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Definitions

• the present invention relates to anti-CXCLl2 antibody molecules and their uses, and more particularly to anti-CXCL12 antibody molecules that are capable of inhibiting a biological activity of CXCL12 and their use for treating cancer.
• CXCL12 also known as stromal cell-derived factor 1 (SDF-1)
• SDF-1 stromal cell-derived factor 1
• CXCL12 plays a role in
• angiogenesis by recruiting endothelial progenitor cells (EPCs) from the bone marrow through a CXCR4 dependent mechanism, making it a significant factor in carcinogenesis and neovascularisation linked to tumour progression.
• EPCs endothelial progenitor cells
• CXCL12 also has a role in organ-specific metastasis of several cancers, where cancer cells that express the receptor CXCR4 are attracted to metastasis target tissues that release the ligand, CXCL12.
• CXCL12 also acts to recruit CXCR4-positive stromal cells and regulates immune cell infiltration.
• CXCL12 may aid the formation of pre-metastatic niches through the
• CAFs cancer associated fibroblasts
• CXCL12 Comito et al, Oncogene, 33: 2423 - 2431, 2014
• High levels of CXCL12 are associated with low numbers of T cells in a pancreatic cancer model and it was possible to increase T cell infiltration through combined treatment with PD-L1 and CXCR4 inhibitors.
• WO 2008/018641 discloses human monoclonal antibodies that specifically bind to SDF-1 and proposes their medical uses for treating various B cell malignancies, including breast cancer, multiple myeloma and non- Hodgkin's lymphoma arid autoimmune disorders. Zhong et al .
• CCR-13-0943 discloses a humanised version of a hamster monoclonal antibody 30D8 and shows that it was capable of binding to human and murine CXCL12 in in vitro assays. Summary of the Invention
• the present invention is based on the affinity
• the antibody molecules are capable of inhibiting CXCR4-induced cancer cell migration and/or are capable of inhibiting VEGF-induced angiogenesis in vitro. These properties enable the antibody molecules of the present invention to be used in the treatment of cancer, in particular by inhibiting metastasis and or tumour neovascularisation.
• the present invention provides an isolated anti-CXCL12 antibody molecule which specifically binds to human, and optionally murine, CXCL12 and inhibits CXCL12- mediated biological activity, wherein the antibody molecule binds to an epitope of human CXCL12 having the amino acid sequence as set out in SEQ ID NO: 24 that comprises amino acids (a) P10 and R12, and optionally one or more of E15, 128, P32, N45 and/or K54 or (b) P10 and Q48, and optionally one of more of K54 and N45.
• the present invention provides an anti- CXCL12 antibody molecule which comprises: (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 with one, two, three or more amino acid
• the present invention provides an anti- CXCL12 antibody molecule which comprises (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 with one, two, three or more amino acid
• substitutions, deletions or insertions and (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13 with one, two, three or more amino acid substitutions, deletions or insertions, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 14 with one, two, three or more amino acid substitutions, deletions or insertions; and optionally (d) a CDR- Ll having the amino acid sequence of SEQ ID NO: 15, or the sequences of SEQ ID NO: 15, with one or more amino acid
• substitutions, deletions or insertions and (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, or the sequences of SEQ ID NO: 16, with one, two, three or more amino acid substitutions, deletions or insertions and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or the sequences of SEQ ID NO: 17, with one, two, three or more amino acid substitutions, deletions or insertions.
• the present invention provides a
• composition comprising an antibody molecule or immunoconjugate as disclosed herein and a pharmaceutically acceptable excipient.
• the present invention provides an antibody molecule or immunoconj ugate as disclosed herein for use in a method of treatment of the human or animal body.
• the present invention provides an antibody molecule or immunoconj ugate as disclosed herein for use in a method of treatment of a CXCLl2-mediated condition.
• the present invention provides the use of an antibody molecule or immunoco j ugate as disclosed herein in the manufacture of a medicament for use in treating a CXCL12-mediated condition .
• the present invention provides a method of treating an individual with a CXCL12-mediated condition
• the present invention provides an antibody molecule of the present invention for use in a method for the diagnosis or prognosis of a patient having a CXCL12-mediated condition.
• the method may comprise
• the CXCL12-mediated condition is cancer, including cancer and/or immune cell migration and/or metastasis.
• the types of cancer that may be treated using the antibodies or immunoconjugates of the present invention include ovarian cancer, breast cancer, bone cancer, prostate cancer, thyroid cancer, pancreatic cancer, multiple myeloma, non-Hodgkin ' s lymphoma, intraocular lymphoma, follicular centre lymphoma, CML, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, kidney cancer, brain cancers, such as glioma and astrocytoma, rhabdmyosarcoma, lung cancer, such as small cell lung cancer, melanoma, B cell malignancies, such as B-cell chronic lymphocytic leukemia (B-CLL) , and leukaemia, such as acute myeloid leukaemia (AML) and acute lymphoblast
• the present invention may be used for the
• Figure 1 Vector systems used for expressing anti-CXCL12
• Restriction sites Ncol , Xhol , Nhel and Afotl facilitate the sub- cloning of variable heavy (VH) and light (VL) chain genes into the Fab and IgG expression vectors.
• Heavy (VH-CH1-CH2-Ch3 ) and light chain expression cassettes are located in two different plasmids. Plasmid pBIOCAM-1 codes for the light chain genes and the pBIOCAM-2 encodes the heavy chain cassette. Transcription of antibody gene is under the control of CMV promoter in both plasmids. Hexa-histidine (6X-His) and tri- FLAG tags fused to the antibody genes in pSANG10-3F and pBIOCAM7 enable purification and immune-detection of the expressed antibodies .
• FIG. 1 SDS-PAGE analysis of anti-CXCL12 Fabs and IgGs .
• Lead anti-CXCL12 antibodies and their parent clones were expressed in HEK-293 cells as Fabs and IgGs.
• Affinity purified antibodies were visualised on a reducing SDS-PAGE gel using SYPRO ⁇ Red staining.
• Clones 093_2D06, 093_2A02, 114_3H1 (labelled as 114_3H01) and 113_1H12 were loaded respectively as Fabs (lanes 1-4) and IgGs (lanes 5-8) .
• Smearing of VH-CHl bands in some of the Fab preparations could be due to the cleavage of the FLAG tag (which often occurs in Flag tagged proteins.
• FIG. 3 Affinity measurement of anti-CXCL12 antibodies using SPR
• B) Binding constants of 114_3H1 (labelled as 114_3H01) were determined using 1:1 Langmuir binding model. The equilibrium dissociation constant (KD) of the parent clone 093_2D06 was calculated using steady state binding model due to its very fast off-rate.
• Human umbilical vein endothelial cells were plated onto fibroblasts that had been grown for 6 days on gelatin coated chamber slides. These two cell types were co-cultured for 7 days in a media containing VEGF and lead anti-CXCL12 antibodies
• PECAM-1 platelet/endothelial adhesion molecule-1
• metastatic melanoma cell line B16F10
• human ovarian
• carcinoma cell line (TOV-21) in the presence of human CXCL12.
• FIG. 10 Results of in vivo experimental metastasis model cell migration assay based B16F10 melanoma cells requiring CXCR4 for migration to the lung and initiation of metastasis.
• B16F10 melanoma cells were introduced into C57B1 mice through tail vein injection on day 0 and treatment commenced on day 1.
• Treatment regimes were either 5 mg/kg of the clinical CXCR4 inhibitor A D3100 (Plerixafor) twice daily or twice a week with either 10, 15 or 20 mg/kg of the anti-CXCLl2 antibody in a chimeric murine IgG2a format.
• Mice in the control arm were treated twice a week with 20 mg/kg of a control antibody. All mice were culled on day 14 and the number of metastatic colonies in the lungs quantified.
