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WO2010000875A1 - Anticorps contre le récepteur d'érythropoïétine et ses utilisations - Google Patents

Anticorps contre le récepteur d'érythropoïétine et ses utilisations Download PDF

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Publication number
WO2010000875A1
WO2010000875A1 PCT/EP2009/058535 EP2009058535W WO2010000875A1 WO 2010000875 A1 WO2010000875 A1 WO 2010000875A1 EP 2009058535 W EP2009058535 W EP 2009058535W WO 2010000875 A1 WO2010000875 A1 WO 2010000875A1
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WIPO (PCT)
Prior art keywords
antibody
fragment
variant
seq
acid sequence
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PCT/EP2009/058535
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English (en)
Inventor
Richard J Buick
Thomas Jaquin
James Johnston
Roberta Burden
Angela Mcclurg
Shane Olwill
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Fusion Antibodies Ltd
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Fusion Antibodies Ltd
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Publication of WO2010000875A1 publication Critical patent/WO2010000875A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • This application relates to antibodies or fragments thereof and to their use in methods of treatment.
  • the application relates to antibodies specific for a fragment of the erythropoietin receptor (hereinafter "EPO-R”) .
  • EPO-R erythropoietin receptor
  • Erythropoietin (EPO) and its receptor EPO-R are essential for the survival, proliferation and differentiation of erythroid precursors. Binding of EPO to EPO-R causes receptor dimerisation and phosphorylation by Janus Kinase 2 (Jak 2), leading to the activation of several downstream signalling pathways including the Stat5-Bcl xL pathway, important for the survival of early erythroblasts, and the phosphatidylinositol-3 (PI3) kinase-AKT pathway and the Ras-mitogen-activated protein kinase pathways.
  • Jak 2 Janus Kinase 2
  • PI3 phosphatidylinositol-3
  • EPO-R contains a -230 amino acid extracellular domain, one transmembrane segment and a ⁇ 230 amino acid cytosolic domain which contains no enzymatic activity (Constantinescu et al . , 1999). Mice homozygous for mutations of the EPO-R gene die at embryonic day 12.5 owing to severe anaemia (Constantinescu et al . , 1999) and EPO-R knockout mice develop an abnormal brain and heart, characterized by massive cellular apoptosis. Transgenic mice that lack EPO-R exhibit severe anaemia die at embryonic day 13.5 (Wu et al . 1995) .
  • rHuEPO recombinant human erythropoietin
  • EPO-R Erythropoietin expression has been identified in a range of different haematological malignancies. EPO-R has been found to be expressed in a proportion of patients with myelodysplastic syndrome (MDS) , acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) and a significant correlation has been found to exist between survival and level of EPO-R expression on leukaemia cells (Takeshita et al., 2002). In addition, 71 leukaemia cell lines have been shown to express EPO-R by a 40 cycle RT-PCR (Chiba et al . , 1997) .
  • EPO-R expression in malignancy involved the study of 24 malignant human cell lines and highlighted that EPO and EPO-R expression in malignant tissue is widespread and autocrine mechanisms may be present. The results also showed a trend towards highly proliferative malignant cell lines secreting a greater amount of EPO than cell lines with a slower proliferation rate.
  • the HMVl, G361, P39, 220, SCH, DLDl, A549, SBC3, HeLa, PC-3 and HepG2 cell lines were injected subcutaneously into nude mice (Yasuda et al . , 2003) .
  • the largest amount of data concerning EPO-R expression in malignant tissue relates to cancers of the breast.
  • EPO-R was shown to be present in the MCF-7, BT-549, T47D, MDA-134 and MDA231 breast carcinoma cell line
  • EPO-R was shown to be functional in the MCF-7 cell line by increased phosphotyrosine levels following EPO stimulation (250 U/ml for 5 min) .
  • low amounts of rHuEpo were able to stimulate DNA synthesis and cell proliferation in both MCF-7 and BT- 549 cells.
  • Their data suggest that cell lines originating from solid human cancers, such as breast carcinoma, express functional EPO-R and can proliferate in response to EPO. They subsequently showed that EPO- R could be detected by Western blot analysis in clinical breast biopsy samples containing cancerous tissue but not in normal breast tissue from regions immediately adjacent to the tumours.
  • exposure to hypoxia for 8 h caused an up-regulation of EPO, and to a lesser extent of EPO- R mRNA, in both MCF-7 and BT-549 cell lines.
  • EPO-R is expressed in primary cultured neurons prepared from hippocampus and cerebral cortex of rat embryos. EPO-R mRNA is abundantly expressed in the brain of mouse early embryos, and its level is dramatically reduced during development, suggesting that EPO might play a role in brain development.
  • Astrocytes have been shown to be responsible for production of brain EPO. More recently it has been shown that neurons produce EPO. It is unlikely that renal EPO crosses the blood brain barrier under physiological conditions. Thus, the CNS has a paracrine EPO/EPO-R system that is independent of the endocrine system for erythropoiesis .
  • EPO and EPO-R expression in malignant brain tissue There are now also reports of EPO and EPO-R expression in malignant brain tissue.
  • the first report of EPO having an effect on malignant brain tissue was in the human neuroblastoma cell line, h-NMB (Wollman et al . , 1996). In this report they observed an inhibition of the cell line when grown in the presence of rHuEpo . rHuEpo also induced a partial differentiation of the cell line as evidenced by two different enzymatic markers and dopamine uptake.
  • EPO and EPO-R have been shown to be up-regulated in human cerebral gliomas (Mohyeldin et al . , 2003) .
  • Immunohistochemical analysis of 28 human glioma biopsies demonstrated prominent EPO and EPO-R expression in all samples, with EPO being specifically associated with malignant cells and EPO-R with malignant cells and tumour vasculature.
  • Normal adult brain tissue displayed very low levels of immunoreactivity for either protein. From cell cultures it was shown that EPO and EPO-R were hypoxia- inducible and the EPO-R was functional due to STAT5 phosphorylation following stimulation with 10 U/ml rHuEpo or 200 U/ml of the clinically used Epoetin-alfa . Finally they showed pre-treatment of glioma cultures with rHuEpo protected the cells against cisplatin cytotoxicity.
  • Prostate cancer EPO may regulate the growth of neoplastic and normal prostate cells in humans (Brower, 2003; Feldman et al . , 2003) .
  • Prostate epithelial cells that are transformed but non-tumourigenic, various cultured neoplastic prostate cell lines and normal prostate cells have all been shown to express EPO-R. All three types of prostate cells grew more rapidly in a dose-dependent manner, when exposed to increasing concentrations of EPO in vitro. The presence of EPO-R raises the possibility that therapeutic doses of EPO may stimulate tumour cell growth especially as a large number of patients receive EPO supplementation to correct chemotherapy-induced anaemia (Brower, 2003) .
  • Angiogenesis occurs actively in embryos, but it is repressed in healthy adults. An exception in adults is the female reproductive system.
  • EPO plays an important role in angiogenesis via EPO-R expressed in vascular endothelial cells of the uterine endometrium. Whereas EPO production in the kidney, liver and brain is hypoxia-inducible, in the endometrium and oviduct, it depends on estradiol-17 (Yasuda et al . , 2003).
