The patent's assignee is Philogen S.p.a.
News editors obtained the following quote from the background information supplied by the inventors: "Tumour necrosis factor alpha (TNF.alpha.) is a cytokine produced by many cell types, mainly activated monocytes and macrophages. It is expressed as a 26 kDa integral transmembrane precursor protein from which a mature protein of approximately 17 kDa is released by proteolytic cleavage. The soluble bioactive TNF.alpha. is a homotrimer that binds cell surface receptors. TNF.alpha. has been shown to induce necrosis of solid tumours. It exerts its effects mainly on the endothelium of the tumour-associated vasculature, with increased permeability, upregulation of tissue factor, fibrin deposition and thrombosis, and massive destruction of the endothelial cells.
"Interleukin-2 (IL2), a four a helix bundle cytokine produced by T helper 1 cells, plays an essential role in the activation phases of both adaptive and innate immune responses. Although it is not believed to have a direct cytotoxic effect on cancer cells, it has been reported to induce tumour regression by stimulating a cell-mediated immune response.
"Intratumoural injections of IL2 have been trialled in metastatic melanoma patients . In that study, treatment was administered three times weekly for at least 2 weeks, and overall 69% of patients were reported to achieve a complete response.
"WO01/66298 described immunoconjugates comprising TNF.alpha. and IL2 respectively, fused to antibody L19. L19 specifically binds the ED-B domain of fibronectin isoform B--FN, which is one of the best known markers angiogenesis (U.S. Ser. No. 10/382,107; WO01/62298). ED-B is an extra domain of 91 amino acids found in the B--FN isoform and is identical in mouse, rat, rabbit, dog and man. B--FN accumulates around neovascular structures in aggressive tumours and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues.
"Carnemolla et al.  described enhancement of the antitumour properties of IL2 by its targeted delivery to the tumour blood vessel extracellular matrix in an L19-IL2 immunoconjugate.
"Christ et al.  described intratumoural administration of an IL2 immunoconjugate, a TNF.alpha. immunoconjugate, or antibody alone. The antibody used was anti-EGFR, which had an anti-tumour effect. An anti-tumour immune response was reported following multiple injections of either fusion protein.
"Borsi et al.  reported a study in which L19-TNF.alpha. and L19-IL2 immunoconjugates were administered intravenously to mice on days 7 and 10 following implantation of tumour cells. L19 was used to concentrate and maximise the anti-tumour effects of the systemically delivered cytokines. The combination of immunocytokines was reported to have a synergistic effect on tumour volume. Mice who received the combination treatment had markedly reduced tumour volume compared with those who received just one immunocytokine."
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "Reported here are unexpected effects on tumours resulting from local administration of a combination of immunocytokines, TNF.alpha.-L19 and IL2-L19, at the tumour site. A single administration of these two immunocytokines promoted the complete eradication of large subcutaneous tumours in mice.
"Mice received a single intratumoural injection of the TNF.alpha.-L19, a single intratumoural injection of IL2-L19, or the combination. No further treatments were given. Tumour volume was measured daily and it was observed that tumours in mice treated with the combination therapy rapidly reduced in size and appeared to be completely eliminated within a period of days and showed no regrowth. Compared with saline-treated control mice, mice treated with one cytokine alone also showed inhibition of tumour growth, but tumours in these mice nevertheless continued to increase slowly in size. These results show a synergistic effect of the combined cytokine therapy, and a remarkable therapeutic effect, in which tumours were eradicated following just a single dose of each cytokine.
"Accordingly, a first aspect of the invention is a method of treating a tumour in a patient by injecting a single dose of a TNF.alpha. immunoconjugate and a single dose of an IL2 immunoconjugate at the tumour site.
"The immunoconjugate comprises the cytokine linked to an antibody molecule, which targets the cytokine to the site of the lesion. The antibody molecule binds a splice isoform of an extracellular matrix component, which is selectively expressed by the extracellular matrix in tumour tissue. By combining this targeting effect with direct administration of the immunoconjugate to the tumour site, a very localised administration is achieved, which concentrates the effect of the cytokines at the tumour site and reduces side effects and toxicity associated with systemic use of the cytokines.
