an anti-CXCL13 binding member with one or more other drugs.
• a binding member for CXCL13 may be administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed herein.
• a binding member according to the present invention may be provided in combination or addition with one or more of the following agents:
• a modulator of B cells e.g. a monoclonal antibody targeting B-lymphocytes (such as CD20 (rituximab,MRA-aILl6R or Belimumab) or T-lymphocytes (e.g. CTLA4-Ig (Abatacept), HuMax Il-15);
• An inhibitor may be specific or may be a mixed inhibitor, e.g. an inhibitor targeting more than one of the molecules (e.g. receptors) or molecular classes mentioned above.
• the binding member could also be used in association with a chemotherapeutic agent or another tyrosine kinase inhibitor in co-administration or in the form of an immunoconjugate. Fragments of said antibody could also be use in bispecific antibodies obtained by recombinant mechanisms or biochemical coupling and then associating the specificity of the above described antibody with the specificity of other antibodies able to recognize other molecules involved in the activity for which CXCL13 is associated.
• a binding member of the invention may be combined with one or more agents, such as non-steroidal anti-inflammatory agents (hereinafter NSAIDs) including non-selective cyclo-oxygenase (COX)-1/COX-2 inhibitors whether applied topically or systemically, such as piroxicam, diclofenac, propionic acids, such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates, such as mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones, such as phenylbutazone, salicylates, such as aspirin); selective COX-2 inhibitors (such as meloxicam
• a binding member of the invention can also be used in combination with an existing therapeutic agent for the treatment of cancer.
• Suitable agents to be used in combination include:
• a binding member of the invention and one or more of the above additional medicinal components may be used in the manufacture of a medicament.
• the medicament may be for separate or combined administration to an individual, and accordingly may comprise the binding member and the additional component as a combined preparation or as separate preparations. Separate preparations may be used to facilitate separate and sequential or simultaneous administration, and allow administration of the components by different routes e.g. oral and parenteral administration.
• compositions provided may be administered to mammals. Administration is normally in a “therapeutically effective amount”, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
• the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of binding member, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated.
• Appropriate doses of antibody are well known in the art [83, 84]. Specific dosages indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used.
• a therapeutically effective amount or suitable dose of a binding member of the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis, prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment or diabody) and the nature of any detectable label or other molecule attached to the antibody.
• a typical antibody dose will be in the range 100 ⁇ g to 1 g for systemic applications, and 1 ⁇ g to 1 mg for topical applications.
• An initial higher loading dose, followed by one or more lower doses, may be administered.
• the antibody will be a whole antibody, e.g. the IgG1 isotype.
• This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight.
• Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. Treatments may be every two to four weeks for subcutaneous administration and every four to eight weeks for intra-venous administration.
• Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment.
• Binding members of the invention may be used ex vivo to determine the level of CXCL13 in a sample as described herein. Such a method generally comprises contacting the sample with a binding member of the invention under conditions that allow binding of the binding member to CXCL13 and measuring the level of bound CXCL13 in the sample. The level of CXCL13 may be compared with a suitable standard or control.
• the method may be used for the diagnosis or prognosis of a disease or condition in the individual from which the sample has been obtained, and for which CXCL13 levels are predictive.
• the method may be used to diagnose or prognose disease severity in, rheumatoid arthritis, such a method comprises determining the level of CXCL13 in a suitable sample, e.g. a serum or synovial fluid sample, obtained from an individual suffering from or suspected to be suffering from rheumatoid arthritis. Elevated levels of CXCL13 compared to a control sample are indicative of disease, and there is a positive correlation between CXCL13 levels and disease severity.
• a suitable sample e.g. a serum or synovial fluid sample
• CHO Gqi5 cells CHO K1 cells expressing G protein Gqi5 (SEQ ID NO: 19).
• CHO Gqi5 hCXCR5 cells or CHO Gqi5 hCXCR5 c4.4 cells CHO Gqi5 cells stably expressing human CXCR5 (SEQ ID NO: 17)
• Minimal essential media (Gibco 31095) containing 0.5 mM 3-Isobutyl-1-methylxanthine (Sigma I7018) 1% Non essential amino acids (Gibco 11140) 0.1% BSA
• cAMP-XL665 solution for HTRF assay a solution comprising XL665 (an allophycocyanin dye, 80 kD in size, coupled directly to cAMP)
• Alexa Fluor®-647 anti-cAMP antibody for LANCE® cAMP assay a cAMP specific antibody labelled with the dye Alexa Fluor®-647, formulated in 50 mM Tris HCl (pH7.8) salt solution, 0.9% sodium chloride, 0.1% BSA, 0.05% sodium azide (preservative).
