US008580267B2

(12) United States Patent (10) Patent No.: US 8,580,267 B2 Pedretti et al. (45) Date of Patent: Nov. 12, 2013

(54) IMMUNOCYTOKINES FORTUMOUR (56) References Cited THERAPY WITH CHEMOTHERAPEUTIC AGENTS FOREIGN PATENT DOCUMENTS (75) Inventors: Marta Pedretti, Zurich (CH): Dario WO O2/O59264 8, 2002 WO O3,O93478 11, 2003 Neri, Buchs (CH) WO 2004/OO2528 1, 2004 WO 2006/026020 3, 2006 (73) Assignee: Philogen S.p.A., Siena (IT) WO 2006/050834 5, 2006 WO 2007/128563 11, 2007 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Paul, William, Fundamental Immunology, 3rd Edition, Raven Press, New York, 1993, pp. 292-295.* (21) Appl. No.: 13/139,655 Vajdos et al., Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning 1-1. mutagenesis. J. Mol. Biol. 320:415-428, 2002.* (22) PCT Filed: Dec. 14, 2009 Bartolomei, M., et al. “Combined treatment of glioblastomapatients with locoregional pre-targeted 90Y-biotin radioimmunotherapy and (86). PCT No.: PCT/EP2009/008920 temozolomide.” QJNuclMed Mol Imaging. Sep. 2004:48(3):220-8. S371 (c)(1) Marlind, J., et al. "Antibody-mediated delivery of interleukin-2 to the (2), (4) Date. Jun. 14, 2011 Stroma of breast cancer strongly enhances the potency of chemo s 9 therapy” Clin Cancer Res. Oct. 15, 2008;14(20):6515-24. Brack, S.S., et al. “Tumor-targeting properties of novel antibodies (87) PCT Pub. No.: WO2010/078916 specific to the large isoform oftenascin-C.” Clin Cancer Res. May 15, PCT Pub. Date: Jul. 15, 2010 2006; 12(10):3200-8. Leins, A., et al. “Expression of tenascin-C in various human brain O O tumors and its relevance for Survival in patients with astrocytoma.” (65) Prior Publication Data Cancer. Dec. 1, 2003:98(11):2430-9. US 2011 FO25O17OA1 Oct. 13, 2011 Ebbinghaus, C., et al. “Engineered vascular-targeting antibody-inter feron-gamma fusion protein for cancer therapy.” IntJ Cancer. Aug. 20, 2005; 116(2):304-13. Related U.S. Application Data * cited by examiner (60) Provisional application No. 61/139,484, filed on Dec. Primary Examiner — Ruixiang Li 19, 2008. (74) Attorney, Agent, or Firm — Dann, Dorfman, Herrell & Skillman; Kathleen D. Rigaut (51) Int. Cl. A 6LX39/395 (2006.01) (57) ABSTRACT A6 IK38/20 (2006.01) Immunocytokine comprising cytokine, e.g. interleukin 2 (IL AOIN 43/73 (2006.01) 2), conjugated to antibody against tumour neovasculature (52) U.S. Cl. antigen, e.g. tenascin-C, for use in combination therapy with USPC ...... 424/1781; 424/85.2, 514/183 chemotherapeutic agent such as temozolomide. Use of immunocytokine and chemotherapy for treatment of tumours (58) Field of Classification Search e.g. glioblastoma and other cancers. None See application file for complete search history. 12 Claims, 6 Drawing Sheets U.S. Patent Nov. 12, 2013 Sheet 1 of 6 US 8,580,267 B2

r/vryvywryyyyyrry, A WVAAAA/UV/J domains subject to alternative splicing damameramnaanamomb

W/AWJVAJAVAJV? s N-S ? EGF like Fibronectin type I like repeats domains

Figure 1 U.S. Patent Nov. 12, 2013 Sheet 2 of 6 US 8,580,267 B2

tumor liver lung spleen heart kidney intestine blood Organs

Figure 2 U.S. Patent Nov. 12, 2013 Sheet 3 of 6 US 8,580,267 B2

2500 as a F6-2--TMZ 2OOO arA-F6-2 -- TMZ

s so e CTRL 10%DMSO tumor 1500 volume (mm) 1000

500

12 A A A A. A days after tumor implantation

Figure 3 U.S. Patent Nov. 12, 2013 Sheet 4 of 6 US 8,580,267 B2

1OO m

80 ak-e TMZ

E.60 d as Stage.cgregaga>e --TMZ F16 s - e 40 H.kara ages F16-L2 ape CTRL 10% DMSO

10 O dke-eeeeeeeeeeeeeeeeee-eeeeee 10:301 11 ft 50 70 90 110 THERAPY days after tumor implantation

Figure 4 U.S. Patent Nov. 12, 2013 Sheet 5 of 6 US 8,580,267 B2

thR$: (NYATI : l ...net

days after tumor implantation U.S. Patent Nov. 12, 2013 Sheet 6 of 6 US 8,580,267 B2

a. Maa awa, was a wa Asa was A. Akas. As Kaas Aaa and aaa.. aka is a was as Aa. Adua aal as a has aa kak aa al- alias a RYGMSWRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKAHNAFDYWGQGTLVTVSR

------ee- or------see -s a SSELTQDPAWSWALGOTWRITCOGDSLRSYYAS i WYQQKPGQAPWLWIYGKNNRPSGIPDRFSGSSscNASTreacAEDEADYdrissiNipp " VLCF16) : WFGGGTKLTVL

wo, rv me -er rvin -rr war err w re rer EFSSSSG SSSSG Linker SSSSG APTssSTKKTQQLEHLLLDLQMILNGINNY--- KNPKLTRMLTFKFYMPKKATELKHLQCLEE ELKPLEEWLNLAQSKNFHLRPRDLISNINVI - IL2 WLELKGSETTFMCEYADETATIVEFLNRWIT FCQSIISTLT