"A number of splice isoforms of tumour extracellular matrix components are known, and antibody molecules targeting any such isoform may be used to selectively target the tumour. These include splice isoforms of fibronectin, such as B--FN. B--FN includes an extra domain ED-B, and antibody molecules of the invention are preferably targeted to this domain. A preferred antibody molecule comprises the complementarity determining regions (CDRs) of antibody L19. These are, as illustrated in FIG. 3:
"TABLE-US-00001 VH CDR1 SFSMS SEQ ID NO: 1 VH CDR 2 SISGSSGTTYYADSVKG SEQ ID NO: 2 VH CDR 3 PFPYFDY SEQ ID NO: 3 VL CDR 1 RASQSVSSSFLA SEQ ID NO: 4 VL CDR 2 YASSRAT SEQ ID NO: 5 VL CDR 3 QQTGRIPPT SEQ ID NO: 6
"The TNF.alpha. immunoconjugate preferably comprises TNF.alpha. linked to an antibody molecule. comprising the L19 CDRs. The IL2 immunoconjugate comprises IL2 linked to an antibody molecule, which may be an identical or different antibody molecule as the TNF.alpha. immunoconjugate. The antibody molecule in each immunoconjugate may bind the same extracellular matrix component, optionally the same splice isoform e.g. they may bind the same domain. Preferably, the IL2 immunoconjugate comprises IL2 linked to an antibody molecule comprising the L19 CDRs.
"Preferably, the antibody molecule (of the TNF.alpha. and/or the IL2 immunoconjugate) comprises the L19 VH domain and/or the L19 VL domain. Amino acid sequences of the L19 VH and VL domains are SEQ ID NO: 7 and SEQ ID NO: 9 respectively (FIG. 3).
"Preferably the antibody molecule is a single chain Fv (scFv) or other antibody fragment of low molecular weight and/or lacking an Fc region. These properties assist with targeting and tissue penetration of the immunoconjugate at the tumour site. A preferred antibody molecule is scFv-L19, which is an scFv comprising an L19 VH domain and an L19 VL domain, wherein the VH and VL are conjoined in a single polypeptide chain by a peptide linker sequence. The VH domain contains VH CDR1, CDR2 and CDR3 sequences, and the VL domain contains VL CDR1, CDR2 and CDR3 sequences. The VH domain may have an amino acid sequence as set out in FIG. 3 (SEQ ID NO: 7). The VL domain may have an amino acid sequence as set out in FIG. 3 (SEQ ID NO: 9). The VH and VL domains are normally joined by a peptide linker such as the 12 residue linker shown in FIG. 3 (SEQ ID NO: 8). Preferably, the scFv-L19 comprises or consists of the amino acid sequence shown in FIG. 3 (SEQ ID NO: 10).
"A molecular linker such as a peptide may be used to join the cytokine to the antibody molecule, facilitating expression of all or part of the immunocytokine as a fusion protein. Where the antibody molecule is also a single chain molecule, such as scFv, the entire immunocytokine polypeptide chain may conveniently be produced as a fusion protein. For the TNF.alpha. immunoconjugate, the fusion proteins are then assembled into trimers, allowing TNF.alpha. to adopt its normal trimeric form .
"Optionally, the immunocytokine carries a detectable and/or functional label, such as a radioactive isotope. Radiolabelled L19, and its use in cancer therapy, has been described before.
"It is generally convenient to provide the IL2 immunoconjugate and the TNF.alpha. immunoconjugate as separate molecules. They may be provided as a combined preparation, or as separate formulations to permit either simultaneous or sequential administration. The clinician can determine the most suitable manner of administering the single dose of each immunocytokine to the patient. For example, the method of treatment may comprise injecting the TNF.alpha. immunoconjugate and the IL2 immunoconjugate in separate injections, simultaneously or sequentially. Where sequential administration is used, the immunocytokines are preferably injected within 24 hours, 12 hours, 1 hour or more preferably within 30 minutes of each other. The two immunocytokines may be injected at the same point in the tumour site, or at different points. A combined injection of both immunocytokines may be administered. It may be preferable to administer a dose in multiple injections, for example to inject multiple locations across the tumour or around the tumour site, or to facilitate administration of a larger volume of immunocytokine.
"The dose is an amount of cytokine, administered at one time, effective to treat the tumour in the combination therapy according to the invention. A single dose may be administered in a treatment period of 1 hour or less, preferably in a period of 30 minutes or less, e.g. 15, 10, 5 or 1 minute or less.
"The quantity of TNF.alpha. or IL2 administered will depend on the size and nature of the tumour, among other factors. For example, the dose of an TNF.alpha.-scFv immunoconjugate may be in the range of 2-20 .mu.g, e.g. 5-10 .mu.g. The dose of IL2-scFv immunoconjugate may be in the range of 10-100 .mu.g, e.g. 20-40 .mu.g. Corresponding doses using other immunoconjugate formats may be straightforwardly calculated to administer an appropriate quantity of cytokine. These are examples only and, of course, different doses may be used. The clinician will determine a therapeutically effective amount for administration.
"As reported here, a single dose of the TNF.alpha. immunoconjugate and a single dose of the IL2 immunoconjugate were sufficient for tumour therapy. Multiple doses were not required, and treatment of a tumour according to the present invention does not comprise repeating the combination therapy. In addition to the advantages this offers to patients, the single dose regimen provides a considerable advantage to clinicians and significant cost savings.