• DMEM Dulbecco's modified Eagle's medium
• DMEM Dulbecco's modified Eagle's medium
• FBS foetal bovine serum
• Invitrogen 1% (v/v) MEM-non-essential amino acids without L-glutamine
• hCXCR5 cells A murine pre-B cell line stably transfected with human CXCR5
• the CXCL13 receptor ligand binding assay measures the interaction between biotinylated human CXCL13 (Almac) and the CXCR5 receptor over-expressed on B300.19 cells (Almac) using Fluorescence Microvolume Assay Technology (FMAT) (Dietz et al 1996, Miraglia et al 1999, Mellentin-Michelotti et al. 1999). This assay may be used to screen crude scFv periplasmic extracts or purified scFv for inhibition of the CXCL13:CXCR5 interaction.
• FMAT Fluorescence Microvolume Assay Technology
• % Specific binding was determined from the FL1 Total data using equation 1 where NSB FL1 Total is the mean value of the non specific binding control wells and Total binding FL1 Total is the mean value of the Total binding control wells.
• % ⁇ ⁇ specific ⁇ ⁇ binding ( Sample ⁇ ⁇ FL ⁇ ⁇ 1 ⁇ ⁇ Total - NSB ⁇ ⁇ FL ⁇ ⁇ 1 ⁇ ⁇ Total ) ( Total ⁇ ⁇ binding ⁇ ⁇ FL ⁇ ⁇ 1 ⁇ ⁇ Total - NSB ⁇ ⁇ FL ⁇ ⁇ 1 ⁇ ⁇ Total ) ⁇ 100 Equation ⁇ ⁇ 1
• 3′, 5′ cyclic adenosine monophosphate (cAMP) levels in cells are increased through the activation of the family of adenylyl cyclase enzymes, which catalyse conversion of adenosine triphosphate (ATP) to cAMP and pyrophosphate.
• adenylyl cyclase enzymes which catalyse conversion of adenosine triphosphate (ATP) to cAMP and pyrophosphate.
• ATP adenosine triphosphate
• HTRF® Homogeneous Time-Resolved Fluorescence
• CXCL13 mediated decreases in cAMP levels in CHO Gqi5 cells stably expressing CXCR5 are measured.
• Co-stimulation of the cells with the adenylyl cyclase activator, NKH477 (Tocris Cookson 1603), a water-soluble forskolin derivative, is carried out to ensure the CXCL13 response is within the linear range of detection of the HTRF® cAMP assay kit.
• This assay may be used to determine the potencies of scFvs for inhibition of the CXCL13 mediated modulation of cellular cAMP levels in CHO Gqi5 hCXCR5 cells.
• CHO Gqi5 hCXCR5 cells were prepared in Assay media (Minimal essential media (Gibco 31095) containing 0.5 mM 3-Isobutyl-1-methylxanthine (Sigma I7018), 1% Non essential amino acids (Gibco 11140) and 0.1% BSA) unless otherwise stated.
• Assay media Minimal essential media (Gibco 31095) containing 0.5 mM 3-Isobutyl-1-methylxanthine (Sigma I7018), 1% Non essential amino acids (Gibco 11140) and 0.1% BSA) unless otherwise stated.
• CHO Gqi5 hCXCR5 cells were harvested from tissue culture flasks and resuspended at 1.2 ⁇ 10 6 cells/ml in Assay media.
• cAMP-XL665 and Anti-cAMP Europium Cryptate were reconstituted in distilled water according to the manufacturer's instructions (CisBio International)
• ScFvs were pre-incubated with 4 nM human (SEQ ID NO: 14) or cynomolgus (SEQ ID NO: 16) CXCL13 (MEL cell derived) for 30 minutes at room temperature in a 384 well low volume plate (Costar 3676). 5 ⁇ l of the pre-incubated sample were transferred to the assay plate (Costar 3676), then 2.5 ⁇ l NKH477 and 2.5 ⁇ L CHO Gqi5 hCXCR5 cell suspension added to give a final reaction volume of 10 ⁇ l containing 0.5 ⁇ M NKH477 and 3000 cells/well.
• NKH477 control CHO Gqi5 hCXCR5 cells with 0.5 ⁇ M NKH477)
• CXCL13 control (2 nM CXCL13, 0.5 ⁇ M NKH477 and CHO Gqi5 hCXCR5 cells)
• basal cAMP control CHO Gqi5 hCXCR5 cells only
• negative control assay media only.
• CXCL13 and NKH477 titrations were also run in each experiment to ensure the concentrations used in the assay were within the EC 50 -EC 85 range and hence within the linear range of detection of HTRF® cAMP assay kit.