Figure 6 US 8,580,267 B2 1. 2 MMUNOCYTOKINES FORTUMIOUR F16 and F16-IL2 have been shown to intensely stain THERAPY WITH CHEMOTHERAPEUTIC aggressive cancer types and to preferentially accumulate at AGENTS the tumour site following intravenous administration 22, 25. Following observation of its excellent safety profile observed This is a national stage application of PCT/EP2009/ 5 in cynomolgus monkeys, F16-IL2 is currently being studied 008920 filed on Dec. 14, 2009 which claims priority to U.S. in two phase Ib clinical trials in combination with doxorubi Provisional patent application No. 61/139,484, filed on Dec. cin or with paclitaxel in patients with metastatic cancer. 19, 2008. The entire disclosure of each of the foregoing Central nervous system tumours rank first among neopla applications is incorporated by reference herein. sia types for the average years of life lost 26. Approximately This invention relates to the treatment of tumours and 10 cancer using a combination of chemotherapeutic agents and 13,000 deaths and 18,000 new cases of central nervous sys immunocytokines. tem tumours occur annually in the US 27. Mortality rates Conventional cytotoxic therapies of cancer often do not are generally similar to incidence rates in most geographical discriminate between tumour and normal tissues. To achieve areas 28. therapeutically relevant concentrations in the tumour mass, 15 The standard of care for patients with glioblastoma large drug doses have to be administered to the patient, lead includes Surgery, radiotherapy and/or temozolomide-based ing to a poor therapeutic index and unacceptable toxicities to pharmacotherapy. Nevertheless, the prognosis of glioblas healthy tissues 1. The selective delivery of therapeutic toma continues to be dismal, in spite of progress made in the agents to the tumour site using antibodies directed against molecular characterisation of the most frequent genetic alter tumour-associated antigens is a strategy to overcome the dis ations found in this disease. advantages of conventional cancer therapies 2, 3, 4. Anti Microvascular proliferation is a characteristic feature of gens that are expressed around the tumour neovasculature are glioblastoma 35. A striking over-expression of the EDB especially attractive targets for antibody-based pharmacode domain of fibronectin in high-grade astrocytomas has been livery applications due to their inherent accessibility for unequivocally established 29:30 and the monoclonal anti blood-borne agents and to the fact that angiogenesis is a 25 body L19 has been shown to target glioblastoma in patients characteristic feature of virtually all aggressive solid tumours 31. Furthermore, radiolabelled preparations of monoclonal 5, 6, 7. antibodies specific to the A1 or to the D domain oftenascin-C Tenascin-C is a large hexameric glycoprotein of the extra have been investigated for the radioimmunotherapy of cellular matrix which modulates cellular adhesion. It is patients with glioblastoma 32. involved in processes such as cell proliferation and cell 30 Bartolomei et al. 33 described treatment of glioblastoma migration and is associated with changes in tissue architec patients with radioimmunotherapy, using biotinylated anti ture as occurring during morphogenesis and embryogenesis tenascin murine monoclonal antibody, avidin and 'Y-biotin, as well as under tumourigenesis orangiogenesis. in combination with temozolomide. Overall survival and pro A strong over-expression of the large isoform of tenascin-C gression free Survival of patients receiving the combined has been reported for a number of tumours, and monoclonal 35 treatment were observed to be higher compared with patients antibodies specific for domains A1 and D, respectively, have who received radioimmunotherapy without temozolomide. been extensively characterised in the clinic 8, 9, 10, 11, 12. The inventors have observed that the combination of an Human monoclonal antibody fragments specific to tenas alkylating agent with an antibody-IL2 conjugate targeted to a cin-C are described in WO2006/050834 and shown to bind tumour-associated antigen of the tumour neovasculature preferentially to tumour tissue relative to normal tissue. These 40 exhibited more effective therapeutic action against the antibodies are useful, for example, in delivering toxins. Such tumour compared with the use of the alkylating agent or the as cytokines, specifically to tumour cells. conjugate alone. Delivery of bioactive agents to the subendothelial extracel F16-IL2 is shown herein to potentiate the therapeutic lular matrix has been demonstrated using derivatives of action of the alkylating agent temozolomide in mice models monoclonal antibodies specific to splice isoforms of 45 of human glioblastoma. A synergistic effect was observed for fibronectin or of tenascin-C 5, 13, 14, 15, 16, 17, 18, 19, 20. the combination of temozolomide and F16-IL2, which pro In particular, promising results obtained with derivatives of duced an anti-tumour effect much greater than would have the human monoclonal antibodies L19 (specific to the alter been expected based on the effects of F16-IL2 or temozolo natively spliced ED-B domain of fibronectin) and F16 (spe mide alone. Subcutaneous U87 glioblastomas were com cific to the extra-domain A1 of tenascin-C) have led to the 50 pletely eradicated in nude mice using the combination treat clinical development of five immunocytokines and radioim ment, compared with only a minor retardation of tumour munoconjugates, based on these antibodies 7, 13, 14, 15, 16. growth observed for F16-IL2 treatment alone and a recur 21, 22. F16 is also known as 4A1-F16. rence of tumour growth following regression in mice treated L19 specifically binds the ED-B domain of fibronectin with temozolomide alone. isoform B-FN, which is one of the best known markers of 55 Aspects of the first development of the invention are as angiogenesis (U.S. Ser. No. 10/382,107, WO01/62298). follows. ED-B is an extra domain of 91 amino acids found in the B-FN Accordingly, a first development of the invention relates to isoform and is identical in mouse, rat, rabbit, dog and man. the combined use of a chemotherapeutic alkylating agent and B-FN accumulates around neovascular structures in aggres an immunocytokine for treating a tumour, where the immu sive tumours and other tissues undergoing angiogenesis. Such 60 nocytokine comprises a cytokine (e.g. an interleukin Such as as the endometrium in the proliferative phase and some ocular IL2) conjugated with an antibody molecule, wherein the anti structures in pathological conditions, but is otherwise unde body molecule binds a target antigen, e.g. tumour-associated tectable in normal adult tissues (19, 23, 24). Schv(L19) has antigen and/or antigen of the tumour neovasculature. been shown to be capable of selective tumour targeting in A first aspect of the invention is a method of treating a patients with cancer 16. Use of immunocytokines compris- 65 tumour in an individual, comprising administering a chemo ing L19 conjugated with IL-12 is described in WO2006/ therapeutic agent and an immunocytokine to the individual, 119897. wherein the chemotherapeutic agent is an alkylating agent US 8,580,267 B2 3 4 and wherein the immunocytokine comprises a cytokine con and an immunocytokine to the individual, wherein the immu jugated to an antibody molecule that binds the target antigen. nocytokine comprises a cytokine conjugated to an antibody A second aspect of the invention is use of a chemothera molecule that binds the target antigen. peutic agent and an immunocytokine for the manufacture of a A second aspect is use of a chemotherapeutic agent and an medicament for treatment of a tumour in an individual, immunocytokine for the manufacture of a medicament for wherein the