"Accordingly, in treating a particular tumour, the method of the invention is not repeated. The method of treating the tumour may comprise:
"(a) injecting a single dose of the TNF.alpha. immunoconjugate and a single dose of the IL2 immunoconjugate at the tumour site, and
"not repeating step (a).
"The tumour is treated without any repeated administration of the combination of immunocytokines to the tumour site. As shown herein, a tumour may be treated without any subsequent injection of a TNF.alpha. immunoconjugate or an IL2 immunoconjugate. Indeed, the tumour may be treated without administering any further anti-cancer agent to the patient. Optionally, the patient has not previously been given either TNF.alpha. or IL2 for the tumour, although in some cases a patient may have received previous therapy with only one of IL2, TNF.alpha. or an immunoconjugate including one of these cytokines, which did not achieve complete treatment of the tumour.
"Accordingly, a method of the invention may comprise treating a tumour in a patient by injecting a dose of the TNF.alpha. immunoconjugate and a dose of the IL2 immunoconjugate at the tumour site, wherein the tumour is treated without administering any subsequent dose of the TNF.alpha. immunoconjugate or the IL2 immunoconjugate to the tumour site.
"Of course, the method of the invention may be used to treat multiple tumours in a patient, by performing the method on each tumour.
"Other treatments that may be used in combination with the invention include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.
"The immunocytokines are injected at the site of the tumour, preferably by intratumoural injection. Peritumoural injection, e.g. local intradermal injection, is another suitable method for administering the immunocytokine locally to a tumour site.
"The treated tumour may be a primary tumour or a metastatic tumour. The invention is particularly suited to treatment of skin tumours, e.g. malignant skin tumour, melanoma or carcinoma, since their location is amenable to direct local injection. Other tumours within the body may also be treated, and injections may be guided to tumours within soft tissue or internal organs, e.g. by sonography W. The methods of the invention may also be used in a surgical context, where injection is performed before, during or after tumour surgery.
"Treatment of a tumour according to the present invention may include complete eradication of the tumour. The disappearance of any evidence of vital tumour after stopping injections represents complete treatment of the tumour. Disappearance of the tumour may be determined when the tumour has no discernable volume or is no longer visible. Treatment may comprise treatment to eradicate the tumour and prevent tumour regrowth.
"A method of treating a tumour according to the present invention may comprise injecting a single dose of the TNF.alpha. immunoconjugate and a single dose of the IL2 immunoconjugate at the tumour site, and observing disappearance of the tumour. Absence of tumour regrowth may also be observed.
"Patients are preferably monitored during a followup period of at least one month, preferably at least six months or at least a year, after administration of the immunocytokine combination therapy. Disappearance of the tumour, and lack of tumour regrowth, may be observed in the followup period.
"In the event of tumour recurrence after the followup period, or if other tumours develop, patients may receive a further treatment with immunocytokine combination therapy according to the invention, to remove the further tumour.
"For example, a method according to the invention may comprise eradicating a tumour in a patient by injecting a single dose of the TNF.alpha. immunoconjugate and a single dose of the IL2 immunoconjugate at the tumour site, wherein the tumour disappears in the absence of further doses of the TNF.alpha. immunoconjugate and/or the IL2 immunoconjugate.
"Further aspects of the invention relate to TNF.alpha. and IL2 immunoconjugates for use in any of the methods of the invention described herein. A composition comprising the TNF.alpha. immunoconjugate and/or the IL2 immunoconjugate may be provided for use in a method as described. Compositions may further comprise additional components, such as pharmaceutically acceptable excipients. A composition may comprise the immunocytokines as separate formulations (e.g. separately packaged, optionally in a kit), or as a combined formulation. The formulation may be adapted for intratumoural administration. Use of the TNF.alpha. immunoconjugate and/or the IL2 immunoconjugate for the manufacture of a medicament for use in a method as described herein is another aspect of the invention.
"Nucleic acid molecules encoding immunoconjugates may be provided. The nucleic acids may be present in host cells. A method of producing the immunoconjugate may comprise by expressing the nucleic acid in cultured host cells, optionally followed by purifying the immunoconjugate from the host cell culture. The IL2 and TNF.alpha. immunoconjugates are preferably produced in separate cell cultures. They may then be individually formulated as medicaments for administration as described."
For additional information on this patent application, see: Schwagen, Kathrin. Immunocytokine Combination Therapy. Filed
Keywords for this news article include: Antibodies, Therapy, Cytokines, Immunology, Amino Acids, Blood Proteins, Immunoproteins, Nanotechnology, Immunoglobulins, Philogen S.p.a., Serum Globulins, Immunoconjugates, Extracellular Space, Extracellular Matrix, Emerging Technologies, Intercellular Signaling Peptides and Proteins.
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