• Delta F % was determined according to equation 2 where CXCL13 control Delta F % is the mean value of the CXCL13 control wells and NKH477 control Delta F% is the mean value of the NKH477 control wells.
• Delta ⁇ ⁇ F ⁇ % ( sample ⁇ ⁇ 665 ⁇ ⁇ nm / 620 ⁇ ⁇ nm ⁇ ⁇ ratio ⁇ ⁇ value ) - ( negative ⁇ ⁇ control ⁇ ⁇ 665 ⁇ ⁇ nm / 620 ⁇ ⁇ nm ⁇ ⁇ ratio ⁇ ⁇ value ) ( negative ⁇ ⁇ control ⁇ ⁇ 665 ⁇ ⁇ nm / 620 ⁇ ⁇ nm ⁇ ⁇ ratio ⁇ ⁇ value ) ⁇ 100 Equation ⁇ ⁇ 2
• % ⁇ ⁇ inhibition ( Sample ⁇ ⁇ Delta ⁇ ⁇ F ⁇ ⁇ % - CXCL ⁇ ⁇ 13 ⁇ ⁇ control ⁇ ⁇ Delta ⁇ ⁇ F ⁇ ⁇ % ) ( NKH ⁇ ⁇ 477 ⁇ ⁇ control ⁇ ⁇ Delta ⁇ ⁇ F ⁇ ⁇ % - CXCL ⁇ ⁇ 13 ⁇ ⁇ Control ⁇ ⁇ Delta ⁇ ⁇ F ⁇ % ) ⁇ 100 Equation ⁇ ⁇ 3
• X is the logarithm of concentration.
• Y is % inhibition
• the principle of the CXCL13 LANCE® cAMP Assay is comparable to that outlined for the CXCL13 HTRF® cAMP assay but cellular cAMP levels are measured using the LANCE® cAMP Assay kit (Perkin Elmer AD0263).
• the CXCL13 LANCE® cAMP Assay may be used to determine the potency of IgGs for inhibition of CXCL13 mediated decreases in cAMP levels in CHO Gqi5 hCXCR5 cells upon co-stimulation with NKH477.
• LANCE® detection mix containing 1:2250 diluted Europium-W8044 Streptavidin and 1:750 fold diluted Biotin-cAMP was prepared at least 15 minutes prior to use as per the manufacturer's instructions.
• Europium-W8044 is a europium chelate with DCA (dichlorotriazinyl) reactive arm.
• IgG samples were pre-incubated with 4 nM human (SEQ ID NO: 14) or cynomolgus (SEQ ID NO: 16) CXCL13 (MEL cell derived) and 2 ⁇ M NKH477 (Tocris Cookson 1603) for 1 hour at room temperature in a 384 well low volume plate (Costar 3676). 5 ⁇ L of the pre-incubated sample was transferred to a white Proxiplate Plus 384 well assay plate (Perkin Elmer Cat no. 6008280).
• Alexa Fluor®-647 anti-cAMP antibody/CHO Gqi5 hCXCR5 cell suspension (5 ⁇ L) was added to give a final reaction volume of 10 ⁇ L containing 1 ⁇ M NKH477, 3000 CHO Gqi5 hCXCR5 cells/well and 1:200 diluted Alexa Fluor®-647 anti-cAMP antibody.
• NKH477 control CHO Gqi5 hCXCR5 cells/Alexa Fluor®-647 anti-cAMP antibody and 1 ⁇ M NKH477) and CXCL13 control (2 nM CXCL13, 1 ⁇ M NKH477 and CHO Gqi5 hCXCR5 cells/Alexa Fluor®-647 anti-cAMP antibody).
• CXCL13 and NKH477 titrations were also run in each experiment to ensure the concentrations used in the assay were within the EC 50 -EC 85 range and hence within the linear range of detection of the LANCE® cAMP assay kit.
• the 665 nm emission data was used to calculate % inhibition as described in equation 7, where CXCL13 control 665 nm emission is the mean value of the CXCL13 control wells and NKH477 control 665 nm emission is the mean value of the NKH477 control wells.
• % ⁇ ⁇ inhibition ( Sample ⁇ ⁇ 665 ⁇ ⁇ nm ⁇ ⁇ emission - CXCL ⁇ ⁇ 13 ⁇ ⁇ control ⁇ ⁇ 665 ⁇ ⁇ nm ⁇ ⁇ emission ) ( NKH ⁇ ⁇ 477 ⁇ ⁇ control ⁇ ⁇ 665 ⁇ ⁇ nm ⁇ ⁇ emission - CXCL ⁇ ⁇ 13 ⁇ ⁇ control ⁇ ⁇ 665 ⁇ ⁇ nm ⁇ ⁇ emission ⁇ ) ⁇ 100 Equation ⁇ ⁇ 7
• IC 50 values were determined using GraphPad Prism software by curve fitting using a four-parameter logistic equation (see Equation 4).