• a level of inhibition equivalent to that of AMD3100 was achieved with the 20 mg/kg dose of 113_1H12.
• the full length amino acid sequence of CXCL12 is set out as SEQ ID NO: 23 and consists of 89 amino acids.
• the amino acid sequence of the synthesized 68 amino acid CXCL12 fragment used to select the exemplified antibodies is set out in SEQ ID No: 24.
• the epitope mapping studies described in the examples below used a wild type mature CXCL12 polypeptide having the amino acid sequence set out in SEQ ID NO: 25 that include a polyhistidine tag and linker sequence.
• the antibody molecules of the present invention are capable of binding to CXCL12
• polypeptides that comprise a polypeptide having at least 90% sequence identity to amino acids 1 to 68 as set out in SEQ ID NO: 23, or a fragment thereof, wherein the fragment is biologically active.
• the biological activities of the antibody molecules or immunoconj ugates of the present invention include binding to CXCL12, for example to block the interaction of CXCL12 with CXCR4 and optionally also the interaction of CXCL12 with CXCR7.
• the biological activities of CXCL12 that may be inhibited (antagonised) by the antibody molecules or
• immunoco j ugates of the present invention include inhibition of VEGF-induced angiogenesis in vitro and/or the inhibition of CXCLl2-induced cancer cell migration and/or spreading and/or metastasis.
• the antibody molecules or immunocon ugates of the present invention may also inhibit the role of CXCL12 in
• the in vitro affinity maturation of primary anti-CXCL12 antibodies was carried out in two steps. Firstly, the primary antibody sequences were diversified by light chain shuffling to create a derivative library. Secondly, tailored selection and screening procedures were used to identify affinity-improved variants from the light chain shuffled library. The rationale for using light chain shuffling for diversifying primary anti-CXCLl2 antibodies was as follows. The original diversity of naive immune repertoires in vivo (pre-immune B-cell repertoire) or in vitro (“McCafferty library”) is derived from the combinatorial rearrangement of germline variable gene segments.
• VL light chain variable region
• J short joining
• VH heavy chain variable region
• D diversity
• a light chain shuffled library of 2xl0 8 was created by combining the heavy chain variable regions of 20 anti-CXCLl2 antibodies with a repertoire of kappa and lambda light chain variable domains. Thus each original heavy chain was paired with approximately 10 million new light chain partners.
• Three rounds of phage display selection were carried out under stringent conditions to enrich for high affinity binders from the chain shuffled library. The stringency of the selection conditions was increased at each round by reducing the antigen concentration or using harsher and longer wash steps. Such selection procedures facilitate preferential enrichment of antibody clones with lower dissociation constants.
• Affinity maturation selections were carried out in solution- phase, allowing precise control of antigen concentration, which is an important parameter in determining the stringency of a selection.
• This process identified two antibodies (114_3H1 and 113_1H12) exhibiting the lowest dissociation constants were selected as lead antibodies for detailed characterisation and optimisation.
• the biological properties of the antibody molecules were tested in cell-based functional assays to determine their likely in vivo potency as this does not necessarily correlate with binding, especially if the antibody molecule is intended to modulate complex biological functions.
• the biological properties of the antibody molecules of the present invention include inhibition of CXCL12-induced cancer cell migration and/or inhibition of VEGF-induced angiogenesis in vitro.
• the biological property of inhibiting CXCL12-induced cancer cell migration may be determined using a transwell migration assay used here was a modified version of the Boyden chamber assay used to study the chemotactic response of leukocytes (Boyden, J. Exp. Med. 115, 453-46, 1962) in which migration of fluorescently labelled cancer cells, such as human ovarian cancer cells TOV- 21G, seeded in an upper chamber across a porous membrane and into a lower chamber containing CXCL12 is determined.
• the biological property of inhibiting angiogenesis may be determined using a cell based assay in which human umbilical vein endothelial cells (HUVECs) and fibroblasts are cultured together in a media containing anti-CXCL12 antibodies and VEGF.
• HUVECs human umbilical vein endothelial cells
• fibroblasts are cultured together in a media containing anti-CXCL12 antibodies and VEGF.
• VEGF vascular endothelial cells
• the interaction of these two cell types in the presence of VEGF results in the formation of three-dimensional tubes that resemble small capillaries in vivo, see Hetheridge et al . (Biochem. Soc. Trans. 39, 1597-1600, 2011) .
• the two lead antibodies, 114_3H1 and 113_1H12 were tested using in vitro cancer cell migration assay and angiogenesis assay in order to evaluate their functional characteristics. Both antibodies inhibited CXCL12-induced migration of ovarian cancer cells, with 114_3H1 outperforming 113_1H12. For both antibodies the ICsoS observed in this assay were comparable to the calculated KD values from SPR analysis. In addition, antibody clone
• this difference in the properties of the antibodies may be a result of the fact that CXCL12 can induce angiogenesis via interaction with both CXCR4 and CXCR7. Therefore, it may be possible that 114_3H1 only blocks CXCL12/CXCR4 interaction, but not CXCL12 /CXCR7 resulting in partial inhibition of angiogenesis. By contrast, 113_1H12 might be blocking CXCL12 binding to both CXCR4 and CXCR7 leading to superior inhibition of CXCL12 induced angiogenesis.
• the present invention provides antibody molecules that are based on the antibody clones 113_1H12 or 114_3H1.
• the present invention provides an anti-CXCL12 antibody molecule comprising at least one, two, three, four, five, or six of the following CDR sequences based on the CDR sequences of antibody 114_3H1:
• the anti-CXCL12 antibody molecules comprise all six CDRs as defined above optionally with one or more amino acid substitutions, deletions or insertions.
• the present invention provides an anti- CXCL12 antibody molecule comprising at least one, two, three, four, five, or six or more of the following CDR sequences based on the CDR sequences of antibody 113_1H12:
• amino acid sequence of SEQ ID NO: 14 with one, two, three or more amino acid substitutions, deletions or insertions; and/or
• the anti-CXCL12 antibody molecules comprise all six CDRs as defined above (SEQ ID NO: 12-17), optionally with one or more amino acid substitutions, deletions or insertions.
• VL light chain variable region
• J short joining
• VH heavy chain variable region
• D diversity
• the present invention provides anti-CXCL12 antibody molecules that comprise the CDRs of the heavy chain of the exemplified antibodies as defined herein, optionally each with one, two, three or more amino acid
• the present invention provides an anti-CXCLl2 antibody that comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-Ll comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 6.
• the antibody molecules of the present invention comprise a VH domain comprising the amino acid sequence set out in SEQ ID NO 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.
• the VH sequence comprises one or more substitutions, insertions, or deletions relative to the reference sequence, while the antibody molecule retains the property of binding to CXCL12, and optionally one or more of the other biological activities of the anti-CXCL12 antibody molecules of the present invention as described herein.
• the VH domain comprises one, two or three CDRs selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3.
• the antibody molecules of the present invention comprise a VL domain comprising the amino acid sequence set out in SEQ ID NO 9, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acid sequence of SEQ ID NO: 9.
• the VH sequence comprises one or more substitutions, insertions, or deletions relative to the reference sequence, while the antibody molecule retains the property of binding to CXCL12, and
• the VL domain comprises one, two or three CDRs selected from (i) CDR-Ll comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.
• the antibody molecules of the present invention comprise a VH domain comprising the amino acid sequence set out in SEQ ID NO 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7, and a VL domain comprising the amino acid sequence set out in SEQ ID NO 9, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.
• an anti-CXCLl2 antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:
• CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 14; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-Ll comprising the amino acid sequence of SEQ ID NO: 15, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
• the antibody molecules of the present invention comprise a VH domain comprising the amino acid sequence set out in SEQ ID NO 18, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.
• the VH sequence comprises one or more substitutions, insertions, or deletions relative to the reference sequence, while the antibody molecule retains the property of binding to CXCL12, and optionally one or more of the other biological activities of the anti-CXCL12 antibody molecules of the present invention as described herein.