chemotherapeutic agent is an alkylating agent treatment of glioma in an individual, wherein the immunocy and wherein the immunocytokine comprises a cytokine con tokine comprises a cytokine conjugated to an antibody mol jugated to an antibody molecule that binds the target antigen. ecule that binds the target antigen. A third aspect of the invention is use of an immunocytokine A third aspect is use of an immunocytokine for the manu for the manufacture of a medicament for treatment of a 10 facture of a medicament for treatment of glioma in an indi tumour in an individual, wherein the treatment comprises vidual, wherein the treatment comprises administering the administering the immunocytokine and a chemotherapeutic immunocytokine and a chemotherapeutic agent to the indi agent to the individual, wherein the chemotherapeutic agent vidual, and wherein the immunocytokine comprises a cytok is an alkylating agent and wherein the immunocytokine com ine conjugated to an antibody molecule that binds the target prises a cytokine conjugated to an antibody molecule that 15 antigen. binds the target antigen. A fourth aspect is use of a chemotherapeutic agent for the A fourth aspect of the invention is use of a chemotherapeu manufacture of a medicament for the treatment of glioma in tic agent for the manufacture of a medicament for the treat an individual, wherein the treatment comprises administering ment of a tumour in an individual, wherein the treatment the chemotherapeutic agent and an immunocytokine to the comprises administering the chemotherapeutic agent and an individual, and wherein the immunocytokine comprises a immunocytokine to the individual, wherein the chemothera cytokine conjugated to an antibody molecule that binds the peutic agent is an alkylating agent and wherein the immuno target antigen. cytokine comprises a cytokine conjugated to an antibody A fifth aspect is an immunocytokine for use in treatment of molecule that binds the target antigen. glioma in an individual, wherein the treatment comprises A fifth aspect of the invention is an immunocytokine for 25 administering the immunocytokine and a chemotherapeutic use in treatment of a tumour in an individual, wherein the agent to the individual, and wherein the immunocytokine treatment comprises administering the immunocytokine and comprises a cytokine conjugated to an antibody molecule that a chemotherapeutic agent to the individual, wherein the che binds the target antigen. motherapeutic agent is an alkylating agent and wherein the A sixth aspect is a chemotherapeutic agent for use in treat immunocytokine comprises a cytokine conjugated to an anti 30 ment of glioma in an individual, wherein the treatment com body molecule that binds the target antigen. prises administering the chemotherapeutic agent and an A sixth aspect of the invention is a chemotherapeutic agent immunocytokine to the individual, and wherein the immuno for use in treatment of a tumour in an individual, wherein the cytokine comprises a cytokine conjugated to an antibody treatment comprises administering the chemotherapeutic molecule that binds the target antigen. agent and an immunocytokine to the individual, wherein the 35 A seventh aspect is a medicament comprising an immuno chemotherapeutic agent is an alkylating agent and wherein cytokine and a chemotherapeutic agent, for use in treatment the immunocytokine comprises a cytokine conjugated to an of glioma in an individual, wherein the treatment comprises antibody molecule that binds the target antigen. administering the medicament to the individual, and wherein A seventh aspect of the invention is a medicament com the immunocytokine comprises a cytokine conjugated to an prising an immunocytokine and a chemotherapeutic agent, 40 antibody molecule that binds the target antigen. for use in treatment of a tumour in an individual, wherein the A third development of the invention relates to an improve treatment comprises administering the medicament to the ment to the known anti-tenascin C antibody 4A1-F16 individual, wherein the chemotherapeutic agent is an alkylat (“F16'). F16 was described in WO2006/050834 and in other ing agent and wherein the immunocytokine comprises a publications cited above. cytokine conjugated to an antibody molecule that binds the 45 As disclosed herein, an improved F16 antibody has now target antigen. been produced, which increases expression yield of the anti A second development of the invention relates to treatment body and facilitates its production, especially large-scale pro of glioma using an immunocytokine, wherein the immuno duction on a commercial scale. cytokine comprises a cytokine conjugated to an antibody The inventors discovered that by making a single amino molecule that binds a target antigen, e.g. tumour-associated 50 acid change in VHCDR1 of the F16 VH domain, yield was antigen and/or antigen of the tumour neovasculature. The significantly improved and a high binding affinity was glioma may be glioblastoma. retained. As disclosed here, the inventors have discovered that a Accordingly, an aspect of the invention is an antibody combination of a chemotherapeutic agent with an antibody molecule comprising a VHCDR1 sequence SEQID NO: 18. cytokine conjugate exhibits more effective therapeutic action 55 The antibody molecule may comprise an antibody VH against glioma compared with the use of the chemotherapeu domain having a set of CDRs VHCDR1, VHCDR2 and VH tic agent or the conjugate alone. By using a therapeutic regi CDR3 wherein VH CDR1 is SEQID NO: 18, VHCDR2 is men of combined administration of an immunocytokine with SEQID NO: 6 and VHCDR3 is SEQID NO: 7. Alternatively chemotherapy, the therapeutic activity of the immunocytok the antibody molecule may be a variant comprising up to five ine and chemotherapeutic agent can be significantly 60 (e.g. one or two) amino acid mutations in the VHCDR2 increased. These findings are of particular value for therapy of and/or VH CDR3 sequence. The antibody molecule may glioblastoma, which is a devastating disease with very poor comprise a VH domain SEQID NO: 17 or a variant thereof prognosis, and for which there is currently no definitive cure. having up to five amino acid mutations (in the framework Aspects of the second development of the invention are as and/or CDR 1 or 2), wherein VHCDR1 is SEQID NO: 18. follows. 65 The antibody molecule may further comprise the F16 VL A first aspect is a method of treating glioma in an indi domain SEQ ID NO. 4 or a variant thereof as described vidual, comprising administering a chemotherapeutic agent herein, e.g. an F16 VL domain with up to five amino acid US 8,580,267 B2 5 6 mutations (e.g. one or two). The antibody molecule is pref renal cancer, stomach cancer and cerebral cancer. Non-can erably an scFv antibody molecule. cerous tumours of any of these tissues may also be treated. These antibody molecules may be employed in other Cancers may be familial or sporadic. aspects of the invention as a whole, and thus may be provided The tumour may be a tumour of the central nervous system. in immunocytokines for use in aspects of the invention The tumour may be a brain tumour, such as glioma, e.g. described herein. glioblastoma. The term “glioma’ refers to tumours of glial Nucleic acids, e.g. Vectors, encoding the antibody mol cell origin and includes astrocytomas, oligodendrogliomas, ecules and immunocytokines described herein are also an ependimomas, and mixed gliomas 35. They account for aspect of the invention. One aspect is a nucleic acid vector more than 70% of all brain tumours and their prognosis is comprising a nucleotide sequence encoding an immunocy 10 very poor. Glioblastoma is the most frequent (65% of all tokine as described herein. gliomas) and also the most malignant histological type 36. Nucleic acid and/or antibody molecules of the present The antibody moiety in the immunocytokine of the present invention may be provided in isolated and/or purified form. invention may bind an antigen of the tumour cells or the Aspects of the first, second and third developments of the tumour neovasculature. The antigen may be an antigen of the invention are described in more detail below. 