• CHO Gqi5 hCXCR5 c4.4 cells were seeded in 96-well black-walled Poly-D-Lysine treated tissue culture assay plates (BD) at 1 ⁇ 105 cells/well. Cells were then cultured overnight in 100 ⁇ L assay media (Dulbecco's modified Eagle's medium (DMEM) (Invitrogen), 10% (v/v) heat inactivated foetal bovine serum (FBS) (Invitrogen), 1% (v/v) MEM-non-essential amino acids without L-glutamine (Invitrogen)) in a humidified atmosphere at 37° C. and 5% CO 2 .
• DMEM Dulbecco's modified Eagle's medium
• FBS heat inactivated foetal bovine serum
• MEM-non-essential amino acids without L-glutamine Invitrogen
• a no-wash FLIPR protocol was employed as follows, using a commercially available kit (Fluo-4 no wash Calcium Assay Kit F36206, Molecular Probes).
• Dye-loading solution was prepared by adding 10 mls of Assay buffer (20 mM HEPES in 1 ⁇ Hanks Balanced salt solution) and 100 ⁇ l probenecid stock solution (250 mM in Assay buffer) to a single vial of Fluo-4 NW dye mix. Media was aspirated from cells, and 70 ⁇ l/well of dye-loading solution was added and incubated at 37° C. for 30 minutes, followed by 30 minutes at room temperature.
• titrations of purified IgGs were prepared in FLIPR buffer (125 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 30 mM Hepes, 2.5 mM probenecid, 5 mM glucose and 1% (v/v) heat-inactivated FBS) and pre-incubated with CXCL13 (100 nM final concentration) also in FLIPR buffer for 30 minutes at 37° C.
• FLIPR buffer 125 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 30 mM Hepes, 2.5 mM probenecid, 5 mM glucose and 1% (v/v) heat-inactivated FBS
• Fluorometric Imaging Plate Reader System FLIPR Fluorescence of the intracellular calcium-sensitive dye was assayed using a 470 to 495 nm excitation filter and 515 to 575 nm emission filter over a reading period of ⁇ 120 seconds.
• the CXCL13 FLISA measures the interaction between biotinylated human CXCL13 (Almac) coupled to a streptavidin bead and CXCL13 binding members in IgG format using Fluorescence Microvolume Assay Technology (Dietz et al 1996, Swartzman et al 1999). This assay may be used to determine the specificity of IgGs for biotinylated-human CXCL13 compared to related chemokine family members in a competition assay format.
• % Specific binding was determined from the FL1 data using equation 8, where NSB FL1 is the mean value of the non specific binding control wells and Total binding FL1 is the mean value of the Total binding control wells.
• IC 50 values were determined using GraphPad Prism software by curve fitting using a four-parameter logistic equation (see Equation 4).
• the Fc 2 Protein G′surface was used to capture 240-385 RUs Antibody 1 (germlined IgG 1 ) by titrating a 0.5 ⁇ L mL-1 HBS-EP solution of Antibody 1 (germlined IgG 1 ) at 5 ⁇ L min-1 for 2 minutes.
• a specified dilution of either human or cynomolgus CXCL13 (0.25, 0.3125, 0.50, 0.625, 1.0, 1.25, 2.5, 5.0, 10.0 and 20.0 nM for Human CXCL13 or 0.125, 0.25, 0.50, 1.0, 2.0, 5.0, 10.0, 20.0 and 40.0 nM for cynomolgous CXCL13) in HBS-EP buffer was then flowed over the chip surface at a flow rate of 100 ⁇ L min-1 (association time of 2.5 minutes, dissociation for 10 minutes and regeneration with a 20 ⁇ L pulse of 10 mM Glycine pH 1.75 followed by a 20 ⁇ L pulse of 10 mM Glycine pH 1.50). Blank injections were made with the HBS-EP alone. All solutions were prepared and stored in polypropylene.
• Antibodies were diluted to 1.0 ⁇ g mL-1 in 10 mM acetate buffer pH 4.5. Standard amine linkage chemistry (using amine coupling kit BR-1000-50) was employed to create blank (Fc 1) and 500 RU surfaces of IgG on a CM3 chip (BIAcore, BR-1005-41, lot: 1160100 expiry April '07). The wash solution employed was Glycine pH 2.0.