  • E 2 estradiol-17
  • OVX ovariectomised mice
  • EPO-R expression has also been observed in human non-small cell lung carcinoma (NSCLC) (Saintigny et al, 2007) .
  • NSCLC human non-small cell lung carcinoma
  • RT-PCR the lung adenocarcinoma cell line, A549, and the small cell lung carcinoma cell line, SBC3, expressed both EPO and EPO-R transcripts (Yasuda et al. , 2003) .
  • Renal cancer Erythropoietin receptor expression has been confirmed in both renal cell carcinoma (RCC) cell lines and patient samples (Westenfelder and Baranowski, 2000). Nephrectomy samples were screened from patients with RCC (one chromophilic, two clear cell) as well as two human cell lines (Caki-2, 786-0) and a mouse renal adenocarcinoma cell line (RAG) and found to be positive for EPO-R transcript and protein. Whereas the cell lines did not show any EPO expression the patient samples exhibited EPO expression suggesting a possible autocrine mechanism.
  • RCC renal cell carcinoma
  • patient samples exhibited EPO expression suggesting a possible autocrine mechanism.
  • EPO stimulated cell proliferation dose dependently, and the individual mitogenic effects of either EPO or 10% calf serum were markedly amplified when both were co-administered.
  • the authors suggested that if the mitogenic effects of EPO were operative in patients with RCC, endogenous EPO or its administration for the treatment of anaemia could potentially hasten proliferation of the malignancy.
  • EPO-R antibodies using immunohistochemical techniques. They found that each of the antibodies were capable of detecting additional proteins by western blot and identified one of these as HSP70 (Elliott et al, 2006) . Additional evidence published studying NSCCL tissue sections reinforced the non-specific nature of antibodies towards EPO-R (Brown et al, 2007) . From these results, they concluded that any immunohistochemical data produced using these particular antibodies should be interpreted with caution.
  • the present inventors have unexpectedly found that useful antibodies can be developed when directed to a specific region of EPO-R. Such antibodies have been found by the inventors to impair the rate of invasion of cancer cells.
  • an antibody comprising: - a) at least one, at least two or three CDRs of the V H domain selected from the group consisting of (i) CDRl consisting essentially of the amino acid sequence of SEQ ID NO 31, (ii) CDR2 consisting essentially of the amino acid sequence of SEQ ID NO 32, and (iii) CDR3 consisting essentially of the amino acid sequence of SEQ ID NO 33, and/or; b) at least one, at least two or three CDRs of the V L domain selected from the group consisting of (i) CDRl consisting essentially of the amino acid sequence of SEQ ID NO 34, (ii) CDR2 consisting essentially of the amino acid sequence of SEQ ID NO 35, and (iii) CDR3 consisting essentially of the amino acid sequence of SEQ ID NO 36, and/or; c) V H domain consisting essentially of the amino acid sequence of SEQ ID NO 1, and/or; d) V L domain
  • amino acid sequence corresponding to SEQ ID No 1 is as follows :-
  • amino acid sequence corresponding to SEQ ID No 31 is as follows: - FIFSDAWMD
  • amino acid sequence corresponding to SEQ ID No 32 is as follows: - IRSKANNHATYYAESVKG
  • amino acid sequence corresponding to SEQ ID No 33 is as follows: - DFDS
  • amino acid sequence corresponding to SEQ ID No 34 is as follows: - KSSQSLFNSRTRKNYLA
  • amino acid sequence corresponding to SEQ ID No 35 is as follows: - WASSRES
  • amino acid sequence corresponding to SEQ ID No 36 is as follows: - KQSYNLRT
  • any one or more of the above CDRs, VH domains or VL domains may consist of the recited sequences.
  • the antibodies, or fragments thereof comprise in sequence a CDRl, CDR2 and CDR3.
  • the use of such antibodies, or fragments or variants thereof, has surprisingly been found not to disrupt erythroid tissue function. Consequently, it can be deduced that the antibodies, or fragments or variants thereof, can be used to attenuate cancer invasion without the side-effect of inducing anaemia in the subject .
  • the antibody, or fragment or variant thereof is capable of:- (a) attenuating invasion of cancer cells, and/or; (b) binding specifically to an epitope on EPO-R (e.g. that defined in SEQ ID NO 37), and/or (c) being administered to a subject without impairing the subject's erythroid function, and/or (d) being administered to a subject without impairing the subject's haematocrit level.
  • the antibody, or fragment or variant thereof comprises an antigen binding domain comprising at least one, at least two, or three of the CDRs.
  • the antibody, or fragment or variant thereof comprises an antibody VH domain, an antibody VL domain, or both.
  • the antibody, or fragment or variant thereof comprise an antibody V H domain which comprises at least one, at least two, or three of the CDRs.
  • the antibody, or fragment or variant thereof is a scFv.
  • the antibody is a whole antibody.
  • the antibody, or fragment or variant thereof is an isolated antibody, or fragment or variant thereof.
  • the antibodies according the present invention which possess the aforementioned therapeutic activities have been developed by being raised against a specific region of EPO-R.
  • amino acid sequence corresponding to SEQ IN No. 37 is as follows :-
  • the polypeptide is derived from the nucleic acid sequence (DNA) corresponding to SEQ ID No. 38, which is as follows :- CCGGACCCCAAGTTCGAGAGCAAAGCGGCCTTGCTGGCGGCCCGGGGGCCCGAAG AGCTTCTGTGCTTCACCGAGCGGTTGGAGGACTTGGTGTGTTTCTGGGAGGAAGC GGCGAGCGCTGGGGTGGGCCCGGGCAACTACAGCTTCTCCTACCAGCTCGAGGAT GAGCCATGGAAGCTGTGTCGCCTGCACCAGGCTCCCACGGCTCGTGGTGCGGTGC GCTTCTGGTGTTCGCTGCCTACAGCCGACACGACACG
  • the antibody, or fragment or variant thereof is capable of:- (a) attenuating invasion of cancer cells, and/or; (b) binding specifically to an epitope on EPO-R (e.g. that defined in SEQ ID NO 37), and/or (c) being administered to a subject without impairing the subject's erythroid function, and/or (d) being administered to a subject without impairing the subject's haematocrit level.
  • the antibody, or fragment or variant thereof comprises an antigen binding domain comprising at least one, at least two, or three of the CDRs.
  • the antibody, or fragment or variant thereof comprises an antibody VH domain, an antibody VL domain, or both.
  • the antibody, or fragment or variant thereof comprise an antibody V H domain which comprises at least one, at least two, or three of the CDRs.
  • the antibody, or fragment or variant thereof is a scFv.
  • the antibody is a whole antibody. In an embodiment the antibody, or fragment or variant thereof, is an isolated antibody, or fragment or variant thereof.
  • an antibody according to the second aspect of the present invention has been characterised by amino acid sequence. Therefore, in an embodiment, the antibody comprises : -
  • SEQ ID NO 35 and (iii) CDR3 consisting essentially of the amino acid sequence of SEQ ID NO 36, and/or; c) V H domain consisting essentially of the amino acid sequence of SEQ ID NO 1, and/or; d) V L domain consisting essentially of the amino acid sequence of SEQ ID NO 2.
  • any one or more of the above CDRs, VH domains or VL domains may consist of the recited sequences.
  • the antibodies, or fragments thereof comprise in sequence a CDRl, CDR2 and CDR3.