• the VH domain comprises one, two or three CDRs selected from (i) CDR-H1 comprising the amino acid sequence of SEQ I D NO: 12, (ii) CDR-H2 comprising the amino acid sequence of SEQ I D NO: 13, and (iii)
• CDR-H3 comprising an amino acid sequence selected from SEQ I D NO: 14.
• the antibody molecules of the present invention comprise a VL domain comprising the amino acid sequence set out in SEQ I D NO 20, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acid sequence of SEQ I D NO: 20.
• the VH sequence comprises one or more substitutions, insertions, or deletions relative to the reference sequence, while the antibody molecule retains the property of binding to CXCL12, and optionally one or more of the other biological activities of the anti-CXCLl2 antibody molecules of the present invention as described herein.
• the VL domain comprises one, two or three CDRs selected from (i) CDR-Ll comprising the amino acid sequence of SEQ I D NO: 15, (ii) CDR-L2 comprising the amino acid sequence of SEQ I D NO: 16, and (c) CDR-L3 comprising the amino acid sequence of SEQ I D NO: 17.
• the antibody molecules of the present invention comprise a VH domain comprising the amino acid sequence set out in SEQ I D NO: 18, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ I D NO: 18, and a VL domain comprising the amino acid sequence set out in SEQ I D NO: 20, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ I D NO: 20.
• the present invention provides anti-CXCL12 antibody molecules capable of binding to an epitope of CXCL12 having the amino acid sequence as set out in SEQ I D NO: 24 or 25 that comprises amino acids (a) P10 and R12, and optionally one or more of E15, 128, P32, N45 and/or K54 or (b) P10 and Q48, optionally one of more of K54 and N45.
• the exemplified antibody 114___3H1 binds to epitope (a) and antibody 113_1H12 binds to epitope (b) .
• E15 is outside of regions that involved in receptor or heparin binding. All other epitope residues are within regions involved in receptor binding, which according to numbering of the full length protein at UniProt P48061 (SDF1_HUMAN) are 29-33, 39-41, 48-50, 60-70) .
• the present invention provides an isolated antibody molecule which binds CXCL12 and which comprises the 114__3H1 VH domain (SEQ ID NO: 7) and/or the 114_3H1 VL domain ( SEQ ID NO: 9) .
• the CXCL12 is human CXCL12, and optionally also murine CXCL12.
• the present invention provides an isolated antibody which binds CXCL12 and which comprises the 113_1H12 VH domain (SEQ ID NO: 18) and/or the 113_1H12 VL domain (SEQ ID NO: 20) .
• the CXCL12 is human CXCL12, and optionally also murine CXCL12.
• VH domain is paired with a VL domain to provide an antibody antigen binding site, although as discussed further below a VH domain alone may be used to bind antigen.
• the 114_3H1 or 113_1H12 VH domain (SEQ ID NO: 7 or 18) is paired with the 114_3H1 or 113_1H12 VL domain (SEQ ID NO: 9 or 20), so that an antibody antigen binding site is formed comprising both the 114_3H1 or 113_1H12 VH and VL domains.
• the 114_3H1 or 113_1H12 VH is paired with a VL domain other than the 114_3H1 or 113_1H12 VL .
• Light-chain promiscuity is well established in the art.
• One or more CDRs may be taken from the 114_3Hl or 113_1H12 VH or VL domain and incorporated into a suitable framework. This is discussed further below.
• 114 3H1 VH CDRs Hi, H2 and H3 are shown in SEQ ID NOs : 1, 2 and 3, respectively.
• 114_3H1 VL CDRs LI, L2 and L3 are shown in SEQ ID NOs: 4, 5 and 6, respectively.
• 113_1H12 VH CDRs Hi, H2 and H3 are shown in SEQ ID NOs: 12, 13 and 14, respectively.
• 113_1H12 VL CDRs LI, L2 and L3 are shown in SEQ ID NOs: 15, 16 and 17, respectively.
• the present invention provides an anti-CXCLl2 antibody that binds CXCL12 and which comprises:
• an antibody VH domain selected from the group consisting of the 114_3H1 VH domain (SEQ ID NO: 7) and a VH domain comprising a
• VH CDR3 with the amino acid sequence of SEQ ID NO: 3 and
• VH CDR' optionally one or more VH CDR' s with an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2; and/or
• an antibody VL domain selected from the group consisting of the 114_3H1 VL domain (SEQ ID NO: 9) and a VL domain comprising one or more VL CDR' s with an amino acid sequence selected from
• the present invention provides an anti-CXCLl2 antibody that binds CXCL12 and which comprises:
• an antibody VH domain selected from the group consisting of the 113_1H12 VH domain (SEQ ID NO: 18) and a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO: 14 and optionally one or more VH CDR' s with an amino acid sequence selected from SEQ ID NO: 12 and SEQ ID NO: 13; and/or
• an antibody VL domain selected from the group consisting of the 113_1H12 VL domain (SEQ ID NO: 20) and a VL domain comprising one or more VL CDR' s with an amino acid sequence selected from SEQ ID NOs: 15, 16 and 17.
• the antibody molecule of the present invention can tolerate a number of amino acid alterations to the sequences of the CDRs, while retaining the properties of the parent antibody.
• the amino acid sequences of the CDRs of the antibody molecule may each comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or insertions as compared to any one of SEQ ID NOs: 1 to 6 and 12 to 17.
• the CDRs may be present in a range of different antibody types or framework regions, optionally involving one or more further sequence alterations to ensure retention of a useful property of the antibody as disclosed herein.
• Figure 11 shows that the antibodies of the present invention are functional in scFc-Fv fusion and human IgG2 formats .
• Each of the VH and VL domains typically comprise three
• the present invention provides antibody molecules which comprise a VH domain comprising a CDR-H1, CDR-H2 and CDR-H3 having the sequences of SEQ ID NOs: 1, 2 and 3, respectively, and/or a VL domain comprising a CDR-L1, CDR-L2 and CDR-L3 having the sequences of SEQ ID NOs: 4, 5 and 6,
• the present invention provides antibody molecules which comprise a VH domain comprising a CDR-Hl, CDR-H2 and CDR-H3 having the sequences of SEQ ID NOs: 12, 13 and 14, respectively, and/or a VL domain comprising a CDR-L1, CDR-L2 and CDR-L3 having the sequences of SEQ ID NOs: 15, 16 and 17, respectively.
• the present invention also provides an anti-CXCLl2 antibody molecule in an scFv format having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 11 or SEQ ID NO: 22.
• the present invention relates to antibody molecules that are capable of inhibiting a biological activity of CXCL12, i.e. antagonist antibody molecules as understood by those skilled in the art.
• the properties may be determined in in vitro cancer cell migration assay and an in vivo
• angiogenesis assay Biological activities include inhibiting CXCLl2-induced cancer cell growth, inhibiting cancer cell migration, inhibiting cancer cell adhesion, inhibiting cancer metastasis and/or angiogenesis , e.g. VEGF-induced angiogenesis .
• the antibody molecules of the present invention function by binding and sequestering CXCL12 thereby preventing it from interacting with the receptor in the biological system in which it is present.
• Binding kinetics and affinity (expressed as the equilibrium dissociation constant Kd) of the anti-CXCL12 antibody molecules may be determined using standard techniques, such as surface plasmon resonance e.g. using BIAcore analysis, for example as described in the experimental examples below.
• Kd may be measured using a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and CXCL12.
• RIA radiolabeled antigen binding assay
• Anti-CXCL12 antibody molecules may have a dissociation constant for CXCL12 of less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than ⁇ , or less than InM.
• an antibody molecule may have an affinity for CXCL12 of 1 to 20 nM, e.g. 9 to 15 nM.