• Clones were converted from scFv to IgG format by sub-cloning the V H and V L domains into vectors expressing whole antibody heavy and light chains respectively.
• the VH domain was cloned into a vector (pEU15.1) containing the human heavy chain constant domains and regulatory elements to express whole IgG 1 heavy chain in mammalian cells.
• the VL domain was cloned into a vector for the expression of the human light chain constant domains and regulatory elements to express whole IgG light chain in mammalian cells (pEU3.4 for kappa light chains, pEU4.4 for lambda).
• Vectors for the expression of heavy chains and light chains were originally described in Persic et al, 1997. Vectors have been engineered to permit episomal replication of the vectors.
• the heavy and light chain IgG expressing vectors were transfected into EBNA-HEK293 mammalian cells. IgGs were expressed and secreted into the serum-free medium. Harvests were pooled and filtered prior to purification. The IgG was purified using Protein A chromatography. Culture supernatants are loaded on a column of appropriate size of Ceramic Protein A (BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH 3.0) and neutralised by the addition of Tris-HCl (pH 9.0).
• the eluted material was buffer exchanged into PBS using Nap10 columns (Amersham, #17-0854-02) and the concentration of IgG was determined spectrophotometrically using an extinction coefficient based on the amino acid sequence of the IgG (Mach 1992).
• the purified IgGs were analysed for aggregation or degradation using SEC-HPLC and by SDS-PAGE.
• the HTRF® epitope competition assay may be used to determine competition between binding members for binding biotinylated CXCL13.
• Antibody molecules tested in this assay may for example be in scFv or IgG format.
• a FRET complex is formed between cryptate labelled ScFv (SEQ ID NO: 11), biotinylated human CXCL13 and streptavidin XLent (streptavidin crosslinked with XL665, coupled under optimised conditions).
• An ScFv or IgG recognising the same (or possibly overlapping) epitope as the cryptate labelled ScFv will compete for binding to the biotinylated CXCL13 and thus reduce the assay signal.
• Optimal concentrations of CXCL13, streptavidin-XLent and cryptate labelled IgG may be titrated in to establish the concentration of each reagent that gives an appropriate assay signal.
• Delta F % values are subsequently used to calculate % specific binding as described in equation 6, where NSB Delta F % is the mean value of the non specific binding control wells and Total binding Delta F % is the mean value of the Total binding control wells.
• IC50 values are determined using GraphPad Prism software by curve fitting using a four-parameter logistic equation (see Equation 4).
• X is the logarithm of concentration.
• Y is % inhibition
• B300.19 hCXCR5 cells were adjusted to 5 ⁇ 10 5 cell/ml for at least 2 days prior to running the assay using culture media (RPMI-1640 Sigma, Cat#R0883) containing 10% foetal calf serum (FCS); 1% penicillin, streptomycin and glutamine (PSG); 1% sodium pyruvate; 1.5 ⁇ g/ml puramycin and 0.1% 2- ⁇ -mercaptoethanol) in a humidified atmosphere at 37° C., 5% CO 2 .
• culture media RPMI-1640 Sigma, Cat#R0883
• FCS foetal calf serum
• PSG penicillin, streptomycin and glutamine
• sodium pyruvate 1.5 ⁇ g/ml puramycin and 0.1% 2- ⁇ -mercaptoethanol
• B300.19 hCXCR5 cells were counted and adjusted to 6 ⁇ 10 6 cell/ml after being spun down and washed once in buffer (RPMI-1640 (Sigma Cat#R0883) +0.35% BSA (Sigma Cat#A2153)).
• IgGs Titrations of IgGs were made in assay buffer.
• the IgGs were pre-incubated with an ED80 concentration of CXCL13 for 30 min in a humidified atmosphere at 37° C., 5% CO 2 .
• the plates were removed from the incubator and washed to remove excess cells off the filter surface. This was done by pouring PBS over the membrane surface and using a cell scraper (Corning, Cat#3011) to remove all excess cells/PBS. The chemotaxis plate was then centrifuged at 1500 rpm for 10 min, leaving the filter on.
• the filter membrane was removed and the contents of the lower chamber of the chemotaxis plate were transferred to a 96-well polystyrene flat bottomed plate (Corning, Cat#3598) (enzymatic plate) containing 80 ⁇ l/well of assay buffer and 15 ⁇ l/well of lysis buffer (H 2 O+9% Triton-X). The plate was incubated for 1 hr in a humidified atmosphere (37° C., 5% CO 2 ).
• the CytoTox 96 non-radioactive cytotoxicity assay (Promega Cat#G1780) consists of the following reagents: 5 vials Substrate Mix; 60 ml Assay Buffer; 25 ul LDH Positive Control (NOT USED); 3 ml Lysis Solution (10 ⁇ ) (NOT USED); 65 ml Stop Solution (1M Acetic Acid); 1 protocol.