  • nucleic acid encoding an antibody, or fragment or variant thereof, according to any one of the preceding aspects of the present invention.
  • the nucleic acid sequence for CDRl of the V H domain may consist essentially of the nucleic acid sequence TTCATTTTTAGTGACGCCTGGATGGAC (SEQ ID NO 47), or variants thereof.
  • the nucleic acid sequence for CDR2 of the V H domain may consist essentially the nucleic acid sequence ATTAGAAGCAAAGCTAACAATCATGCAACATACTATGCTGAGTCTGTG AAAGGG (SEQ ID NO 48), or variants thereof.
  • the nucleic acid sequence for CDR3 of the V H domain may consist essentially the nucleic acid sequence GACTTTGACTCC (SEQ ID NO 49), or variants thereof.
  • the nucleic acid sequence for CDRl of the V L domain may consist essentially the nucleic acid sequence AAATCCAGTCAGAGTCTGTTCAACAGTAGAACCCGAAAGAACTACTTG
  • GCT SEQ ID NO 50, or variants thereof.
  • the nucleic acid sequence for CDR2 of the V L domain may consist essentially the nucleic acid sequence
  • the nucleic acid sequence for CDR3 of the V L domain may consist essentially the nucleic acid sequence AAGCAATCTTATAATCTTCGGACG (SEQ ID NO 52), or variants thereof.
  • any one or more of the above nucleic acid sequences may consist of the recited sequences.
  • nucleic acid sequence is an isolated nucleic acid sequence.
  • the present inventors have surprisingly shown that aforementioned antibody, or fragment or variant thereof, is able to attenuate cancer cell invasion. This demonstrates that the antibodies, and fragments and variants thereof, may be used to treat a proliferative disorder, such as cancer.
  • a pharmaceutical composition comprising the antibody, or fragment or variant thereof, or the nucleic acid, according to earlier aspects of the present invention.
  • a method of attenuating cancer cell invasion comprising administration of the antibody, or fragment or variant thereof, or the nucleic acid, according to earlier aspects of the present invention.
  • the antibody, or fragment or variant thereof may attenuate cancer cell invasion by inhibiting proteolytic activity of proteases associated with cancer invasion.
  • a method of attenuating protease activity associated with cancer cell invasion comprising administration of the antibody, or fragment or variant thereof, or the nucleic acid, according to earlier aspects of the present invention.
  • the methods according the above aspects of the present invention may be performed in vitro, in vivo, or ex vivo, as required.
  • a method of treating a proliferative disorder in a patient in need of treatment thereof comprising administration of the antibody, or fragment or variant thereof, the nucleic acid, or the compositions, according to earlier aspects of the present invention.
  • the antibody, or fragment or variant thereof, the nucleic acid, or the compositions, according to earlier aspects of the present invention for use in medicine.
  • the use is the use in a method of treating a proliferative disorder.
  • the uses and methods of the earlier aspects of the present invention may further comprise the separate, sequential or simultaneous administration of an additional therapeutic agent for treating cancer.
  • the additional therapeutic agents may be antibody therapeutics, for example EvizonTM, PTK787, RetaaneTM, AG-13958, CAND5, CombretastatinTM and/or VEGF TrapTM.
  • the proliferative disorder may be cancer or an inflammatory disorder (such as asthma, allergy etc.).
  • EPO erythropoiesis
  • a method of treating anaemia in a patient in need of treatment thereof comprising administration to the patient of the antibody, or fragment or variant thereof, the nucleic acid, or the compositions, according to earlier aspects of the present invention separately, sequentially or simultaneously with EPO.
  • the antibody, or fragment or variant thereof, the nucleic acid, or the compositions for use in a method of treating anaemia in which the antibody, or fragment or variant thereof, the nucleic acid, or the compositions, are administered separately, sequentially or simultaneously with EPO.
  • the antibody, or fragment or variant thereof, the nucleic acid, or the compositions according to earlier aspects of the present invention in the preparation of a medicament for the treatment of anaemia, in which the antibody, or fragment or variant thereof, the nucleic acid, or the compositions, are administered separately, sequentially or simultaneously with EPO.
  • the anaemia may be anaemia associated with cancer, or induced by therapeutic regimes for treating cancer. Consequently, the patient may be a patient suffering from cancer.
  • composition comprising the antibody, or fragment or variant thereof, or the nucleic acid, according to earlier aspects of the present invention and EPO.
  • a method of producing the antibody, or fragment or variant thereof, according to earlier aspects of the present invention comprising expressing the nucleic acid according to the earlier aspect of the present invention in a host cell and isolating said antibody, or fragment or variant thereof .
  • the attenuation of the invasion of a cancer cell is demonstrated by a reduction in the number of cancer cells that invade for example tissue, or a matrigel, or the like, than in the absence of the antibody, or fragment or variant thereof.
  • the ability of the antibody, or fragment or variant thereof, to attenuate cancer cell invasion may be tested using any suitable method. For example, such ability may be tested using a modified Boyden chamber as is known in the art.
  • the antibody or fragment thereof may be tested using any suitable tumour cell line, for example a prostate carcinoma cell line, e.g. pC3, an astrocytoma cell line e.g.U251mg, a colorectal carcinoma cell line, e.g.
  • HCT116 or a breast cancer cell line, e.g. MDA-MB-231 or MCF7.
  • An antibody or fragment thereof may be considered to attenuate cancer cell invasion if it has the ability to inhibit invasion by a statistically significant amount. For example, wherein the number of cells invading is at least 20% fewer, for example at least 50% fewer, for example at least 70% fewer, for example at least 80% fewer, or for example at least 90 % fewer in the presence of said antibody or fragment thereof.
  • the antibody, and fragments and variants thereof may induce ADCC/CDC. This may be achieved by binding to a EPO-R on a cancer cell.
  • the antibody, or fragments or variants thereof used to induce ADCC/CDC may be humanised antibodies.
  • an "antibody” should be understood to refer to an immunoglobulin or part thereof or any polypeptide comprising a binding domain which is, or is homologous to, an antibody binding domain.
  • Antibodies include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies, humanised antibodies, and fragments thereof and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide.
  • the antibodies or fragments thereof further encompasses the use of mimetic propeptides which can be used as therapeutic peptides.
  • Mimetic pro peptides are short peptides which mimic the biological activity of the antibody or fragment thereof described herein.
  • Such mimetic peptides can be obtained from methods known in the art such as, but not limited to, phage display or combinatorial chemistry. For example, the method disclosed by Wrighton, et al . , Science 273:458-463 (1996) can be used to generate mimetic QUB 698.8 peptides .
  • Intact (whole) antibodies comprise an immunoglobulin molecule consisting of heavy chains and light chains, each of which carries a variable region designated VH and VL, respectively.
  • the variable region consists of three complementarity determining regions (CDRs, also known as hypervariable regions) and four framework regions (FR) or scaffolds.
  • CDRs complementarity determining regions
  • FR framework regions
  • the CDR forms a complementary steric structure with the antigen molecule and determines the specificity of the antibody.