• antibody molecules of the present invention have affinity constants (K D ) of less than 10 nM, more preferably less than 5 nM and most preferably less than 3 nM for human CXCL12.
• the antibodies of the present invention preferably also bind to CXCL12 of other species, such as murine CXCL12, making them compatible with animal models of disease.
• the affinity constants for binding to CXCL12 can be determined using techniques well known in the art, such as
• Anti-CXCL12 antibody molecule of the present invention is a monoclonal antibody, including a chimeric, humanized or human antibody.
• the antibody molecule may be an antibody fragment, e.g., a Fv, Fab, Fab', scFv, scFv-Fc, diabody, or F(ab' )2 complete antibody, a triabody, a bispecific antibody or a chimeric antibody.
• the anti-CXCLl2 antibody molecule may be a whole antibody.
• the anti-CXCL12 antibody molecules may be monoclonal antibodies. Antibody molecules and methods for their construction and use are
• Antibody molecules normally comprise an antigen binding domain comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) , although antigen binding domains comprising only a heavy chain variable domain (VH) are also possible (e.g. camelid or shark antibodies) . Such antibodies are included within the scope of the present invention .
• antibody molecules of the present invention may be modified to alter the extent to which the antibody molecule glycosylated. This may be accomplished by altering the amino acid sequence such that one or more of the glycosylation sites present in a parent antibody is created or removed.
• an antibody molecule comprises an Fc region
• alteration of the carbohydrates attached to the Fc region can change the properties of the antibody molecule, in particular by reducing the fucosylation of the Fc region, it is possible to increase ADCC function.
• Anti-CXCL12 antibody molecules as described herein may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides and/or serum
• Monoclonal antibodies are preferred for most purposes, though polyclonal antibodies may also be employed.
• Methods of producing anti-CXCL12 antibody molecules include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof.
• Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , 1992, Nature 357: 80-82) . Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
• an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces.
• the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
• the method described in the examples may be employed to screen for further examples of anti-CXCL12 antibodies having antagonistic properties.
• the biological activity of an anti-CXCL12 antibody molecule may be tested.
• one or more biological activities may be determined that are selected from inhibiting CXCLl2-induced cancer cell growth, inhibiting cancer cell migration, inhibiting cancer cell adhesion, inhibiting cancer metastasis and/or angiogenesis , e.g. VEGF-induced angiogenesis .
• Competition between antibody molecules may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules to enable identification of antibody molecules which bind the same epitope or an overlapping epitope.
• ELISA electrospray sorbent assay
• tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules to enable identification of antibody molecules which bind the same epitope or an overlapping epitope.
• the present invention also provides nucleic acid molecules encoding the antibody molecules of the present invention.
• the nucleic acid molecules are useful for expressing the anti-CXCL12 antibody molecules, for example by incorporating the nucleic acid sequences into an expression vector having control sequences operably linked to the nucleic acid encoding the anti-CXCL12 antibody molecule to control its expression.
• the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the anti-CXCLl2 antibody molecule is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
• Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as
• Vectors may be plasmids or viral, e.g. 'phage, or phagemid, as appropriate.
• vectors may be plasmids or viral, e.g. 'phage, or phagemid, as appropriate.
• Anti-CXCL12 antibody molecules can be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the anti-CXCL12 antibody molecule is produced and recovering the anti-CXCL12 antibody molecule from the host cells or the surrounding medium.
• Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, insect cells (e.g. transformed with baculovirus) , yeast, and eukaryotic cells such as COS or CHO cells.
• the choice of host cell can be used to control the properties of the anti-CXCL12 antibody molecule expressed in those cells, e.g. controlling where the polypeptide is deposited in the host cells or affecting properties such as its
• the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
• the antibody molecule of the present invention may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components such as a carrier as described elsewhere in the present application.
• the present invention provides nucleic acid encoding an anti-CXCLl2 antibody molecule of the present invention, an expression vector comprising the nucleic acid encoding an anti-CXCL12 antibody molecule, operably linked to control sequences to direct its expression, and host cells transformed with this expression vector.
• the present invention provides a method of producing an anti-CXCL12 antibody molecule of the present invention, the method comprising culturing the host cells and isolating the anti-CXCL12 antibody molecule thus produced.
• the antibody molecules of the present invention may also be derivatised to modify their properties, and in particular their pharmacological properties, such as half-life (e.g. increasing half-life) .
• pharmacological properties such as half-life (e.g. increasing half-life)
• An example is the conjugation of antibody molecules to poly (alkylene glycol) molecules, in particular polyethylene glycol (PEG) molecules, that may be used to enhance the half-life or other pharmacological properties of polypeptide therapeutics.
• Pegylation is a known strategy for modifying the properties of therapeutic polypeptides, such as peptides, proteins and
• PEG molecules In general, the attachment of PEG molecules to polypeptides is used to alter their conformation, electrostatic or hydrophobic properties, and lead to improvements in their biological and pharmacological properties, such as increasing drug solubility, reducing dosage frequency, modulating
• Pegylation works by increasing the molecular weight of the therapeutic polypeptide by conjugating the polypeptide to one or more PEG polymer molecules. This is particularly applicable to types of antibody molecules that are fragments of complete antibodies, such as Fab fragments.
• poly ( alkylene oxide) molecules are polyethers.
• Poly ( alkylene glycol) molecules may have linear, branched, comb or star structures and generally are highly water soluble.
• the basic poly (alkylene glycol) structure may be provided with one or more reactive functional groups such as hydroxy, amine, carboxylic acid, alkyl halide or thiol groups to facilitate the reaction of the
• poly (alkylene glycol) molecule with other species such as polypeptides.
• Preferred poly (alkylene glycol) molecules include those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbon atoms.
• Preferred poly ( alkylene glycol) molecules for use in accordance with the present invention are polyethylene glycol ("PEG") molecules, although the skilled person would be able to derivatise antibody molecules of the present invention using other poly ( alkylene glycol) molecules, such as
• Polypropylene glycol or polyethylene-polypropylene glycol copolymers typically have molecular weights between about 400 Da and about 80 kDa, more preferably between about 1 kDa and about 60 kDa, and more preferably between about 5 kDa and about 50 kDa, e.g.
• molecular weights of 10 kDa, 20 kDa, 30 kDa or 40 kDa.
• Poly ( alkylene glycol) molecules that may be used in accordance with the present invention are well known in the art and publicly available, for example from commercially available sources such as SigmaAldrich .
• the present invention also provides immunoconjugates comprising an anti-CXCLl2 antibody molecule as described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
• cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
• an immunoconjugate of the present invention is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, for example a chemotherapeutic drug.
• ADC antibody-drug conjugate
• the antibody moiety is optionally linked to the drug via a linker.
• the antibody molecules of the present invention may additionally be labelled to enable them to be employed for imaging, either in conjunction with or independent of their therapeutic uses.
• One particular example of the use of the antibody molecules for imaging involves the use of radionuclide labels in nuclear medicine imaging techniques, such as Single Photon Emission Computed Tomography (SPECT) , an imaging technique that detects gamma rays emitted from a radionuclide to produce a two
• nuclear medicine imaging techniques such as Single Photon Emission Computed Tomography (SPECT)
• SPECT Single Photon Emission Computed Tomography
• PET Positron Emission Tomography
• Antibody molecules having radionuclide labels may also be employed for multi-modal studies in which imaging techniques are combined, either by selecting radionuclides that are active in more than one imaging technique or by labelling the antibody molecules with more than one type of label.
• the antibody molecules of the present invention may be labelled with a radionuclide, for example a radionuclide provided as a complex, or conjugated to a second molecule, such as a linker, that is can be associated with the label.