• the Assay Buffer was thawed; 12 ml was removed and the unused portion promptly stored at ⁇ 20° C. A 37° C. water bath may be used to thaw the Assay Buffer.
• the 12 ml of Assay Buffer was warmed to room temperature (keep protected from light).
• the 12 ml of room temperature Assay Buffer was added to one bottle of Substrate Mix. This was inverted and shaken gently to dissolve the Substrate Mix.
• the cells at the interface of lymphoprep and tissue culture medium were removed with a plastic pasteur pipette and transferred to a 15 ml centrifuge tube.
• the cell suspension was made up to a volume of 15 ml with cold tissue culture medium and centrifuged at 1500 rpm for 5 mins. The supernatant was discarded and the cell pellet resuspended in 10 ml of cold tissue culture medium.
• a viable cell count is performed using a coulter counter. Cells were adjusted to a concentration of 5 ⁇ 10 6 cells per ml and cultured overnight with 1 ⁇ g/ml LPS approximately 20 hrs.
• the lymphocyte layer was removed using a Pasteur pipette and transferred into a fresh Fisher 50 ml tube.
• the cells were then washed with pre-warmed RPMI-1640 (Sigma: R0883) media.
• the cells were then washed by centrifugation at 1500 rpm for 10 min with the brake on.
• the supernatant was discarded and the cells were then washed a twice more by centrifugation using RPMI at 1500 rpm for 10 min with the brake on.
• the pellet was resuspended in 50 ml of RPMI and a 200 ⁇ l sample removed for counting using the Coulter Counter.
• the cells were resuspended in the appropriate amount of culture media (RPMI-1640 (Sigma: R0883) media containing 10% Foetal Calf Serum, 1% Penicillin 4 (10,000 units/ml)+Streptomycin (10 mg/ml)+Glutamine (200 mM) solution (Sigma: G1146) to gives a final concentration of 100 Units/ml Penicillin+0.1 mg/ml Streptomycin+2 mM Glutamine) to obtain a cell concentration of 3.3 ⁇ 10 6 cell/ml.
• RPMI-1640 Sigma: R0883
• Penicillin 4 10,000 units/ml
• Streptomycin 10 mg/ml
• Glutamine 200 mM
• G1146 G1146
• Human M-CSF (R&D Systems: 216-MC-025) to obtain a final concentration of 50 ng/ml+human IL-4 (R&D Systems: 204-MC-025) to obtain a final concentration of 25 ng/ml.
• the supernatant containing native CXCL13 was then purified on a cation exchange resin and dialysed into PBS and requantified using by ELISA.
• maxi-sorp high binding 96-well plates were coated with CAZ-1040 (5 ⁇ g/ml 200 ⁇ l per well). Also, within the ELISA kit, plates were provided coated with unspecified anti-CXCL-13 antibody. The purified native CXCL13 was then added to both Antibody 1-coated and kit supplied plates. The ELISA was then carried out according to manufacturers instructions (R&D systems: Quantikine Human CXCL13/BLC/BCA-1 Immunoassay. Cat. N o :DCX130).
• Antibody 1 binds to both native CXCL13 and recombinant CXCL13 (standards provided in kit) with equivalent concentrations of ligands giving an equivalent signal in the assay.
• CXCL13 was used at 0.5 mg/ml in 50 mM Tris.HCl pH 7.4, 150 mM NaCl.
• Antibody was used at 10.6 mg/ml in PBS (1.54 mM KH2PO4, 2.71 mM Na2HPO4, 155 mM NaCl pH7.2).
• Antibody was coupled to POROS AL resin (Applied Biosystems) according to the manufacturer's instructions to prepare a 100 ⁇ l column which was kept at 1° C.
• the column was washed in 50 mM Tris.HCl pH7.5, 150 mM NaCl prepared in 75% D2O.
• CXCL13 was diluted in buffer prepared with 75% D2O to 0.125 mg/ml and incubated for 150, 500, 1500 and 5000 s before injection onto the antibody column.
• the column was quickly washed with 0.2 ml buffer in H2O and then incubated for the same period of time i.e. the sample that was exchanged into D2O for 150 s was exchanged back to H2O for 150 s and similarly for the samples at 500, 1500 and 5000 s.
• the different time points were eluted by injecting first 80 ⁇ l and then 40 ⁇ l 0.8% formic acid.