  • antibody fragments may retain the binding and/or attenuation of cancer cell invasion abilities of the intact antibody and may be used in place of the intact antibody. Accordingly, for the purposes of the present invention, unless the context demands otherwise, the term "antibodies” should be understood to encompass antibody fragments. Examples of antibody fragments include Fab, Fab', F (ab')2, Fd, dAb, and Fv fragments, scFvs, bispecific scFvs, diabodies, linear antibodies (see US patent 5, 641, 870, Example 2 ; Zapata et al . , Protein Eng 8 (10) : 1057-1062 [1995]) ; single-chain antibody molecules ; and multispecific antibodies formed from antibody fragments.
  • the Fab fragment consists of an entire L chain (VL and CL), together with VH and CHl.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHl domain including one or more cysteines from the antibody hinge region.
  • the F (ab') 2 fragment comprises two disulfide linked Fab fragments .
  • Fd fragments consist of the VH and CHl domains.
  • Fv fragments consist of the VL and VH domains of a single antibody.
  • Single-chain Fv (scFv) fragments are antibody fragments that comprise the VH and VL domains connected by a linker which enables the scFv to form an antigen binding site, (see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
  • Diabodies are small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra- chain pairing of the V domains is achieved, resulting in a multivalent fragment, i.e.
  • the antibody molecules, or fragments thereof, for use in any one of the above described aspects of the present invention are not limited to antibodies having the specific sequences defined in the attached SEQ IDs.
  • the present invention extends to variants of the antibodies, or fragments thereof, defined by sequence above on the condition that the variants are capable of: - (a) attenuating invasion of cancer cells, and/or; (b) binding specifically to an epitope on EPO-R (e.g. that defined in SEQ ID NO 37), and/or (c) being administered to a subject without impairing the subject's erythroid function, and/or (d) being administered to a subject without impairing the subject's haematocrit level.
  • variants of the antibody, or fragments thereof defined by sequence that maintain any one or more of the abilities defined in (a) to (d) but that include additions, substitutions or deletions to any part of the aforementioned sequences.
  • an antibody or fragment thereof including any of the CDR amino acid sequences, V H amino acid sequences, and/or V L amino acid sequences described in the aspects of the present invention, in which one or more amino acid residues are modified (e.g. by addition, substitution or deletion) is a variant of the antibody on the condition that any one or more of the abilities defined in (a) to (d) are maintained.
  • the modified amino acid residues in the amino acid sequences are preferably 30% or less, more preferably 20% or less, most preferably 10% or less, within the entire modified amino acid sequence of the V H domain, V L domain and/or CDR.
  • Variants may include antibodies with the selection of CDRs defined in the first and second aspects of the present invention, in which at least one, two, three, four, five or six of the CDRs include five or less, four or less, three or less, two or one amino acid addition, deletion or substitution and retain the abilities defined in (a) to (d) .
  • Such variants may be provided using the teaching of the present application and techniques known in the art.
  • the CDRs may be carried in a framework structure comprising an antibody heavy or light chain sequence or part thereof.
  • Such CDRs are positioned in a location corresponding to the position of the CDR (s) of naturally occurring VH and VL domains.
  • the positions of such CDRs may be determined as described in Rabat et al, Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, Public Health
  • nucleic acids for use in any one of the above described aspects of the present invention are not limited to nucleic acids having the specific sequences defined in the attached SEQ IDs.
  • the present invention extends to variants of the nucleic acids defined by sequence above on the condition that the variants code for amino acids that are capable of:- (a) attenuating invasion of cancer cells, and/or; (b) binding specifically to an epitope on EPO-R (e.g. that defined in SEQ ID NO 37), and/or (c) being administered to a subject without impairing the subject's erythroid function, and/or (d) being administered to a subject without impairing the subject's haematocrit level.
  • variants may code for the specified amino acids that possess any of the abilities of (a) to (d) .
  • a nucleic acid sequence described in the aspects of the present invention in which one or more nucleic acid residues are modified (e.g. by addition, substitution or deletion) , is a variant of the nucleic acid on the condition that any one or more of the abilities defined in (a) to (d) are maintained.
  • the modified nucleic acid residues in the nucleic acid sequences are preferably 30% or less, more preferably 20% or less, most preferably 10% or less, within the entire modified nucleic acid sequence of the V H domain, V L domain or CDR.
  • a polypeptide may have additional features or elements beyond those described provided that such additional features or elements do not materially affect the abilities defined in (a) to (d) above. That is, the antibody or antibody fragments comprising the polypeptides may have additional features or elements that do not interfere with the ability of the antibody or antibody fragments to function as described in (a) to (d) .
  • a polypeptide consisting essentially of a specified sequence may contain one, two, three, four, five or more additional amino acids, at either end or at both ends of the sequence provided that the additional amino acids do not interfere with, inhibit, block or interrupt the role of the polypeptide in an antibody or antibody fragment.
  • a polypeptide molecule which contributes to the aforementioned abilities of the invention may be chemically modified with one or more functional groups provided that such chemical groups do not interfere with the abilities.
  • Antibodies for use in the invention herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U. S. Patent No. 4, 816, 567 ; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 : 6851-6855 (1984)) .
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e. g. Old World Monkey, Ape etc), and human constant region sequences.
  • Antibodies, or fragments or variants thereof may be conjugated to cytotoxic agents, thereby enabling targeted administration of the cytotoxic agents to tissues expressing the epitope to which the antibodies, or fragments or varients thereof, bind.
  • isolated means isolated 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 origin other than the sequence encoding a polypeptide with the required function.
  • Antibody molecules for use in the present invention may be produced in any suitable way, either naturally or synthetically. Such methods may include, for example, traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256 :495-499), recombinant DNA techniques (see e.g. U. S. Patent No. 4,816, 567), or phage display techniques using antibody libraries (see e.g. Clackson et al . (1991) Nature, 352: 624-628 and Marks et al . (1992) Bio/ Technology, 10: 779-783).
  • lymphocytes capable of binding the antigen.
  • the lymphocytes are isolated and fused with a myeloma cell line to form hybridoma cells which are then cultured in conditions which inhibit the growth of the parental myeloma cells but allow growth of the antibody producing cells.
  • the hybridoma may be subject to genetic mutation, which may or may not alter the binding specificity of antibodies produced. Synthetic antibodies can be made using techniques known in the art (see, for example, Knappik et al, J. MoI. Biol. (2000) 296, 57-86 and Krebs et al, J. Immunol. Meth. (2001) 2154 67-84.
  • variable VH and/or VL domains may be produced by introducing a CDR, e.g. CDR3 into a VH or VL domain lacking such a CDR.
  • CDR e.g. CDR3
  • VH or VL domain lacking such a CDR Marks et al . (1992) Bio/ Technology, 10: 779-783 describe a shuffling technique in which a repertoire of VH variable domains lacking CDR3 is generated and is then combined with a CDR3 of a particular antibody to produce novel VH regions.
  • novel VH and VL domains comprising CDR derived sequences of the present invention may be produced.
  • Alternative techniques of producing antibodies for use in the invention may involve random mutagenesis of gene(s) encoding the VH or VL domain using, for example, error prone PCR (see Gram et al, 1992, P. N. A. S. 89 3576-3580. Additionally or alternatively, CDRs may be targeted for mutagenesis e.g. using the molecular evolution approaches described by Barbas et al 1991 PNAS 3809-3813 and Scier 1996 J MoI Biol 263 551-567.