• a radionuclide for example a radionuclide provided as a complex
• a second molecule such as a linker
• radionuclides for use in imaging techniques or therapy include technetium, rhenium, copper, cobalt, gallium and indium isotopes such as Tc-99m, Re-186, Re-188, Co-57, Ga-67, In-Ill (SPECT) , Cu- 64, Cu-60, Cu-61, Cu-62, Cu-67, Tc-94m, Ga-68, Co-55 (PET) .
• technetium isotopes are employed for imaging purposes, rhenium isotopes for therapeutic purposes and copper isotopes for both imaging and therapy.
• CXCL12 has been reported to be involved in angiogenesis by recruiting endothelial progenitor cells (EPCs) from the bone marrow through a CXCR4 dependent mechanism, making it an
• CXCL12 also has a role in tumour metastasis where cancer cells that express the receptor CXCR4 are attracted to metastasis target tissues that release the ligand, CXCL12.
• the CXCL12 /CXCR /CXCR7 pathway has therefore generated considerable interest as a potential therapeutic target given its role in tumour growth, survival and angiogenesis. Accordingly, CXCL12 has been shown to be important in the organ- specific metastasis of tumours, as reviewed in Balkwill et al., Seminars in Cancer Biology, 14: 171-179, 2004.
• Tumours that express CXCL12/CXCR4 include ovarian cancer, breast cancer, bone cancer, prostate cancer, thyroid cancer, pancreatic cancer, multiple myeloma, non-Hodgkin ' s lymphoma, intraocular lymphoma, follicular center lymphoma, CML, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, kidney cancer, brain cancers such as glioma and astrocytoma, rhabdmyosarcoma, lung cancer, such as small cell lung cancer, melanoma, B cell
• B- CLL B-cell chronic lymphocytic leukemia
• AML acute myeloid leukaemia
• CAFs are known to secrete CXCL12 and this increases angiogenesis and tumour growth directly in breast cancer (Orimo et al . ,
• Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF- 1/CXCL12 secretion, Cell, 121(3) : 335-348, 2005) and pancreatic cancer (Feig et al., Targeting CXCL12 from FAP-expressing carcinoma associated fibroblasts synergizes with anti-PD-Ll immunotherapy in pancreatic cancer. P.N.A.S., 110: 50: 20212- 20217, 2013) .
• high levels of CXCL12 are associated with low numbers of T cells in a pancreatic cancer model and it was possible to increase T cell infiltration through combined treatment with PD-L1 and CXCR4 inhibitors.
• the antibody molecules or immunoconj ugates of the present invention may be used in the treatment of WHIM Syndrome (Warts, Hypogammaglobulinemia, Infections, and
• Myelokathexis syndrome is a rare congenital immunodeficiency disorder characterized by chronic noncyclic neutropenia which results from mutations in the chemokine receptor CXCR4.
• the antibody molecules or immunoconj ugates of the present invention may be administered in conjunction with a further cancer therapy or in conjunction with radiotherapy.
• the antibody molecules or immunoconjugates of the present invention may be administered in conjunction with a chemotherapeutic agent, an antibody therapy, immune modulatory therapy, surgery or in conjunction with radiotherapy, or in conjunction with cell mediated therapy.
• the antibody molecule and the further cancer therapy are administered together, optionally as a combined formulation.
• the antibody molecule and the further cancer therapy may be administered by alternation, with either the further cancer therapy administered before the antibody molecule, or the further cancer therapy administered after the antibody molecule.
• the combination may be administered in accordance with clinical practice, for example being administered at intervals from about one week to three weeks.
• the antibody molecules of the present invention are administered in conjunction with an angiogenesis inhibitor. Based on the mode of action of the antibody molecules of the present invention, combination
• angiogenesis inhibitors as used herein include agents that inhibit angiogenesis, vasculogenesis , or undesirable vascular permeability, either directly or indirectly. It should be understood that the angiogenesis inhibitors include those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor. See, e.g., Grothey and Galanis (2009) Nat. Rev. Clin. Oncol.
• Angiogenesis inhibitors include antibodies or peptide-antibody fusions targeted to angiogenesis-promoting growth factor
• Bevacizumab (Avastin®)
• Cetuximab (Erbitux®)
• Ramucirumab (Cyramza®)
• Icrucumab HuMV833, 2C3, Aflibercept
• angiogenesis inhibitors include small molecule kinase inhibitors, e.g. Sorafenib (Nexavar®) , Sunitinib (Sutent®) , Pazopanib (Votrient®) , Everolimus
• angiogenesis inhibitors examples include endostatin and angiostatin.
• Other drugs used to inhibit angiogenesis include thalidomide, squalamine and angiozyme.
• Bevacizumab (Avastin®) is a recombinant humanised anti-VEGF monoclonal antibody generated according to Presta et al . (1997) Cancer Res. 57 ( 20 ) : 4593-4599.
• Afibercept is a recombinant peptide-antibody fusion consisting of VEGF-binding portions fused to the Fc portion of human IgGl.
• Sorafenib (Nexavar®) is a multikinase inhibitor that blocks the receptor tyrosine kinases VEGFR, PDGFR (Platelet Derived Growth Factor Receptor), RAF serine/threonine kinases and c-KIT.
• sorafenib is named 4- [4- [ [4-chloro-3-
• Sunitinib (Sutent®) is a small molecule multi-targeted receptor tyrosine kinase inhibitor used in the treatment of cancer. It inhibits cellular signalling by targeting PDGFR and VEGFR.
• sunitinib is named N- (2-diethylaminoethyl ) -5- [ ( Z) (5-fluoro-2-oxo-lH-indol-3-ylidene) mWethyl] -2, 4-dimethyl-lfi- pyrrole-3-carboxamide and has the structure:
• Pazopanib (Votrient®) is a multikmase inhibitor used m the treatment of cancer. It is known to target c-KIT, PDGFR and VEGFR. In chemical terms it is named 5- [ [4- [ (2 , 3-Dimethyl-2H indazol-6-yl ) methylamino] -2-pyrimidinyl] amino] -2- methylbenzolsulfonamide and has the structure:
• Everolimus is a signal transduction inhibitor targeting mTOR (mammalian target of rapamycin) .
• mTOR mimmalian target of rapamycin
• AEE788 is a small molecule drug being evaluated for treatment for cancer. It is a combined inhibitor of both the EGFR (epidermal growth factor receptor) and VEGFR family members. In chemical terms it is named 6- [ - [( 4-Ethylpiperazin-l-yl ) methyl] henyl ] -N- [ (1R) -1-phenylethyl] -7H-pyrrolo [2, 3-d] yrimidin-4-amine and has the structure:
• PTK787 (Vatalanib) is a protein kinase inhibitor that inhibits angiogenesis being developed for cancer treatment. It inhibits VEGF receptors, PDGFR-beta and c-kit . In chemical terms it is named N- ( 4-chlorophenyl) -4- (pyridin-4-ylmethyl ) phthalazin- 1- amine and has the structure:
• additional chemotherapeutic agents include an EGFR pathway inhibitor, such as an anti-EGFR antibody or an EGFR kinase inhibitor, such as cetuximab, panitumumab, Iressa
• chemotherapeutic agents include alkylating agents, such as cisplatin, carboplatin and oxaliplatin, anthracyclines , plant alkaloids such as taxanes and vinca alkaloids, and topoisomerase inhibitors such as irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate and
• the antibody molecules of the present invention may be administered with an immune therapeutic, for example an immune pathway agent, small molecule agent or an antibody specific for PD-1, PDL-1, CTLA-4 or OX40.
• the antibody molecules of the present invention may be antibody-drug conjugates in which the antibody molecule is linked to a drug or toxin. This may be done to direct the drug or toxin to a target site in a biological system where CXCL12 is present.
• This approach may entail engineering the antibody molecule to provide a functional group capable of reacting with the drug or toxin, or alternatively providing the antibody molecule with a linker group that is capable of reacting with the drug or toxin.
• the drug may also be a pro-drug for conversion to active drug at a target site in a patient.