• the latter 40 ⁇ l was collected and 20 ⁇ l of 2 M urea, 1 M TCEP pH3 at 1° C. added. The whole mixture was injected onto a 100 ⁇ l column containing pepsin to digest the protein into peptides that were separated by rpHPLC using a gradient of acetonitrile from 12-28.5% and the masses determined on both a Thermo Finnigan LCQ electrospray mass spectrometer and a Micromass Q-TOF mass spectrometer.
• the SEQUEST software program (Thermo Finnigan San Jose, Calif.) was used to identify sequences of the parent peptide ions.
• the effect of the antibody on the rate of exchange of different parts of CXCL13 was determined essentially as described above, with the following exceptions: the CXCL13 was first diluted to 0.125 mg/ml in H2O containing buffer, then injected onto the column which had been pre-washed with 50 mM Tris.HCl pH7.5, 150 mM NaCl prepared in H2O. After binding, the column was washed with 50 mM Tris.HCl pH7.5, 150 mM NaCl prepared in H2O and then incubated with 50 mM Tris.HCl pH7.5, 150 mM NaCl prepared in 75% D2O for 150, 500, 1500 and 5000 s before being exchanged back into H2O and treated as above.
• Human spleen cDNA was used as a template to PCR the open reading frame of human CXCL13. This was cloned into a mammalian expression vector (pEV3) for stable transfection of MEL cells. The highest CXCL13-expressing clonal MEL cell line was identified by western blot of cell culture supernatants using a commercially available anti-CXCL13 antibody. Recombinant CXCL13 was purified from the culture medium of MEL cells expressing the protein by adjusting the pH to 6 and then loading it onto a cation exchange column. CXCL13 was eluted in a MES buffer at pH6 containing 1M NaCl.
• the sample was loaded directly onto a Resource rpc column and eluted with a gradient of up to 90% acetonitrile in 0.1% TFA. Fractions containing CXCL13 were pooled, concentrated and purified by chromatography on a Superdex75 column to generate material of >95% purity. The purified protein was analysed by SDS-PAGE and LCMS and shown to have a mass of 9690 Da.
• scFv Large single chain Fv (scFv) human antibody libraries cloned into a phagemid vector based on filamentous phage M13 were used for selections (Vaughan 1996, Hutchings 2001). Anti-CXCL13 specific scFv antibodies were isolated from the phage display libraries using a series of selection cycles on chemically synthesised biotinylated human CXCL13 (Almac) essentially as previously described (Vaughan 1996). In brief purified phage in PBS-Marvel (3% w/v) were allowed bind to biotinylated human CXCL13 in solution for 1 h.
• ScFv-phage bound to antigen were then captured on streptavidin-coated paramagnetic beads (Dynabeads® M-280) following the manufacturer's recommendations. Unbound phage particles were removed by a series of wash cycles using PBS-Tween (0.1% v/v) and PBS. Bound phage particles were eluted, infected into bacteria and rescued for the next round of selection (Vaughan 1996).
• ScFvs were expressed in the bacterial periplasm and screened for their ability to inhibit binding of biotinylated human CXCL13 (Almac) to its receptor CXCR5 expressed on B300.19 hCXCR5 cells using Fluorescence Microvolume Assay Technology (FMAT). ScFvs which showed a significant inhibitory effect as crude periprep samples on the CXCL13:CXCR5 interaction in the assay were subjected to DNA sequencing (Vaughan 1996, Osbourn 1996).
• scFvs were expressed again in bacteria and purified by affinity chromatography (as described in Bannister 2006). IC50 values were determined by testing dilution series of purified scFvs in a HTRF cAMP (3′, 5′ cyclic adenosine monophosphate) assay against human and cynomolgus (cyno) non-human primate CXCL13 (both MEL cell derived).
• HTRF cAMP 3′, 5′ cyclic adenosine monophosphate
• IgGs were tested in a chemotaxis assay using B300.19 hCXCR5 cells, a murine pre-B cell line that has been transfected with human recombinant CXCR5. IgGs were titrated against a concentration of unglycosylated human CXCL13 which gave an approximately ED80 response (12.32 nM)
• the table below illustrates IC50 values (nM) ⁇ SEM for Antibody 1 IgG (non-germlined). Each IgG was tested at least 3 times.
• IgG activity on CXCL13 induced calcium release was evaluated using a CHO cell line transfected with the G-protein Gqi5 and human CXCR5. Stimulation of these cells with human CXCL13 gives rise to increases in cytoplasmic Ca 2+ in a concentration dependent manner by inducing release from intracellular stores and influx from extracellular media. This can be measured using calcium-sensitive dyes in a Fluorescence Imaging Plate Reader (FLIPR).
• FLIPR Fluorescence Imaging Plate Reader
• the inhibitory activity, as determined by their IC 50 values, of anti-CXCL13 IgG was determined against 100 nM human CXCL13 (MEL cell derived).