15 extracellular matrix, e.g. the subendothelial extracellular matrix. The antigen may be expressed on cells of the tumour DETAILED DESCRIPTION or tumour vasculature. Thus, antigens exposed on the mem brane of tumour cells may be targeted using antibody mol A chemotherapeutic agent is a cytotoxic compound which ecules of the invention. inhibits the proliferation of tumour or cancer cells. Chemo The target antigen may be a tumour associated antigen therapeutic agents may, in some circumstances, have a cyto and/or an antigen of the tumour neovasculature. Preferably, toxic effect on normal (non-cancerous and non-tumour) cells the antigen is an antigen of the tumour neovasculature, e.g. in a patient. the Subendothelial extracellular matrix. The target antigen is Alkylating agents area known category of chemotherapeu preferably differentially expressed in the tumour or tumour tic agents, which exert their cytotoxic effect by attaching an 25 neovasculature compared with normal tissue. The antigen alkyl group to DNA. Alkylating agents may attach alkyl may be an isoform of a protein, wherein the isoform is dif groups, e.g. methyl groups, to DNA bases. This may result in ferentially expressed in the tumour or tumour neovasculature DNA fragmentation, cross-linking between DNA strands compared with normal tissue. and/or DNA mutation. Examples of alkylating agents Examples of antigens of the tumour neovasculature such as include: 30 tenascin-C and fibronectin are described in detail elsewhere nitrogen mustards (e.g. chlorambucil, chlormethine, cyclo herein and any may be a target for the antibody of the present phosphamide, ifosfamide, melphalan, bendamustine, tro invention. fosfamide, uramustine, mechlorethamine), hydrazines Preferred antibodies bind preferentially to tumour tissue (e.g. procarbazine), relative to normal tissue. Antibodies may, for example, bind ethyleneimines and methylmelamines (e.g. hexameth 35 to stroma and/or neo- and peri-vascular structures of tumour ylmelamine, thiotepa) alkyl Sulphonates (e.g. buSulfan, tissue preferentially to normal tissue. mannosulfan, treosulfan), The antibody molecule may bind to fibronectin, e.g. an triaZenes and imidazotetrazines (e.g. dacarbazine, procarba isoform of fibronectin that is differentially expressed in the Zine, temozolomide, mitoZolomide), tumour neovasculature compared with normal tissue. For aziridines (carboquone, thioTEPA, triaziquone, triethylen 40 example, the antibody molecule may bind a domain of emelamine) fibronectin that is expressed in isoforms associated with neo nitroSureas (e.g. carmustine, fotemustine, lomustine, nimus plasms, e.g. extra domain B (ED-B). tine, prednimustine, ranimustine, Semustine, Streptozocin) The antibody may bind tenascin-C, e.g. the large isoform platinum compounds (e.g. carboplatin, cisplatin, nedaplatin, of tenascin-C. The antibody may bind preferentially to tena Oxaliplatin, triplatin tetranitrate and satraplatin). 45 Scin-C large isoform relative to tenascin-C Small isoform. Chemotherapeutic agents may be active cytotoxic agents Preferably the antibody binds a domain of tenascin-C which they may be or prodrugs that are hydrolysed or metabolised to are Subject to alternative splicing and are expressed only in active cytotoxic agents. the large isoform, e.g. any of domains A1 to D (see FIG. 1). Preferably the alkylating agent is a triaZene or imidazotet The antibody moiety of the immunocytokine may bind razine. As exemplified in the experimental examples herein, 50 domain A1 of tenascin-C large isoform. the alkylating agent may be temozolomide, i.e. (8-carbamoyl Examples of Suitable antibodies for use in immunocytok 3-methylimidazo[5,1-d-1,2,3,5-tetrazin-4(3H)-one). Temo ines of the invention are disclosed in WO2006/050834. Zolomide is a prodrug and is chemically related to dacarba In some embodiments, the antibody moiety of an immu Zine but, unlike dacarbazine, it spontaneously hydrolyzes to nocytokine as described herein competes for binding to tena the intermediate species methyltriaZen-1-yl imidazole-4-car 55 scin-C with an antibody comprising the 4A1-F16VH domain boxamide (MTIC) MTIC above pH 7 without requiring SEQ ID NO: 2 and the 4A1-F16 VL domain SEQID NO: 4. metabolism by the liver. MTIC degrades to an active cation, Competition between antibodies may be assayed easily in which methylates guanines in DNA at the O position. Temo vitro, for example using ELISA and/or by tagging a specific Zolomide and its use as a chemotherapeutic agent is described reporter molecule to one antibody which can be detected in in ref. 34. 60 the presence of other untagged antibody(s), to enable identi Tumours that may be treated using the present invention fication of antibodies which bind the same epitope or an may be cancerous tumours. The tumour or cancer may be in overlapping epitope. any area of the body, for example the brain. Cancers that may The antibody 4A1-F16 has VHand VL domain amino acid be treated include sarcomas, skin cancer, bladder cancer, sequences and CDRS as shown in the appended sequence breast cancer, uterine cancer, ovarian cancer, prostate cancer, 65 listing, as follows: lung cancer, colorectal cancer, cervical cancer, liver cancer, VH domain SEQID NO: 2 or SEQID NO: 17 head and neck cancer, oesophageal cancer, pancreatic cancer, VL domain SEQID NO: 4 US 8,580,267 B2 7 8 VHCDR1 SEQID NO. 5 or SEQ ID NO: 18 The Sclv may be connected to the constant domain via a VHCDR2 SEQID NO: 6 linker peptide. Dimerisation of the constant domains forms VHCDR3 SEQID NO: 7 the homodimer. VLCDR1 SEQID NO: 8 The antibody molecule may be a SIP comprising a VLCDR2 SEQID NO: 9 homodimer of scFv fused to a human CH4 domain of the VLCDR3 SEQID NO: 10 secretory isoform S2 of human IgE. CH4 is the domain that The alternativeVH domain sequences of F16, SEQID NO: allows dimerisation in the IgE molecule and the eS2 isoform 2 and SEQID NO: 17, differ by one amino acid in VHCDR1. contains a cysteine at the carboxyterminal end, which stabi Thus, alternative VHCDR1 sequences of F16 are SEQ ID lises the IgE dimer through an interchain disulphide bond. NO: 5 and SEQID NO: 18. 10 Thus, a covalent homodimer may be produced. SEQ ID NO: 5, RYGAS, was the original VH CDR1 Preferably, the antibody molecule is an schv antibody mol sequence of the clone 4A1-F16. However, the inventors dis ecule, consisting of a VH domain fused to a VL domain by a covered that by mutating the Ala to Met in position 4 of the peptide linker. The VH domain is normally the N terminal VH CDR1, the expression yield of the antibody could be domain, with the VL domain being the C terminal domain. significantly increased while still retaining high affinity bind 15 The VH and VL domains are preferably connected by a pep ing to tenascin-C. Accordingly, 4A1-F16 having the Ala to tide linker. The linker may be a sequence of about 5 to 10 Met mutation in SEQ ID NO: 5, to provide the VHCDR1 amino acids. An example peptide linker sequence is SEQID sequence SEQID NO: 18, represents an improved variant of NO: 22. F16. In the present application, either of these variants of F16 Preferably, the immunocytokine for use as described may be used. herein comprises IL2 conjugated to the antibody molecule. A Suitable antibody for use in an immunocytokine as IL2 is a secreted cytokine which is involved in immuno described herein comprises an antibody antigen binding site regulation and the