• the present invention provides an immunoconj ugate which comprises an antibody molecule of the present invention conjugated to a cytotoxic moiety or an immunostimmulatory moiety.
• the cytotoxic moiety may be an alkylating agent, an alkaloid, a platinum coordination complex, a cytotoxic peptide, a radioactive agent, or a pro-drug capable of conversion into a cytotoxic moiety.
• the present invention relates to antibody molecules for use in a method of diagnosis or prognosis of a condition in which CXCL12 is implicated.
• the antibody molecules of the present invention may be used in assays for identifying patients who are likely to be more responsive to treatment than a wider class of patients considered as a whole. This in turn may enable therapy, for example using antibody molecules of the present invention, to be directed to those patients most likely to respond, while providing the patients for whom treatment is less likely to be successful with alternative forms of therapy.
• the present invention provide a method of assaying for the presence of CXCL12 in a sample, the method comprising contacting the sample with antibody molecules of the present invention so that the CXCL12 binds to the antibody molecules to form a complex and detecting the complex thus produced.
• the methods may also employ antibody molecules of the present invention as reagents for detecting the binding of CXCL12 to a capture antibody.
• the method comprises determining the presence or amount of CXCL12 in the sample using the antibody and correlating the presence or amount of CXCL12 with the likely outcome of treating the patient with a CXCL12 inhibitor.
• the antibody molecules may be used in an ELISA-type format or otherwise labelled linked to a detectable molecule such as, but not limited to, radioactive or fluorescent labels or to enzymes which utilise a chromogenic substrate.
• a detectable molecule such as, but not limited to, radioactive or fluorescent labels or to enzymes which utilise a chromogenic substrate.
• radiolabels of use in this technique are 32 P, 3 H or 14 C.
• fluorescent molecules of use in this technique are green fluorescent protein, Fluorescein IsoThioCyanate (FITC), Rhodamine IsoThioCyanate (TRICT) Cy3 and Cy5 Dyes.
• FITC Fluorescein IsoThioCyanate
• TRICT Rhodamine IsoThioCyanate
• Cy3 Cy5 Dyes.
• enzymes with chromagenic substrates of possible use in this technique are peroxidase, alkaline phosphatase or glucose oxida
• the method of the present invention is an in vitro method carried out on a sample obtained from said individual.
• the method may therefore comprise an initial step of obtaining a sample from the individual in question and/or preparing the sample for analysis .
• samples for use in the method include blood samples, tissue samples or cell samples.
• the anti-CXCLl2 antibody molecules or immunoconjugates of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.
• a pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the anti-CXCLl2 antibody molecule, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution.
• These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the anti-CXCLl2 antibody molecule.
• anti-CXCLl2 antibody molecules or
• immunoconjugates may be provided in a lyophilised form for reconstitution prior to administration.
• lyophilised antibody molecules may be re-constituted in sterile water and mixed with saline prior to administration to an individual.
• Anti-CXCL12 antibody molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule.
• pharmaceutical compositions may comprise, in addition to the anti-CXCL12 antibody molecule, 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 anti- CXCL12 antibody molecule.
• the precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.
• composition comprising the anti-CXCL12 antibody molecule
• 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.
• Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and
• 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 lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions, such as sodium; metal complexes (e.g. Zn-protein
• non-ionic surfactants such as TWEENTM
• PLURONICSTM or polyethylene glycol (PEG) are examples of PLURONICSTM or polyethylene glycol (PEG) .
• a pharmaceutical composition comprising an anti-CXCLl2 antibody molecule may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• An anti-CXCLl2 antibody molecule as described herein may be used in a method of treatment of the human or animal body, including prophylactic treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset) .
• the method of treatment may comprise administering an anti-CXCL12 antibody molecule to an individual in need thereof.
• 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 method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of
• a therapeutically effective amount or suitable dose of an antibody molecule may 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 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) 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 iig 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 IgGl, IgG2, IgG3 or IgG4 isotype, according to differences in amino acid sequence in the hinge and Fc regions. These different isotypes affect the in vivo half- lives of the antibody molecules and their ability to induce effector functions. Hence, the choice of IgG isotype may be used to engineer the in vivo properties of the antibody molecules of the present invention.
• effector functions are less critical and so the use of an antibody isotype lacking effector function (such as IgG2) may be preferred to determine the benefits of CXCL12 neutralisation without interference of the host immune system.
• 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.
• the therapeutic effect of the anti-CXCL12 antibody molecule may persist for several half-lives, depending on the dose.
• the therapeutic effect of a single dose of anti-CXCLl2 antibody molecule may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.
• the 'McCafferty naive antibody library' which contains more than 10 billion antibody clones in scFv format, was used for isolating anti-CXCL12 antibodies and the resulting antibodies were then screened in a cell-based assay to assess potential to block the CXCL12-CXCR4 interaction.
• two rounds of panning were carried out on biotinylated CXCL12 immobilised on
• streptavidin or neutravidin In order to avoid enriching for antibody clones that bind to streptavidin or neutravidin two strategies were employed. Round 1 panning was carried out on biotinylated CXCL12 immobilised on streptavidin using the phage library that has been depleted of any streptavidin binders (known as e-selection; or ⁇ subtractive selection') . For Round 2, neutravidin was used (instead of streptavidin) to immobilise CXCL12. Polyclonal phage prepared from round 2 panning output was tested in a TRF binding assay against CXCL12, streptavidin, neutravidin and a non-specific antigen (NCKl) to confirm specific enrichment. The binding signal observed was specific to CXCL12 and no detectable binding was observed for streptavidin,
• the scFv population from the Round 2 panning output was PCR amplified and cloned into the pSANGl0-3F expression vector and transformed into BL21.
• the psANGl0-3F encodes a hexa-histidine tag (for Ni-affinity purification) and tri-FLAG tag (for
• trans formants were picked into 10 x 96 well culture plates and antibody expression was induced using auto-induction media.
• biotinylated-CXCL12 (immobilised on streptavidin in 96 well plates) was detected using an anti-FLAG antibody conjugated to europium. Clones with signal above 1000 FU (100 fold above background) were considered as positive for CXCL12 binding.
• framework residues bringing 38 more unique sequences, i.e. 156 unique sequences from 184 sequenced.
• Detailed analysis of the framework regions revealed a preference for certain heavy chain and light chain germline families. Vh3 (62%) and Vhl (43%) are the most frequently found heavy chain families while VKI (58%) followed by VK2 (17%) dominate the light chain sequences.
• Table 1 A snapshot of sequence analysis of the primary CXCL12 antibodies.
• the framework and the CDR regions were analysed using BLAZE software and the antibodies were clustered based on similarity in VH and VL CDR3 sequence (for example, 093_E11 and 093_E10) .
• a large panel of unique CXCL12 binders were identified from the primary screening and sequence analysis. In order to identify antibodies that block CXCL12-CXCR4 interaction, a cell based CXCL12-CXCR4 binding assay using flow cytometry was established. A human acute lymphoblastic leukemia cell line (MOLT-4) was identified as an ideal CXCR4 expressing cell line for CXCL12- CXCR4 binding assay as they were found to be negative for CXCR7 expression.
• MOLT-4 human acute lymphoblastic leukemia cell line
• a one-step binding detection method was tested and compared to a two-step binding detection method.
• the one step binding detection method involved direct staining of MOLT-4 cells with biotinylated-CXCL12 that has been pre-complexed with streptavidin-PE and detecting the binding by flow cytometry.
• the two-step binding detection method involved incubation of MOLT-4 cells with biotinylated CXCL12, washing, and then staining with
• Table 2 Top 20 blocking antibodies from the cell-based CXCL12- CXCR4 binding assay.
• antibodies with affinity ranging from 10 ⁇ -1 ⁇ Such antibodies are often affinity matured in vitro to attain sufficient affinity for the given application.