• the IgG potencies obtained for Antibody 1 (non-germlined) are shown in Table 4. Positive assay controls were included in all assays. For full details of the assay method see “Assay materials and methods”.
• VH and VL domains of the anti-CXCL13 antibodies were aligned to the known human germline sequences in the VBASE database (Tomlinson 1997), and the closest germline was identified by sequence similarity. For the VH domains of Antibody 1 this was VH3-23. For the VL domains it was VK1 L12.
• the parent and germlined Antibody 1 IgG showed comparable activities in this assay.
• IgGs Fully germlined Antibody 1 IgGs were tested in the chemotaxis assay using B300.19 hCXCR5 cells. IgGs were titrated against ED80 concentrations of unglycosylated and glycosylated human and glycosylated cyno CXCL13. The results are shown in Table 6 below as IC 50 values (nM) ⁇ SEM. IgGs were tested at least 3 times.
• the potency of the germlined and parent (non-germlined) Antibody 1 IgG was compared in the FLIPR assay as outlined in section 1.5.
• Standard amine linkage chemistry was employed to create blank and 265 RU surfaces of Protein G′ (Sigma P4689) on Flow cells (Fc) 1 and 2, respectively, of a normalized CM5 chip (BIAcore, BR-1000-14). This Fc 2 Protein G′ surface was used to capture 240-385 RUs Antibody 1 (germlined IgG 1 ).
• Double blank subtracted (blank flow cell subtracted from IgG flow cell data and a blank injection (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, HBS-EP) was subtracted from the rest of data set). Bulk RI contributions were all set to zero locally.
• HBS-EP subtraction and RI set to zero; the following parameters were obtained for the IgG, based on the 0.125-40 nM cyno CXCL13 curves (12 and 9 concentrations).
• Standard amine linkage chemistry was employed to create blank (Fc 1) and 228 and 303 RU surfaces of Streptavidin (Perbio/Pierce 21125) on Flow cells (Fc) 2 and 3, respectively, of a normalized CM5 chip (BIAcore, BR-1000-14). This Fc 3 streptavidin surface was used to capture 28 RUs human CXCL13 biotinylated near the C-terminus (Almac custom synthesis).
• Double blank subtracted (blank flow cell subtracted from IgG flow cell data and a blank injection (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, HBS-EP) was subtracted from the rest of data set). Bulk RI contributions were all set to zero locally.
• Standard amine linkage chemistry was employed to create blank (Fc 1) and 500 RU surfaces of Antibody l(germlined IgG 1 ) diluted in 10 mM acetate buffer pH 4.5 on a CM3 chip (BIAcore, BR-1005-41).
• Double blank subtracted (blank flow cell subtracted from IgG flow cell data and a blank injection (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, HBS-EP) was subtracted from rest of data set). Bulk RI contribution all set to zero locally.
• HBS-EP subtraction and RI set to zero; the following parameters were obtained for each IgG, based on the 0.10-0.60 nM cyno CXCL13 curves.
• Antibody 1 (germlined IgG 1 ) binds to both native CXCL13 and recombinant CXCL13 with equivalent concentrations of ligands giving an equivalent signal in the assay.
• the region that showed the greatest perturbation in the rate of H/D exchange was between amino acids 31-42 of human CXCL-13 (SEQ ID No. 20).
• Table 18 shows the percentage difference in deuteration levels comparing deuteration and exchange back to protons when bound to antibody 1 with deuteration in solution and exchange back to protons when bound to antibody. Differences of more than 10% when averaging the four time points were considered to be significant. Residue numbering is with the sequence shown in SEQ ID NO. 14.
• Time point (s) start end 150 500 1500 5000 3 5 4 3 ⁇ 3 1 3 6 4 2 1 ⁇ 1 6 9 4 ⁇ 1 ⁇ 5 ⁇ 4 7 9 4 3 ⁇ 2 ⁇ 2 16 20 25 13 2 ⁇ 5 21 24 5 6 11 6 21 28 6 16 25 20 23 28 11 18 27 19 31 42 29 28 19 14 33 42 31 30 31 16 45 53 24 11 15 6 45 54 24 15 11 6 56 61 11 21 30 24 57 61 10 23 31 22 63 65 24 12 ⁇ 5 ⁇ 4 64 67 30 11 ⁇ 4 ⁇ 3 68 69 48 23 2 ⁇ 2 68 82 13 3 ⁇ 5 ⁇ 5 70 82 3 1 ⁇ 5 ⁇ 4 78 82 11 5 ⁇ 5 ⁇ 5

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