proliferation of T and B lymphocytes. IL2 comprising a VH domain and a VL domain, has been shown to have a cytotoxic effect on tumour cells and the VH domain comprising a VHCDR1 SEQID NO: 5 or recombinant human IL2 (aldesleukin: Proleukin R) has FDA SEQID NO: 18, a VHCDR2 SEQID NO: 6 and a VH 25 approval for treatment of metastatic renal carcinoma and CDR3 SEQID NO: 7; and metastatic melanoma. Tumour-targeting immunocytokines the VL domain comprising a VLCDR1 SEQID NO: 8, a based on IL2 have been shown to mediate a massive infiltra VLCDR2 SEQID NO:9 and a VLCDR3 SEQID NO: tion of leukocytes into the tumour mass, with NK cells as the 10. main mediator of therapeutic activity 14, 22.37. The inven The antibody may comprise an antibody antigen binding 30 tors have recognised that the properties of IL2 may be par site comprising the 4A1-F16VH domain of SEQID NO. 2 or ticularly beneficial for treatment of glioma e.g. for treating SEQID NO: 17 and the 4A1-F16VL domain of SEQID NO. glioblastoma 38, 39. The experiments described herein 4. demonstrate that infiltration of cytotoxic effector cells includ In a preferred embodiment, the antibody molecule com ing NK cells, macrophages and leukocytes into the tumour prises a VH domain comprising a VHCDR1, VHCDR2 and 35 was markedly increased in glioblastoma treated with a com VH CDR3 wherein: bination of IL2-antibody conjugate and chemotherapy. VHCDR1 is SEQID NO: 18, The sequence of human IL2 is set out in SEQID NO: 11 VHCDR2 is SEQID NO: 6, and and publicly available under the Swiss Prot database as VHCDR3 is SEQID NO: 7. P60568. SEQID NO: 11 is the precursor sequence including Preferably, an antibody molecule in the present invention 40 an N-terminal signal peptide of 20 amino acids. Mature comprises a VL domain comprising a VLCDR1, VLCDR2 human IL2, lacking the signal peptide is residues 21 to 153 of and a VL CDR3 wherein SEQID NO: 11, and this mature sequence is set out in SEQID VLCDR1 is SEQID NO: 8, NO: 19. The IL2 moiety of the immunocytokine may com VLCDR2 is SEQID NO:9, and prise a sequence of all or part of the IL2 amino acid sequence VLCDR3 is SEQID NO: 10. 45 shown in SEQID NO: 11, e.g. all or part of the IL2 amino acid Variants of these VH and VL domains and CDRs may also sequence shown in SEQID NO: 19. A preferred IL2 sequence be employed in antibodies for use in immunocytokines as comprises SEQID NO: 19. described hereinas described herein. Suitable variants can be Variants of the SEQID NO: 19 amino acid sequence may obtained by means of methods of sequence alteration or be used, e.g. natural variants encoded by human alleles and/or mutation and Screening. 50 variants with one or two amino acid mutations. A mutation Particular variants for use as described herein may include may be deletion, Substitution, addition or insertion of an one or more amino acid sequence alterations (addition, dele amino acid residue. tion, Substitution and/or insertion of an amino acid residue). An IL2 amino acid sequence used in the present invention maybe less than about 20 alterations, less than about 15 may have at least 90% sequence identity, at least 95% alterations, less than about 10 alterations or less than about 5 55 sequence identity or at least 98% sequence identity to the alterations, 4, 3, 2 or 1. Alterations may be made in one or mature human IL2 sequence set out in SEQ ID NO: 19. more framework regions and/or one or more CDRS. In par Sequence identity is commonly defined with reference to the ticular, alterations may be made in VH CDR1, VH CDR2 algorithm GAP (Wisconsin GCG package. Accelerys Inc. and/or VHCDR3, especially VHCDR3. San Diego USA). GAP uses the Needleman and Wunsch A number of antibody molecule formats are known and any 60 algorithm to align two complete sequences that maximizes suitable format may be used for the antibody moiety of the the number of matches and minimizes the number of gaps. immunocytokine. Preferably, the immunocytokine comprises Generally, default parameters are used, with a gap creation the cytokine conjugated to an Schv antibody molecule. penalty=12 and gap extension penalty=4. Use of GAP may be The antibody molecule may consist of scFv, or it may be a preferred but other algorithms may be used, e.g. BLAST Small immunoprotein (SIP) comprising a homodimer of two 65 (which uses the method of Altschul et al. 40), FASTA polypeptides, each polypeptide comprising an ScFv fused to (which uses the method of Pearson and Lipman 41), or the an antibody heavy chain constant domain e.g. CH3 or CH4. Smith-Waterman algorithm 42, or the TBLASTN program, US 8,580,267 B2 10 of Altschul et al. 40, generally employing default param The individual to be treated using the present invention eters. In particular, the psi-Blast algorithm 43 may be used. may be a mammal, preferably a human. Sequence identity may be determined with reference to the Treatment of the tumour or cancer in the individual may full length of a sequence set out herein. comprise eradication of the tumour. However, for many forms Preferably the IL2 moiety of the immunocytokine com of tumours, especially malignant cancers and aggressive prises or consists of the sequence of mature human IL2 set out forms such as glioblastoma, complete cure may not be pos in SEQID NO: 19. sible. Treatment may comprise retarding tumour growth and/ The IL2 moiety may be fused upstream (N-terminal) or or reducing tumour Volume. Treatment may comprise length downstream (C-terminal) of the antibody molecule or ening the overall survival or progression free survival of the 10 individual. Treatment may comprise improving quality of life polypeptide component thereof. of the individual, e.g. by reducing one or more symptoms The cytokine moiety may be connected or attached to the caused by the tumour. Treatment may comprise inhibiting antibody molecule by any Suitable covalent or non-covalent regrowth of the tumour following tumour regression. As dem means. In preferred embodiments, the immunocytokine may onstrated in the examples herein, the combination of immu be a fusion protein comprising the cytokine, e.g. IL2, and the 15 nocytokine treatment with the chemotherapeutic agent antibody molecule or a polypeptide component thereof (e.g. a increased overall Survival time, increased progression-free heavy chain or a light chain of an antibody or multi-chain Survival time, retarded tumour growth, eradicated tumours antibody fragment, such as a Fab). Thus, for example, the and inhibited regrowth of tumours following regression. cytokine moiety may be fused to a VH domain or VL domain Treatment according to the present invention may be used to of the antibody. Typically the antibody molecule, or compo achieve any or all of these therapeutic effects. nent thereof, and cytokine moiety are joined via a peptide The dose of chemotherapeutic agent and immunocytokine linker, e.g. a peptide of about 5-25 residues, e.g. 10-20 resi administered to the individual will depend upon a number of dues, preferably about 15 residues. Suitable examples of pep factors, the size and location of the area to be treated, the tide linkers are well known in the art. In some embodiments, nature of the immunocytokine (e.g. whole antibody, fragment a linker may have an amino acid sequence as set out in SEQ 25 or diabody). A typical immunocytokine dose will be in the ID NO: 12. Normally, the linker has an amino acid sequence range 0.5 mg to 100 g for systemic applications, and 10 ug to comprising one or more tandem repeats of a motif. Typically 1 mg for local applications. This is a dose for a single treat the motif is a five residue sequence, and preferably at least 4 ment of an adult patient, which may be proportionally of the residues are Gly or Ser. Where four of the five residues adjusted for children and infants, and also