• In vitro affinity maturation of antibodies can be achieved by mimicking the in vivo process that occurs during a humoral immune response.
• the initial response to antigen stimulation involves the selection of antigen specific B-cells from a large and diverse pre-immune repertoire of B-cells expressing low affinity antibodies.
• These primary, low affinity antibodies then undergo a process called somatic hypermutation in which they accumulate point mutations in the heavy and light variable regions.
• the B- cells expressing high affinity antibodies compete with B-cells expressing low affinity antibodies for antigen stimulation to survive and proliferate.
• maturation strategies involve two key steps, diversification of the primary antibody sequence and the selective enrichment of affinity improved antibodies using a selection platform such as phage display technology.
• Diversification of primary antibody sequence can be done by introducing mutations to the variable regions using random or targeted mutagenesis.
• new combinations of heavy and light variable regions can be made by recombining selected heavy or light chains with a repertoire of partner chains by a process known as chain shuffling.
• heavy chain variable domains commonly play the dominant role in antigen binding and defining the epitope specificity
• light chain shuffling is preferred over heavy chain shuffling to preserve the binding epitope of the primary antibodies.
• the antibody heavy chain regions from the top 20 blocking antibodies were amplified by PCR.
• the resultant PCR products were cloned into a phage display light chain library preparation encompassing a repertoire of naive lambda and kappa light chain variable region partners (in vector pSANG4) .
• the ensuing plasmid population was transformed into E.Coli TGI to yield a library containing 2.6 x 10 8 scFv clones.
• each original heavy chain was paired with approximately 10 million new light chain partners.
• 20 random clones from the light chain shuffled library were analysed by colony PCR screen. 19 out of 20 clones showed the presence of a full-length scFv gene (not shown) suggesting that approximately 95% of clones in the library are light chain shuffled recombinants.
• Antibodies with high affinities can be enriched by iterative rounds of phage selection using diminishing antigen concentrations. This method relies on the competition for limiting amounts of antigen and the preferential enrichment of variants with lower dissociation constants. For precise control of antigen concentration, phage display
• antibodies are allowed to bind to a biotinylated antigen in solution and the bound phage is subsequently captured using a streptavidin-coated surface for washing and elution.
• the stringency of the selection can be further increased at this step by including a number of harsh and long washing steps.
• the scFv populations from round 3 selection outputs were PCR amplified and cloned into the pSANGlO-3F expression vector and the resulting plasmid DNA was transformed into E.coli BL21 DE3 cells. 960 individual transformants were picked into 10 x 96 well culture plates and the antibody expression was induced using auto-induction media. Recombinant monoclonal antibodies secreted into the culture supernatant after overnight induction, were screened for their ability to bind CXCL12 in a TRF binding assay. 48% (458/960) of the clones screened from various selection outputs were found to be positive for CXCL12 binding.
• binding signals exhibited by these clones were significantly better than those of the clones isolated from the naive library. 37% of the clones screened from the chain shuffled selection outputs showed binding signal above 10,000 fluorescent units (FU) . In contrast, only 12% of the clones isolated from the naive library exhibited binding signals exceeding 10,000 FU.
• the CXCL12 binding clones were and analysed using BLAZE antibody sequence analysis software. Analysis of the heavy and light chain CDR3 diversity identified 227 unique clones. Although 20 different VH genes were used in the construction of the chain shuffled library, only 9 VH genes were represented amongst the 227 unique clones identified. Within this set there were four major clone families. These were derived from the VH gene of primary clones 093_2A02 (122/227), 093_2D06 (38/227), 093_2G07 (24/227) and 093_2A10 (22/227) .
• VL germline usages of these chain shuffled clones were dominated by VK I (63%) and VK2 (22%) germline families.
• the CXCL12 binding signal observed for a particular clone in the primary screen was dependent on the combined effect of antibody expression and affinity. Since the antibody expression varies significantly from clone-to-clone, ranking antibodies by their binding signal in the primary screen do not necessarily correlate with their affinities. Therefore, an expression-independent secondary screening assay was used to identify high affinity anti-CXCLl2 antibodies with superior binding kinetics.
• dissociation constants (off-rates) of the top 150 anti-CXCL12 scFv antibodies were analysed using surface plasmon resonance (SPR) .
• SPR surface plasmon resonance
• 093_2D06 and 113_1H12 (derived from 093_2A02) showed the best off-rates (i.e. the lowest dissociation constants) amongst the 24 Fab antibodies tested. These two clones were selected as lead anti-CXCLl2 antibodies for detailed characterisation. The sequences of these antibodies are provided in the sequence listing .
• ScFv format is well suited for phage display selections and subsequent screening of large number clones to identify a panel of lead antibodies due to the efficient expression in E.coli.
• this format there are a number of limitations associated with this format that make it sub- optimal for downstream biophysical and biological
• screening assays are usually reformatted to larger and more stable antibody formats and are expressed in mammalian cells for downstream characterisation and in vivo testing.
• the two lead anti-CXCL12 antibodies and their parent clones were reformatted to Fabs and IgGs.
• Fabs are optimal for accurate determination of the binding constants.
• the IgG is the preferred format for majority of clinically approved antibodies and the antibodies in development. Their superior in vivo half-life, and the ability to engage the host immune system are ideal for the treatment of diseases, such as cancer.
• the bivalent nature of an IgG molecule greatly enhances its antigen
• sequences of the anti-CXCL12 antibodies of the present invention could be used in any appropriate expression systems designed for the generation of antibody molecules. All scFv antibodies discussed in this work were expressed from the pSANG10-3F vector, in which a single T7 promoter drives the expression of heavy and light variable domains that are connected by a glycine-serine linker ( Figure 1A) . In this system, the scFv gene is transcribed as a single mRNA and translated as a single protein. In contrast, commonly used mammalian Fab and IgG expression systems use expression cassettes that transcribe and translate heavy and light chain genes separately.
• pBIOCAM-7 bicistronic vector
• Figure IB a bicistronic vector
• VL+CL antibody light
• VH+CH heavy chain
• P2A peptide porcine teschovirus-1
• the ribosome skips the synthesis of the glycyl-prolyl peptide bond at the C-terminus of P2A peptide resulting in the release of the polypeptide chain immediately downstream of it.
• the heavy and light chain polypeptides are then folded and assembled
• the furin cleavage site at the C-terminus of light chain facilitates the posttranslational removal of the P2A peptide from the Fab protein.
• IgG antibodies are divided into 4 isotypes (IgGl, IgG2, IgG3 and IgG4) according to differences in amino acid sequence in the hinge and Fc regions. These different isotypes affect the in vivo half-lives of the antibody molecules and their ability to induce effector functions. Hence, the choice of IgG isotype may be used to engineer the in vivo properties of the antibody molecules of the present invention. For neutralising soluble antigens such as CXCL12, effector functions are less critical.
• IgG2 Isotype which exhibits reduced antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) .
• variable regions of the lead antibodies (114_3H1 and 113_1H12) and their parent clones
• Fab and IgG antibodies were purified from the cell culture supernatants (6 days post transfection) using Nickel and Protein-G affinity chromatography methods respectively. As illustrated in Figure 2, purified Fabs and IgGs were composed of a heavy and a light chain domains of the expected sizes.
• the lead anti-CXCL12 antibodies and their parent clones were subjected to a full kinetic analysis to determine the improvement in binding constants after affinity maturation.
• the binding of the Fab antibodies to biotinylated CXCL12 immobilised on a streptavidin chip was analysed using SPR ( Figure 3) .
• Equilibrium dissociation constants (KD) of these antibodies were determined using a binding model appropriate for each binding interaction (Figure 3B and C) .
• KD Equilibrium dissociation constants
• affinities of 113 1H12 and 093 2D06 were 3.7nM and 16.7 nM respectively, which represents a 4.5-fold improvement in affinity following light chain shuffling.