  • antibodies and fragments may be tested for the ability to (a) attenuate invasion of cancer cells, and/or; (b) bind specifically to an epitope on EPO-R (e.g. that defined in SEQ ID NO 37), and/or (c) be administered to a subject without impairing their erythroid function, and/or (d) be administered to an individual without impairing the subject's haematocrit level.
  • EPO-R epitope on EPO-R
  • binding specifically refers to the ability of the antibodies, or fragments or variants thereof, to a target an epitope present on EPO-R (e.g. that defined in SEQ ID NO 37) with a greater affinity than that which results when bound to a non-target epitope.
  • the specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target epitope. In certain embodiments, this affinity is determined by an affinity ELISA assay.
  • affinity can be determined by a BIAcore assay.
  • affinity can be determined by a kinetic method.
  • affinity can be determined by an equilibrium/solution method.
  • Nucleic acid for use in the present invention may comprise DNA or RNA. It may be produced recombinantly, synthetically, or by any means available to those in the art, including cloning using standard techniques.
  • the nucleic acid may be inserted into any appropriate vector, for example a virus (e. g. vaccinia virus, adenovirus, etc .), baculovirus; yeast vector, phage, chromosome, artificial chromosome, plasmid, or cosmid DNA.
  • Vectors may be used to introduce the nucleic acids into a host cell, which may be prokaryotic or eukaryotic .
  • Treatment includes any regime that can benefit a human or non-human animal .
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment) .
  • Treatment may include curative, alleviation or prophylactic effects.
  • tumours include treatment of conditions caused by cancerous growth and/or vascularisation and includes the treatment of neoplastic growths or tumours.
  • tumours that can be treated using the invention are, for instance, sarcomas, including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , cervical and ovarian carcinoma, non-small cell lung cancer, hepatocellular carcinoma, lymphomas, including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, and leukemias, including acute lymphoblastic leukaemia and acute myeloblastic leukaemia, astrocytomas, gliomas and retinoblastomas .
  • the present invention may be used to treat breast, colon and lung cancer.
  • the invention may be particularly useful in the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.
  • compositions according to the present invention may comprise, in addition to active ingredients, a pharmaceutically acceptable excipient, a carrier, buffer stabiliser or other materials well known to those skilled in the art (see, for example, (Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro AR, et al, eds . , Lippincott Williams & Wilkins, 2005.).
  • Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such aspolyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants.
  • buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such aspolyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants.
  • compositions may also contain one or more further active compounds selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the composition of the invention.
  • the formulation may comprise an additional component, for example a second or further antibody or fragment thereof according the present invention, a second or further chemotherapeutic agent, or an antibody to a target other than the amino acid sequence of SEQ ID No. 37 (for example to a growth factor which affects the growth of a particular cancer) .
  • the active ingredients may be administered via microspheres, microcapsules liposomes, other microparticulate delivery systems.
  • active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for further details, see Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro AR, et al, eds . , Lippincott Williams & Wilkins, 2005.
  • Sustained-release preparations may be used for delivery of active agents.
  • suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e. g. films, suppositories or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate) , or poly (vinylalcohol) ) , polylactides (U. S. Pat. No.
  • copolymers of L-glutamic acid and ethyl-L glutamate copolymers of L-glutamic acid and ethyl-L glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-) -3-hydroxybutyric acid.
  • nucleic acids may also be used in methods of treatment.
  • Nucleic acid for use in the invention may be delivered to cells of interest using any suitable technique known in the art.
  • Nucleic acid (optionally contained in a vector) may be delivered to a patient's cells using in vivo or ex vivo techniques.
  • in vivo techniques transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno- associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example) may be used (see for example, Anderson et al . , Science 256 : 808-813 (1992) .
  • nucleic acid is introduced into isolated cells of the patient with the modified cells being administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e. g. U. S. Patent Nos. 4, 892, 538 and 5, 283, 187).
  • Techniques available for introducing nucleic acids into viable cells may include the use of retroviral vectors, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the antibody or fragment thereof, nucleic acid, or composition may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells.
  • Targeting therapies may be used to deliver the active agents more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • the antibodies or fragments thereof, nucleic acid, or compositions of and for use in the invention, as appropriate, are suitably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
  • the actual dosage regimen will depend on a number of factors including the condition being treated, its severity, the patient being treated, the agents being used, and will be at the discretion of the physician.
  • Figure 1 illustrates EPO-R domain structure and antigen fragment 1 (i.e. SEQ ID NO. 37).
  • Figure 2 illustrates Western blot analysis of fragment 1 of EPO-R as identified in Figure 1 when amplified by PCR from an image clone.
  • Figure 3 illustrates SDS-PAGE analysis of lysates from the expression of fragment 1 of EPO-R by pQE40 (A, B and C represent cultures of different densities) .
  • Figure 4a illustrates a purification profile of fragment 1 of EPO-R by virtue of its hexahistidine tag using immobilised metal ion affinity chromatography.
  • Figure 4b illustrates a magnification of the purification profile showing elution of the recombinant protein from the column.
  • Figure 5 illustrates a study of the integrity and purity of the EPO-R recombinant protein by SDS-PAGE analysis. Lanes 5-11 contain purified EPO-R recombinant protein after purification, whereas lane 13 contains a sample of lysate prior to purification. Lanes 1 and 12 are molecular weight markers.
  • Figure 6 illustrates results of an analysis of 40 hybridoma clones for expression of anti-EPO-R antibodies using ELISA.
  • Figure 7 illustrates results of analysis by ELISA of final EPO-R clones found to be specific for EPO-R compared to negative control antigen produced in the same manner.
  • Figure 8 illustrates a purification trace of the antibody according to the present invention (i.e. AB1E3 antibody) .
  • Figure 9 illustrates a Western blot analysis using the antibody according to the present invention (i.e. AB1E3) showing positive detection of the EPO-R recombinant protein.
  • Figure 10 illustrates RT-PCR analysis of a panel of human and murine carcinoma cell lines for EPO-R expression using primers specific for murine EPO-R.
  • Figure 11 illustrates results of an in vitro invasion assay using 4Tl murine breast cancer cell line.
  • Figure 12 illustrates RT-PCR analysis of a panel of human carcinoma cell lines and endothelial cells for EPO-R expression using primers specific for human EPO- R.
  • Figure 13 illustrates whole cell lysate analysis of a panel of human carcinoma cell lines and endothelial cells for EPO-R expression.
  • Figure 14 illustrates confocal microscopy performed on HCT116 after staining EPO-R antibody binding (a) and with an isotype control in the absence of the antibody (b) .
  • Figure 15 illustrates flow cytometry analysis of MDA- Mb231 breast cancer cells incubated with (a) isotype control and Jak2 inhibitor (b) EPO-R antibody (c) EPO-R and Jak2 inhibitor.
  • Figure 16 illustrates a wound scratch assay on MDA231 (breast) and U251 (glioblastoma) cells (data not shown) , where (a) shows a picture of a wound inflicted on MDA 231 cells after incubation with an isotype control, (b) shows a picture of a wound inflicted on MDA 231 cells after incubation with the anti-EPO-R of the present invention, and (c) shows the results of a number of such assays.