adjusted for other is Gly or Ser, the other residue may be Ala. More preferably 30 antibody formats in proportion to molecular weight. each of the five residues is Gly or Ser. Preferred motifs are Appropriate doses and regimens for chemotherapeutic GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the agents are well known in the art. For temozolomide, the daily motifs are adjacent in the sequence, with no intervening dose may be about 75 mg per square meter of body Surface nucleotides between the repeats. The linker sequence may area, followed by a maintenance regimen of 150-200 mg/m comprise or consist of between one and five, preferably three 35 daily for 5 days of every 28 day cycle, repeated for 6 cycles or four, repeats of the motif. For example, a linker with three 44. Treatments may be repeated at daily, twice-weekly, tandem repeats may have one of the following amino acid weekly or monthly intervals, at the discretion of the physi Sequences: cian. The immunocytokine and the chemotherapeutic agent may 40 be administered separately, and administration may be GGGGSGGGGSGGGGS SEO ID NO: 13 sequential or simultaneous, in accordance with any Suitable SSSSGSSSSGSSSSG SEO ID NO: 14 regimen. The immunocytokine and the chemotherapeutic agent will usually be administered to an individual in the form GSGSAGSGSAGSGSA SEO ID NO: 15 of pharmaceutical compositions, which may comprise at least 45 one component in addition to the active compound. GGSGGGGSGGGGSGG. SEO ID NO: 16 Suitable components include a pharmaceutically accept The linker may comprise additional residues at the N and/ able excipient, carrier, buffer, stabiliser or other materials or C terminal end. well known to those skilled in the art. Such materials should A preferred linker sequence is SEQ ID NO: 20 EF be non-toxic and should not interfere with the efficacy of the SSSSGSSSSGSSSSG. 50 active ingredient. The precise nature of the carrier or other Preferably, the IL2 moiety is fused to the C-terminal end of material will depend on the route of administration, which the VL domain of an scFv antibody molecule, preferably by may be oral, or by injection, e.g. intravenous. The immuno means of a linker peptide. cytokine and the chemotherapeutic agent may be formulated In a preferred embodiment, the amino acid sequence of the in separate pharmaceutical compositions or, where appropri immunocytokine is at least 80%, 90%. 95% or 98% identical 55 ate, in the same pharmaceutical composition. to the amino acid sequence SEQ ID NO: 21. This is the Medicaments or pharmaceutical compositions comprising sequence of the F16(scFv)-IL2 sequence shown in FIG. 6. an immunocytokine and a chemotherapeutic agent according Administration of the chemotherapeutic agent, immuno to the present invention can be produced for use in treatment cytokine and compositions comprising one or both of these of a tumour in an individual. A methods of making a medi molecules is preferably in a “therapeutically effective 60 cament or pharmaceutical composition for use in the treat amount', this being sufficient to show benefit to a patient. ment of a tumour is an aspect of the invention. The method Such benefit may be at least amelioration of at least one may comprise formulating the chemotherapeutic agent and symptom. The actual amount administered, and rate and the immunocytokine into a composition comprising one or time-course of administration, will depend on the nature and more pharmaceutically acceptable excipients. The composi severity of what is being treated. Prescription of treatment, 65 tion may comprise separate formulations of the chemothera e.g. decisions on dosage etc., is within the responsibility of peutic agent and immunocytokine, or a combined formula general practitioners and other medical doctors. tion. US 8,580,267 B2 11 12 Pharmaceutical compositions for oral administration may another antibody class or subclass) are therefore included. be in tablet, capsule, powder or liquid form. A tablet may Cloning and expression of chimeric antibodies are described comprise a solid carrier Such as gelatin oran adjuvant. Liquid in EP-A-0120694 and EP-A-0125023, and a large body of pharmaceutical compositions generally comprise a liquid Subsequent literature. carrier Such as water, petroleum, animal or vegetable oils, Preferably, the antibody molecules used in the invention mineral oil or synthetic oil. Physiological saline solution, are human or humanised antibody molecules. dextrose or other saccharide solution or glycols such as eth It has been shown that fragments of a whole antibody can ylene glycol, propylene glycolor polyethylene glycol may be perform the function of binding antigens. Examples of bind included. ing fragments are (i) the Fab fragment consisting of VL, VH. For intravenous injection, or injection at the site of afflic 10 CL and CH1 domains; (ii) the Fd fragment consisting of the tion, the active ingredient will be in the form of a parenterally VH and CH1 domains; (iii) the Fv fragment consisting of the acceptable aqueous solution which is pyrogen-free and has VL and VH domains of a single antibody; (iv) the dAb frag suitable pH, isotonicity and stability. Those of relevant skill in ment 46, 47, 48, which consists of a VH or a VL domain; (v) the art are well able to prepare Suitable solutions using, for isolated CDR regions; (vi) F(ab')2 fragments, a bivalent frag example, isotonic vehicles Such as Sodium Chloride Injec 15 ment comprising two linked Fab fragments (vii) single chain tion, Ringer's Injection, Lactated Ringer's Injection. Preser Fv molecules (sclv), wherein a VH domain and a VL domain vatives, stabilisers, buffers, antioxidants and/or other addi are linked by a peptide linker which allows the two domains tives may be included, as required. to associate to form an antigen binding site 49, 50, (viii) A kit may be provided for use in the treatment of a tumour bispecific single chain Fv dimers (PCT/US92/09965) and (ix) in an individual, the kit comprising a chemotherapeutic agent "diabodies', multivalent or multispecific fragments con according to the invention and an immunocytokine according structed by gene fusion (WO94/13804:51). Fv, scFv or to the invention. The components of the kit (i.e. the chemo diabody molecules may be stabilized by the incorporation of therapeutic agent and immunocytokine) are preferably sterile disulphide bridges linking the VH and VL domains 52. and in sealed phials or other containers. A kit may further Minibodies comprising a scFv joined to a CH3 domain may comprise instructions for use of the components in a method 25 also be made 53. Other examples of binding fragments are described herein. The components of the kit may be sepa Fab', which differs from Fab fragments by the addition of a rately packaged. The components may be comprised or pack few residues at the carboxyl terminus of the heavy chain CH1 aged in a container, for example a bag, box, jar, tin or blister domain, including one or more cysteines from the antibody pack. hinge region, and Fab'-SH, which is a Fab' fragment in which 30 the cysteine residue(s) of the constant domains bear a free TERMINOLOGY thiol group. Qui et al. 54 described antibody molecules containing Antibody Molecule just two CDRs linked by a framework region. CDR3 from the This describes an immunoglobulin whether natural or VH or VL domain was linked to the CDR1 or CDR2 loop of partly or wholly synthetically produced. The term also covers 35 the other domain. Linkage was through the C terminus of the any polypeptide or protein comprising an antibody antigen selected CDR1 or CDR2 to the N terminus of the CDR3, via binding site. a FR region. Qui et al. selected the FR region having the Antibody fragments that comprise an antibody antigen fewest hydrophobic patches. The best combination for the binding site include, but are not limited to, molecules such as antibody tested was found to be VLCDR1 linked by VHFR2 Fab, Fab', Fab'-SH, scEv, Fv, dAb and Fd. Various other 40 to VHCDR3 (VHCDR1-VHFR2-VLCDR3). At a molecular antibody molecules including one or more antibody antigen weight of around 3 kDa, these antibody molecules offer binding sites have been engineered, including for example advantages in terms of improved tissue penetration as com Fab Fab, diabodies, triabodies, tetrabodies and minibodies. pared with full immunoglobulins (approx. 