• CXCR4 expressing cancer cells towards CXCL12 rich environments is one of the key factors promoting metastasis in many malignancies.
• the inhibition of CXCL12/CXCR4 dependent cancer cell migration is an important biological property of the therapeutic anti-CXCL12 antibody molecules of the present invention.
• the lead antibodies 114_3H1 and 113_1H12 were tested for their ability to block CXCL12 induced migration of ovarian cancer cells using a transwell migration assay.
• the transwell migration assay used here was a modified version of the Boyden chamber assay used to study the chemotactic response of leukocytes.
• CXCL12/CXCR4 axis plays a key role in promoting the formation of new blood vessels (angiogenesis ) to establish a tumour supportive vasculature.
• CXCL12 and CXCR4 form a positive feed back loop with VEGF, a well-known pro-angiogenic factor. In this loop, VEGF stimulates the expression of both CXCL12 and CXCR .
• VEGF a well-known pro-angiogenic factor.
• CXCL12 induced activation of CXCR4 up-regulates production of VEGF by endothelial cells.
• angiogenesis assay to evaluate their ability to inhibit the formation and branching of tubules.
• human umbilical vein endothelial cells (HOVECs) and fibroblasts were cultured together in a media containing anti-CXCLl2 antibodies and VEGF.
• HOVECs human umbilical vein endothelial cells
• fibroblasts were cultured together in a media containing anti-CXCLl2 antibodies and VEGF.
• the interaction of these two cell types in the presence of VEGF results in the formation of three-dimensional tubes that resemble small capillaries in vivo.
• Inhibitory effects of anti-CXCLl2 antibodies were analysed after 7 days of co-culture by immunohistochemistry ( Figure 6) .
• WO 2008/018641 describes two pairs of anti-CXCL12 antibodies, 1D3 and 1H2 and 1C6 and 2A5.
• the pairs of antibodies recognise common epitopes in CXCL12.
• the sequences of the heavy and light chains of the antibodies of WO 2008/018641 were aligned with the sequences of the antibodies of the present invention and the alignment is shown in Figure 8. This shows that there are significant differences in the CDR sequences of these antibodies as compared to antibodies 114_H01 and 113_1H12 of the present invention.
• the affinity of the antibodies was also compared.
• the affinity constant (K D ) for human CXCL12 of antibody 114_H01 was 2.4 nM and that of antibody 113_1H12 was 4.2 nM.
• variable regions of the 113_1H12 and 114_3Hl were cloned into an murine IgG2a expression system, permitting production of the chimeric antibodies, with murine constant regions and human variable regions.
• This type of chimeric antibody could be preferable for in vivo testing in immunocompetent animals to reduce immune response towards the antibody which could occur upon administration of a fully human antibody.
• the ability of the antibodies in a chimeric murine IgG2a and a fully human IgG2 formats to block cellular migration of human and murine cell lines was investigated using a murine metastatic melanoma cell line (B16F10) and a human ovarian carcinoma cell line (T0V21G) .
• the collagen mix was composed of sodium bicarbonate. 10X MEM and Collagen (3mg/ml, Sigma) in a 1:2:15 ratio. The antibodies were added to the cell-collagen mix and the resulting mixture was inserted into the migration chamber where the collagen was polymerised by incubation at 37 °C for 30 minutes. Once polymerised the CXCL12 ligand was added to produce the chemokine gradient and movement of the cells towards CXCL12 was determined using time-lapse imaging. Images were taken at an interval of 1 picture/min over a period of 3.5 - 4 hours. Time lapse imagining permit the visualisation of cellular migration tracks which were them analysed using Image J software to determine the extent of cellular migration.
• 113_1H12 in a chimeric murine IgG2a format (mAB113) and 114_3H1 in a chimeric murine IgG2a format (mAB114), were effective at substantially blocking cellular migration in B16F10 cells and to some degree also in TOV-21 cells.
• 113_1H12 in a human IgG2 format (hAB113) was inactive in terms of blocking
• the serum stability of the antibodies 114_3H1 and 113_1H12 in a chimeric murine IgG2a format was tested. Serum stability assays were performed using human serum. Antibodies were incubated in serum at a concentration of 100 ⁇ g/ml for 6, 12, 24 and 48 hours. Serum containing antibodies were diluted to a concentration of 50 ]ig/ml in DMEM or RPMI and used for the time-lapse migration assay as described above in A. These experiments found that the antibodies in a chimeric murine IgG2a format were stable over 48 hours in serum and were still active at inhibiting CXCL12- dependent B16F10 cell migration after 48 hours in serum. This confirms that both antibodies are very stable in serum.
• B16F10 melanoma cells require CXCR4 for migration to the lung and initiation of metastasis.
• a simple experimental metastasis in vivo model was used to evaluate the ability of the chimeric murine IgG2a antibodies to interfere with CXCL12 and therefore block metastatic development.
• B16F10 melanoma cells were introduced into C57B1 mice through tail vein injection on day 0 and treatment commenced on day 1. Treatment regimes were either 5 mg/kg of the clinical CXCR4 inhibitor AMD3100 (Plerixafor) twice daily or twice a week with either 10, 15 or 20 mg/kg of the anti- CXCL12 antibody in a murine IgG2a format.
• mice in the control arm were treated twice a week with 20 mg/kg of a murine IgG2a control antibody. All mice were culled on day 14 and the number of metastatic colonies in the lungs quantified by manual counting. As shown in Figure 10, both chimeric murine 114_3H1 and 113_1H12 antibodies were active at inhibiting metastatic development. In particular, antibody 113_1H12 was more potent and resulted in and inhibition at 20mg/kg equivalent to that seen with 5 mg/kg of the small molecule antagonist of CXCR4 , AMD3100. Thus, both chimeric murine 114_3H1 and 113_1H12 antibodies were active at inhibiting metastatic development. In particular, antibody 113_1H12 was more potent and resulted in and inhibition at 20mg/kg equivalent to that seen with 5 mg/kg of the small molecule antagonist of CXCR4 , AMD3100. Thus, both
• antibodies are active in vivo in inhibiting CXCR4 dependent- pulmonary metastasis.
• SEQ ID NO: 1 CDR-H1 amino acid sequence (from Abll4__3Hl)
• SEQ ID NO: 2 CDR-H2 amino acid sequence (from Abll4_3Hl)
• SEQ ID NO: 3 CDR-H3 amino acid sequence (from Abll4_3Hl)
• SEQ ID NO: 4 CDR-L1 amino acid sequence (from Abll4_3Hl)
• RASQSISDYVN SEQ ID NO: 5 CDR-L2 amino acid sequence (from Abll4_3Hl)
• SEQ ID NO: 6 CDR-L3 amino acid sequence (from Abll4_3Hl)
• SEQ ID NO: 7 VH domain amino acid sequence
• SEQ ID NO: 8 VH domain nucleic acid sequence
• SEQ ID NO: 12 CDR-H1 amino acid sequence 113_1H12
• SEQ ID NO: 13 CDR-H2 amino acid sequence 113_1H12
• SEQ ID NO: 14 CDR-H3 amino acid sequence 113_1H12
• SEQ ID NO: 15 CDR-L1 amino acid sequence 113_1H12
• SEQ ID NO: 17 CDR-L3 amino acid sequence 113_1H12
• SEQ ID NO: 18 VH domain amino acid sequence
• SEQ ID NO: 20 VL domain amino acid sequence
• SEQ ID NO: 21 VL domain nucleic acid sequence
• SEQ ID NO: 24 Synthesised CXCL12 amino acid sequence used for antibody selection. Corresponds to amino acids 22 to 89 of full length CXCL12 protein
• SEQ ID NO: 25 recombinant "wild-type" CXCL12 expressed in E.coli for epitope mapping (including His tag and linker)

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