  • Figure 17 illustrates a cancer cell invasion assay on U251 cells, where (a) shows a picture of a matrigel after incubation with an isotype control, (b) shows a picture of a matrigel after incubation with the antibody according to the present invention, and (c) shows the results of a number of such assays.
  • Figure 18 illustrates results of haematocrit levels taken from untreated mice, mice treated with an isotype control and mice treated with the antibody according to the present invention.
  • Figure 19 illustrates the growth/weight of untreated mice, mice treated with an isotype control and mice treated with the antibody according to the present invention .
  • Figure 20 illustrates an agar gel with amplified product of the V L regions of the antibody according to the present invention.
  • Figure 21 illustrates an agar gel with amplified product of the V H regions of the antibody according to the present invention.
  • Figure 22 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 4 of Figure 21 (SEQ ID NO 16), and in (b) the corresponding amino acid sequence (SEQ ID NO 15) .
  • Figure 23 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 4 of Figure 21 (SEQ ID NO 14), and in (b) the corresponding amino acid sequence (SEQ ID NO 13) .
  • Figure 24 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 4 of Figure 21 (SEQ ID NO 12), and in (b) the corresponding amino acid sequence (SEQ ID NO 11) .
  • Figure 25 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 4 of Figure 21 (SEQ ID NO 10), and in (b) the corresponding amino acid sequence (SEQ ID NO 9) .
  • Figure 26 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 4 of Figure 21 (SEQ ID NO 8), and in (b) the corresponding amino acid sequence (SEQ ID NO 7) .
  • Figure 27 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 5 of Figure 21 (SEQ ID NO 6), and in (b) the corresponding amino acid sequence (SEQ ID NO 5) .
  • Figure 28 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 5 of Figure 21 (SEQ ID NO 4), and in (b) the corresponding amino acid sequence (SEQ ID NO 3) .
  • Figure 29 illustrates in (a) an alignment analysis data of the amino acid sequence of Figures 22 to 28 (the variant domain underlined) , and in (b) the consensus sequence derived from the analysis (SEQ ID NO 1) .
  • Figure 30 illustrates the CDR notation based on the consensus sequence of Figure 29 (b) using IMGT System.
  • Figure 31 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 30), and in (b) the corresponding amino acid sequence (SEQ ID NO 29) .
  • Figure 32 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 28), and in (b) the corresponding amino acid sequence (SEQ ID NO 27) .
  • Figure 33 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 26), and in (b) the corresponding amino acid sequence (SEQ ID NO 25) .
  • Figure 34 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 24), and in (b) the corresponding amino acid sequence (SEQ ID NO 23) .
  • Figure 35 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 20), and in (b) the corresponding amino acid sequence (SEQ ID NO 19) .
  • Figure 36 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 22), and in (b) the corresponding amino acid sequence (SEQ ID NO 21) .
  • Figure 37 illustrates in (a) the nucleic acid sequence for a product of a clone transformed with the product of lane 3 of Figure 20 (SEQ ID NO 18), and in (b) the corresponding amino acid sequence (SEQ ID NO 17) .
  • Figure 38 illustrates in (a) an alignment analysis data of the amino acid sequence of Figures 31 to 37 (the variant domain underlined) , and in (b) the consensus sequence derived from the analysis (SEQ ID NO 2) .
  • Figure 39 illustrates the CDR notation based on the consensus sequence of Figure 38 (b) using IMGT System. Primers were designed that would enable the subsequent amplification by PCR of the desired fragment of EPO-R (i.e. fragment 1 shown in Figure 1, the amino acid sequence of which is provided in SEQ ID No. 1)
  • the DNA sequence encoding the EPO-R protein fragment 1 was amplified by PCR from an IMAGE clone using gene- specific primers encoding BamHl and Sail restriction sites (denoted by lower case letters) .
  • TTTTTTgtcgacACGTGTCGGCTGTAGGCAGCGAACAC SEQ ID No 40
  • the EPO-R DNA sequence was cloned into the bacterial expression vector pQE40 allowing the incorporation of a hexahistidine tag onto the N-terminal of the recombinant protein. This construct was then used to transform competent TOPlOF' E.coli cells. Positive transformants were selected by colony PCR using vector- specific primers flanking the multiple cloning sites.
  • the positive clones were propagated overnight at 37 0 C in 5 mis of Luria-Bertani (LB) broth supplemented with 50 ⁇ M ampicillin. A 300 ⁇ l aliquot of this culture was retained for inoculation of secondary cultures and the remainder of the sample was miniprepped and the sequence verified by DNA sequencing.
  • LB Luria-Bertani
  • the recombinant EPO-R protein was then expressed in 500 mis of LB broth supplemented with ampicillin, using the secondary culture as an inoculant and induced with IPTG once the culture had reached the optimal optical density.
  • the culture was centrifuged at 5000 rpm for 15 mins and the pellet retained for protein purification.
  • the induced recombinant protein was solubilised in 50 mis of 8 M urea buffer (48Og Urea, 29g NaCl, 3.12g
  • the protein was purified by its N-terminal hexahistidine tag and refolded using on-column refolding by immobilized metal affinity chromatography. Chelating Hi-Trap columns were charged using 100 mM nickel sulphate before attachment to the Aktaprime.
  • Refolding takes place by the exchange of the 8 M urea buffer with a 5 mM imidazole wash buffer (29g NaCl, 3.12g NaH2PO4 (dihydrate) 0.34g Imidazole, pH 8.0 ) and elution of the protein using a 500 mM imidazole elution buffer (29g NaCl, 3.12g NaH2PO4 (dihydrate), 34g Imidazole) .
  • the elution profile of the purified recombinant protein was recorded (see Figures 4a and b) .
  • the eluted fractions were then subjected to SDS-PAGE analysis to confirm recombinant protein presence in eluted fractions.
  • the gels were stained with coomassie blue overnight and subsequently destained to determine the fractions containing the EPO-R protein (see Figure 5) .
  • the refolded protein was used as an immunogen to generate monoclonal antibodies.
  • Five BALB/C mice were immunized at three weekly intervals with 150 ⁇ g of purified recombinant protein and the antibody titre was analysed after boosts three and five.
  • a test bleed was taken from each animal and tested at 1:1000 dilutions in western blotting against 100 ng of antigen. Blots were developed using 3, 3 ' -diaminobenzidine (DAB).
  • DAB 3, 3 ' -diaminobenzidine
  • the spleen was removed from the mouse and the antibody producing B cells were fused with SP2 myeloma cells following standard protocols. Five days after the hybridoma fusion, the HAT media was refreshed and after a further five days, the plates were examined for cell growth. Clones were screened by ELISA against recombinant protein and selected positive hybridomas were cloned twice by limiting dilution.
  • the monoclonal antibodies were screened by ELISA to determine which clones should be expanded.
  • Maxi Sorb 96 well plates were coated with recombinant antigen by adding 100 ⁇ l of coating buffer (Buffer A: 0.42g sodium bicarbonate/lOO ⁇ l H20, Buffer B: 0.53g sodium carbonate/100 ⁇ l H 2 O, pH 9.5) containing the screening antigen to each well (100 ng/well) .
  • a control antigen was also used to eliminate non-specific clones.
  • the plates were incubated at 37 °C for 1 hr to allow the antigen to bind to the well and then blocked for 1 hr at room temperature by adding 200 ⁇ l PBS/3 % BSA to each well.