150 kDa) or schv Antibody molecules and methods for their construction and (approx. 28 kDa). use are described in ref. 45. 45 Antibody fragments of the invention can be obtained start It is possible to take monoclonal and other antibodies and ing from a parent antibody molecule by methods such as use techniques of recombinant DNA technology to produce digestion by enzymes e.g. pepsin or papain and/or by cleav other antibodies or chimeric molecules that bind the target age of the disulfide bridges by chemical reduction. In another antigen. Such techniques may involve introducing DNA manner, the antibody fragments comprised in the present encoding the immunoglobulin variable region, or the CDRS, 50 invention can be obtained by techniques of genetic recombi of an antibody to the constant regions, or constant regions nation likewise well known to the person skilled in the art or plus framework regions, of a different immunoglobulin. See, else by peptide synthesis by means of for example, automatic for instance, EP-A-184187, GB 2188638A or EP-A-2394.00, peptide synthesizers, such as those Supplied by the company and a large body of Subsequent literature. A hybridoma or Applied Biosystems, etc., or by nucleic acid synthesis and other cell producing an antibody may be subject to genetic 55 expression. mutation or other changes, which may or may not alter the Functional antibody fragments according to the present binding specificity of antibodies produced. invention include any functional fragment whose half-life is As antibodies can be modified in a number of ways, the increased by a chemical modification, especially by PEGyla term “antibody molecule' should be construed as covering tion, or by incorporation in a liposome. any binding member or Substance having an antibody anti 60 A dAb (domain antibody) is a small monomeric antigen gen-binding site with the required specificity and/or binding binding fragment of an antibody, namely the variable region to antigen. Thus, this term covers antibody fragments and of an antibody heavy or light chain 48. VH dAbs occur derivatives, including any polypeptide comprising an anti naturally in camelids (e.g. camel, llama) and may be pro body antigen-binding site, whether natural or wholly or par duced by immunizing a camelid with a target antigen, isolat tially synthetic. Chimeric molecules comprising an antibody 65 ing antigen-specific B cells and directly cloning dAb genes antigen-binding site, or equivalent, fused to another polypep from individual B cells. dAbs are also producible in cell tide (e.g. derived from another species or belonging to culture. Their small size, good solubility and temperature US 8,580,267 B2 13 14 stability makes them particularly physiologically useful and lar in the manual Antibodies’ 63 or to the technique of suitable for selection and affinity maturation. Camelid VH preparation from hybridomas described by Köhler and Mil dAbs are being developed for therapeutic use under the name stein 64. “NanobodiesTM. An antibody molecule of the present inven Monoclonal antibodies can be obtained, for example, from tion may be a dAb. The antibody molecule comprise a VH or an animal cell immunised against the target antigen or one of VL domain substantially as set out herein, or a VH or VL its fragments containing the epitope recognised by the mono domain comprising a set of CDRS substantially as set out clonal antibodies. Suitable fragments and peptides or herein. polypeptides comprising them are described herein, and may As used herein, the phrase “substantially as set out” refers be used to immunise animals to generate antibodies against a to the characteristic(s) of the relevant CDRs of the VH or VL 10 target antigen. Said antigen, or one of its fragments, can domain of antibody molecules described herein will be either especially be produced according to the usual working meth identical or highly similar to the specified regions of which ods, by genetic recombination starting with a nucleic acid the sequence is set out herein. As described herein, the phrase sequence contained in the cDNA sequence coding for the “highly similar with respect to specified region(s) of one or 15 antigen or fragment thereof, by peptide synthesis starting more variable domains, it is contemplated that from 1 to about from a sequence of amino acids comprised in the peptide 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4, amino sequence of the antigen and/or fragment thereof. acid substitutions may be made in the CDR and/or VH or VL The monoclonal antibodies can, for example, be purified domain. on an affinity column on which the antigen or one of its Bispecific or bifunctional antibodies form a second gen fragments containing the epitope recognised by said mono eration of monoclonal antibodies in which two different vari clonal antibodies, has previously been immobilised. More able regions are combined in the same molecule 55. Their particularly, the monoclonal antibodies can be purified by use has been demonstrated both in the diagnostic field and in chromatography on protein A and/or G, followed or not fol the therapy field from their capacity to recruit new effector lowed by ion-exchange chromatography aimed at eliminating functions or to target several molecules on the Surface of 25 the residual protein contaminants as well as the DNA and the tumour cells. Where bispecificantibodies are to be used, these LPS, in itself, followed or not followed by exclusion chroma may be conventional bispecific antibodies, which can be tography on Sepharose gel in order to eliminate the potential manufactured in a variety of ways 56, e.g. prepared chemi aggregates due to the presence of dimers or of other multim cally or from hybrid hybridomas, or may be any of the bispe ers. In one embodiment, the whole of these techniques can be cific antibody fragments mentioned above. These antibodies 30 used simultaneously or Successively. can be obtained by chemical methods 57, 58 or somatic methods 59, 60 but likewise and preferentially by genetic Antigen-Binding Site engineering techniques which allow the heterodimerization This describes the part of a molecule that binds to and is to be forced and thus facilitate the process of purification of complementary to all or part of the target antigen. In an the antibody sought 61. Examples of bispecific antibodies 35 antibody molecule it is referred to as the antibody antigen include those of the BiTETM technology in which the binding binding site, and comprises the part of the antibody that binds domains of two antibodies with different specificity can be to and is complementary to all or part of the target antigen. used and directly linked via short flexible peptides. This com Where an antigen is large, an antibody may only bind to a bines two antibodies on a short single polypeptide chain. particular part of the antigen, which part is termed an epitope. Diabodies and scFv can be constructed without an Fc region, 40 An antibody antigen-binding site may be provided by one or using only variable domains, potentially reducing the effects more antibody variable domains. An antibody antigen-bind of anti-idiotypic reaction. ing site may comprise an antibody light