  • the blocking solution was removed from the plates and 100 ⁇ l of hybridoma supernatant was added to a positive antigen and a control antigen well.
  • the screening plates were incubated with supernatant for 1 hr on a rocker at room temperature.
  • the plates were washed three times with PBS-T, after which 100 ⁇ l of goat anti-mouse HRP conjugated secondary antibody (1:3000) was added to each well and incubated for 1 hr at room temperature.
  • the plates were washed three times with PBS-T and 100 ⁇ l of 3, 3' , 5, 5' -tetramethylbenzidine (TMB) was added to each well and incubated for 5 mins at 37 °C .
  • TMB 5' -tetramethylbenzidine
  • the supernatant from positive hybridoma cell lines were purified using a protein G column.
  • the hybridoma supernatant was passed through the HiTrap column and after washing the column with PBS, the bound antibody was eluted with 0.5 M glycine (pH 3) as described previously for the polyclonal antibody purification.
  • the eluted fractions were collected for BCA analysis and fractions with a concentration greater than 1 mg/ml were dialysed in PBS overnight.
  • the final product was subjected to BCA analysis once again to determine the final concentration of purified monoclonal antibody (see Figure 8) .
  • the final antibody selected for further analysis and discussed hereinafter was antibody 1E3 (hereinafter referred to as the "anit-EpoR”) .
  • RT-PCR was performed using the One-Step RT-PCR kit (Qiagen) under the following conditions: 50 0 C for 30 min, 95°C for 15 min, and 40 cycles of 94°C for 1 min, 55°C for 1 min 30 sec and
  • EPO-R F CTCTGTCTCCTACTTGCTGGGGCAGC (SEQ ID NO 41)
  • EPO-R R AAGATCTGGCCTGGCATCCCAAGC (SEQ ID NO 42)
  • GAPDH F ACCACAGTCCATGCCATCAC
  • GAPDH R TCCACCACCCTGTTGCTGTA (SEQ ID NO 44)
  • RT-PCR products were analysed by agarose gel electrophoresis.
  • Figure 10 shows strong EPO-R expression in the 3LL and 4Tl cell lines and to a lesser extent in the CHO cell lines. Amplification of GAPDH was used as an internal control.
  • Epo-R F ACCGTGTCATCCACATCAAT (SEQ ID NO 45)
  • EpoR R GCCTTCAAACTCGCTCTCTG (SEQ ID NO 46)
  • the supernatants from the hybridoma cell lines were analysed by western blotting to determine the ability of the monoclonal antibodies to detect endogenous native protein in a range of cancer cell lines.
  • 30 ⁇ g/ml whole cell lysates were separated by SDS-PAGE and transferred onto Hybond-C Extra nitrocellulose membrane (Amersham Biosciences) .
  • the membrane was blocked by incubation in PBS / 5 % admire for 1 hr at room temperature, after which it was rinsed briefly in PBS.
  • the monoclonal antibodies were used at a 1:400 dilutions in PBS / 5% admire and incubated on the membrane overnight at 4°C while gently rocking.
  • the blot were then rinsed three times with PBS / 1 % admire and 0.1% Tween-20 and then incubated with the goat anti-mouse HRP conjugated secondary antibody at a 1:3000 dilution for 1 hr at room temperature while shaking. The blots were then rinsed three times with the PBS / 1 % admire and 0.1 % Tween-20 solution, followed by a short rinse in PBS. The blots were incubated with ECL plus substrate (Amersham Bioscicences) for 5 mins at room temperature prior to analysis on the Kodak imager.
  • ECL plus substrate Amersham Bioscicences
  • Cells were grown on glass coverslips, fixed using ice- cold acetone and incubated with an isotype antibody at a 1:100 dilution. Coverslips were rinsed with PBS and incubated with antimouse AlexaFluor® 488 labelled secondary antibody. Coverslips were mounted using PermaFluor mounting media and visualised by confocal microscopy.
  • Actin was used to counter-stain and visualise the cell surface in both the coverslips incubated with the isotype and anti-EPO-R.
  • the Isotype control includes only a little staining around the edges of the cells from the actin (shown in red in the colour pictures) , whilst the cells incubated with the anti-epo-R includes a substantial staining relating of bound anti-epo-R (shown in green in the colour pictures) in addition to the small amount of staining around the edges of the cells from the actin. Similar staining patters have been observed in U251, MDAA231, HUVECs.
  • MDA-MB231 cells were treated for 1 hour with or without 5 ⁇ M Jak2 inhibitor, and then washed in PBS.
  • 5x105 cells were incubated with anti-EpoR antibody or isotype control for 2 hours and washed in PBS.
  • the cells were incubated with a FITC conjugated goat anti-mouse antibody for 1 hour and washed in PBS before analysis on BD FACS canto.
  • Jak2 inhibitor; 15b show the results of cells incubated with anti-EpoR, and; 15c show the results of cells incubated with anti-EpoR and Jak2 inhibitor. 11.5% of cells were observed in 15c, compared with only 0.1-0.5% for untreated cells (15 a and b) .
  • MB231 and U251mg cells in sterile coverslips in 12 well tissue culture plates were seeded at 1 x 106 cells and cultured in medium containing 10% FBS to nearly confluent cell monolayers, which were then carefully wounded by use of a 200- ⁇ l sterile pipette tip, and any cellular debris was removed by washing with PBS.
  • the wound monolayers were then incubated in medium containing 10% FBS, 5 ⁇ g/ml mitomycin C and various concentrations of anti-EpoR antibody for 18h.
  • the coverslips were then removed and fixed with 4% paraformaldehyde before being photographed under a light microscope ( ⁇ 4) .
  • the experiments were repeated in duplicate wells at least three times.
  • In-vitro invasion assays were performed using a modified Boyden chamber with 12-mm pore membranes (Costar Transwell plates, Corning Costar Corp., Cambridge, MA, USA) . The membranes were coated with Matrigel (100 mg/cm2) (Becton Dickinson, Oxford, UK) and allowed to dry overnight in a laminar flow hood. Cells were added to each well in 500 ml of serum-free medium in the presence of predetermined concentrations of the EPO-R 6A8 1D7 1E3 monoclonal antibody or isotype control antibodies.
  • mice 21
  • 21 Balb/c female mice were randomly assigned to three groups :
  • a first extraction of mRNA was extracted from the hybridoma cell pellet. Extraction of total RNA was carried out according to conventional techniques.
  • cDNA was created from the mRNA of the first and second extractions by reverse-transcription with an oligo (dT) primer.
  • PCR reactions with variable domain primers were used to amplify the V L regions of the monoclonal antibody DNA derived from the first extraction and the amplified products run on a Southern Blott (see Fig. 20) .
  • PCR reactions with variable domain primers were used to amplify the V H regions of the monoclonal antibody DNA from the second extraction and the amplified products run on agar gel (see Fig. 21) .
  • PCR produces from lane 3 shown in Fig. 20 were cloned into the Invitrogen sequencing vector pCR2.1 and transformed into TOPlO for positive transformants . Selected colonies were picked and sequenced using conventional techniques. Sequencing analysis was carried out on the clones transformed with the product of lane 3 seven times; providing nucleic acid SEQ ID Nos 30, 28, 26, 24, 20, 22, 18, which correspond to amino acid SEQ ID Nos 29, 27, 25, 23, 19, 21, 17, respectively (see Figs. 31 to 37). Alignment analysis of each of the amino acid sequences was carried out (see Fig. 38a) and a consensus sequence derived therefrom shown in SEQ ID NO 2 (see Fig. 38b).
  • Fig. 39 shows the V L CDR annotation according to IMGT system.
  • CDRs were identified according to a system devised by Rabat (Rabat EA et al . (1991) Sequences of proteins of immunological interest, 5th edition. Bethesda: US Department of Health and Human Services) .
  • V H Domain PCR reactions 4 and 5 shown in Fig. 21 were cloned into the Invitrogen sequencing vector pCR2.1 and transformed into TOPlO cells. Positive clones identified through colony PCR were selected and sequenced. Sequencing analysis was carried out on the clones transformed with the product of lane 4 five times; providing nucleic acid SEQ ID Nos 16, 14, 12, 10, 8, which correspond to amino acid SEQ ID Nos 15, 13, 11, 9, 7, respectively (see Figs. 22-26). Sequencing analysis was carried out on the clones transformed with the product of lane 5 twice, providing nucleic acid SEQ ID Nos 6, 4, which correspond to amino acid SEQ ID Nos 5, 3, respectively (see Figs. 27 and 28) .
  • Fig. 29a Alignment analysis of each of the amino acid sequences was carried out (see Fig. 29a) and a consensus sequence derived therefrom and shown as SEQ ID No 1 (see Fig. 29b) .
  • Fig. 30 shows the V L CDR annotation according to IMGT system.
  • CDRs were identified according to a system devised by Rabat (Rabat EA et al . (1991) Sequences of proteins of immunological interest, 5th edition. Bethesda: US Department of Health and Human Services) .
  • the Rabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) . This numbering system is used in the present specification except where otherwise stated.
  • the Rabat residue designations do not always correspond directly with the linear numbering of the amino acid residues of the heavy and light chain variable regions of the present invention.
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Rabat numbering corresponding to a shortening of, or insertion into, a structural component, whether a framework region or complementarity determining region (CDR) , of the basic variable domain structure of the heavy or light chain.
  • the correct Rabat numbering of residues may be determined for any given antibody by alignment of residues in the sequence of the antibody with a standard sequence to which the Rabat numbering has been applied.
  • nucleic acid and amino acid sequences for the V H and V L Domains, or variants thereof may be used to define the antibody, or fragments or variants thereof, according to the present invention .

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Abstract

La présente invention concerne un anticorps spécifique d'un épitope particulier du récepteur d'érythropoïétine ou un de ses fragments ou variants. L'anticorps est défini par une séquence et l'épitope auquel il se lie. L'anticorps ou l'un de ses fragments est capable d'atténuer l'invasion de cellules cancéreuses. L'invention concerne également une composition pharmaceutique comprenant l'anticorps, et des procédés et des utilisations de l'anticorps, notamment des procédés de traitement d'affections prolifératives et de l'anémie.
PCT/EP2009/058535 2008-07-04 2009-07-06 Anticorps contre le récepteur d'érythropoïétine et ses utilisations Ceased WO2010000875A1 (fr)

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GBGB0812277.2A GB0812277D0 (en) 2008-07-04 2008-07-04 Antibody and uses thereof

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015189813A1 (fr) 2014-06-12 2015-12-17 Andremacon S.R.L. Utilisation thérapeutique de modulateurs fonctionnels inhibant l'érythropoïétine
CN105542006A (zh) * 2015-12-11 2016-05-04 北京大学第一医院 抗epor融合蛋白抗体的制备方法和用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003438A1 (fr) * 1994-07-26 1996-02-08 Amgen Inc. Anticorps activant un recepteur de l'erythropoietine
WO1999055735A1 (fr) * 1998-04-30 1999-11-04 Tanox, Inc. Anticorps agonistes pour le recepteur g-fsc et procede de criblage associe
WO2000061637A1 (fr) * 1999-04-14 2000-10-19 Smithkline Beecham Corporation Anticorps du recepteur d'erythropoietine
WO2008019290A2 (fr) * 2006-08-04 2008-02-14 Astrazeneca Ab Anticorps anti-erbb2
WO2008028686A2 (fr) * 2006-09-10 2008-03-13 Glycotope Gmbh Système de production à haut rendement, entièrement humain, pour anticorps et protéines améliorés
WO2008035894A1 (fr) * 2006-09-19 2008-03-27 Sungkyunkwan University Foundation For Corporate Collaboration Agent antiviral agissant contre des virus animaux

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003438A1 (fr) * 1994-07-26 1996-02-08 Amgen Inc. Anticorps activant un recepteur de l'erythropoietine
WO1999055735A1 (fr) * 1998-04-30 1999-11-04 Tanox, Inc. Anticorps agonistes pour le recepteur g-fsc et procede de criblage associe
WO2000061637A1 (fr) * 1999-04-14 2000-10-19 Smithkline Beecham Corporation Anticorps du recepteur d'erythropoietine
WO2008019290A2 (fr) * 2006-08-04 2008-02-14 Astrazeneca Ab Anticorps anti-erbb2
WO2008028686A2 (fr) * 2006-09-10 2008-03-13 Glycotope Gmbh Système de production à haut rendement, entièrement humain, pour anticorps et protéines améliorés
WO2008035894A1 (fr) * 2006-09-19 2008-03-27 Sungkyunkwan University Foundation For Corporate Collaboration Agent antiviral agissant contre des virus animaux

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EPO Proteins [online] 19 September 2007 (2007-09-19), "Sequence 143 from Patent WO2007059997.", XP002545998, retrieved from EBI accession no. EPOP:CS669829 Database accession no. CS669829 *
DATABASE Geneseq [online] 19 April 2007 (2007-04-19), "Sp35 antibody, 2F3 heavy chain variable region.", XP002545997, retrieved from EBI accession no. GSP:AER27195 Database accession no. AER27195 *
HARDEE MATTHEW E ET AL: "Erythropoietin biology in cancer.", CLINICAL CANCER RESEARCH : AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH 15 JAN 2006, vol. 12, no. 2, 15 January 2006 (2006-01-15), pages 332 - 339, XP002545996, ISSN: 1078-0432 *
UDUPA KODETTHOOR-B: "Functional significance of erythropoietin receptor on tumor cells.", WORLD JOURNAL OF GASTROENTEROLOGY : WJG 14 DEC 2006, vol. 12, no. 46, 14 December 2006 (2006-12-14), pages 7460 - 7462, XP002545995, ISSN: 1007-9327 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015189813A1 (fr) 2014-06-12 2015-12-17 Andremacon S.R.L. Utilisation thérapeutique de modulateurs fonctionnels inhibant l'érythropoïétine
AU2015273031B2 (en) * 2014-06-12 2021-01-07 Andremacon S.R.L. Use of negative functional modulators of erythropoietin for therapy
US11078270B2 (en) 2014-06-12 2021-08-03 Andremacon S.R.L. Use of negative functional modulators of erythropoietin for therapy
CN105542006A (zh) * 2015-12-11 2016-05-04 北京大学第一医院 抗epor融合蛋白抗体的制备方法和用途

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