chain variable region Bispecific antibodies can be constructed as entire IgG, as (VL) and an antibody heavy chain variable region (VH). bispecific Fab'2, as Fab'PEG, as diabodies or else as bispecific WO2006/072620 describes engineering of antigenbinding sch v. Further, two bispecific antibodies can be linked using 45 sites in structural (non-CDR) loops extending between beta routine methods known in the art to form tetravalent antibod Strands of immunoglobulin domains. An antigenbinding site 1CS may be engineered in a region of an antibody molecule sepa Bispecific diabodies, as opposed to bispecific whole anti rate from the natural location of the CDRs, e.g. in a frame bodies, may also be particularly useful because they can be work region of a VH or VL domain, or in an antibody constant readily constructed and expressed in E. coli. Diabodies (and 50 domain e.g. CH1 and/or CH3. An antigen binding site engi many other polypeptides, such as antibody fragments) of neered in a structural region may be additional to, or instead appropriate binding specificities can be readily selected using of an antigen binding site formed by sets of CDRs of a VH phage display (WO94/13804) from libraries. If one arm of the and VL domain. Where multiple antigen binding sites are diabody is to be kept constant, for instance, with a specificity present in an antibody molecule, they may bind the same directed against the antigen of the tumour neovasculature, 55 then a library can be made where the other arm is varied and antigen (target antigen), thereby increasing Valency of the an antibody of appropriate specificity selected. Bispecific antibody molecule. Alternatively, multiple antigen binding whole antibodies may be made by alternative engineering sites may bind different antigens (the target antigen and one methods as described in Ridgeway et al., 1996 (62. or more anotherantigen), and this may be used to add effector Various methods are available in the art for obtaining anti 60 functions, prolong half-life or improve in vivo delivery of the bodies against a target antigen. The antibodies may be mono antibody molecule. clonal antibodies, especially of human, murine, chimeric or It is convenient to point out here that “and/or where used humanized origin, which can be obtained according to the herein is to be taken as specific disclosure of each of the two standard methods well known to the person skilled in the art. specified features or components with or without the other. In general, for the preparation of monoclonal antibodies or 65 For example A and/or B is to be taken as specific disclosure their functional fragments, especially of murine origin, it is of each of (i) A, (ii) Band (iii) A and B, just as if each is set out possible to refer to techniques which are described in particu individually herein. US 8,580,267 B2 15 16 Certain aspects and embodiments of the invention will now isoform S2 of human IgE (as exemplified for antibody L19 in be illustrated by way of example. All documents and database ref. 65). This domain promotes the formation of entries mentioned in this specification are incorporated herein homodimers that are further stabilized by disulfide bonds by reference in their entirety. between the C-terminal cysteine residues, resulting in a 75 kDa homobivalent miniantibody. This SIP antibody was fur BRIEF DESCRIPTION OF THE DRAWINGS ther biotinylated in order to avoid background staining given by the human endogenous IgE. FIG. 1 shows a schematic representation of the small (A) Sections of 10 um thickness were treated with ice-cold and large (B) tenascin-C isoform. Several fibronectin type III acetone, rehydrated in TBS (50 mM Tris, 100 mM. NaCl, like domains are subject to alternative splicing, either being 10 0.001% Aprotinin, pH 7.4) and blocked with TBS 20% fetal included (B) or omitted (A) in the molecule. The amino acid calf serum. Biot-(SIP) F16 antibody was added onto the sec sequence and encoding nucleotide sequence oftenascin Care tions in a final concentration of 2 g/ml. Bound antibody was publically available under sequence database references detected with Streptavidin-alkaline phosphatase complex NP 002151.1 GI:4504549 and NM 002160.1 GI:4504548, respectively. 15 (Biospa). Fast Red (Tablets Set, Sigma) was used as phos FIG.2 shows radioactivity content of representative organs phatase Substrate and sections were counterstained with was expressed as the percentage of the injected dose per gram Gill's hematoxylin no. 2 (Sigma). For every immunohis of tissue (% ID/g), following intravenous injection of mice tochemical experiment, in addition to staining with the F16 with a radioiodinated preparation of F16-IL2. antibody, a negative control was performed by omitting the FIG. 3 shows volume of tumours over time in the mouse primary antibody. Finally, L19, a proven glioma-targeting Subcutaneous model of human glioblastoma. antibody against the extra-domain B of fibronectin 30, was FIG. 4 shows the Kaplan-Meier survival curve of the mice used as positive control on the same tissue sections and at in the experiments with the mouse Subcutaneous model of identical final concentration (2 g/ml). human glioblastoma. An optic microscope (Zeiss) was used to evaluate the FIG. 5 shows body weight of mice in the four treatment 25 expression of the A1 domain of tenascin C and of the EDB groups in the Subcutaneous model of human glioblastoma. domain offibronectin, as revealed by the staining given by the FIG. 6 shows the amino acid sequence SEQID NO: 21 of F16 and L19 antibodies respectively. an F16-IL2 immunocytokine, composed of VH(F16) SEQID Cell Lines and Animals NO: 17, linker SEQ ID NO: 22, VL(F16) SEQ ID NO: 4, Human Subcutaneous glioblastoma xenografts were cre linker SEQID NO: 20, and IL2 SEQID NO: 19. 30 ated with the U87 cell line (already present in the lab). Cells were cultured in MEM (Invitrogen, Basel, Switzerland), EXPERIMENTS supplemented with 10% FCS (Invitrogen), 2 mM Summary L-glutamine, 1 mM sodium pyruvate, 100 U/mL amplicillin The experiments below describe therapeutic properties of 35 and incubated at 37° C. in 5% CO. For the subcutaneous temozolomide in combination with F16-IL2 for the therapy study, eight-week-old female Balb/c nude mice were pur of experimental murine models of human glioblastoma. The chased from Charles River Laboratories (Sulzfeld, Ger F16-IL2 used in this study is a clinical-stage immunocytokine many). consisting of human IL-2 fused to the human antibody F16, Antibodies and Therapeutic Agents specific to the A1 domain of tenascin-C. 40 The F16 antibody, specific to the extra-domain A1 of tena A radiolabelled preparation of the fusion protein F16-IL2 scin-C, has been described before 25. The expression, puri was shown to preferentially accumulate in Subcutaneously fication and characterization of the F16-IL2 fusion protein grafted human U87 gliomas in nude mice, using quantitative has also been described 22. F16-IL2 was purchased from biodistribution analysis. When co-administered with temozo Philogen (Siena, Italy) and temozolomide from ABCR lomide, F16-IL2 induced a complete remission of established 45 GmbH & Co. KG (Karlsruhe, Germany). Temozolomide was Subcutaneous human U87 gliomas in nude mice, which dissolved in saline 10% DMSO solution in a concentration of remained tumour-free for over 200 days. 3.5 mg/ml. Biodistribution Experiments Materials and Methods The in vivo targeting performance of F16-IL2 was evalu 50 ated by quantitative biodistribution analysis 14. Briefly, Immunohistochemistry on Human Glioblastoma Samples female BALB/c nude mice bearing subcutaneous U87 and on Glioblastoma Xenografts tumours (obtained by a s.c. flank injection of 5x106 U87 Freshly frozen human glioblastomatissues were received cells) were grouped (n=5) when tumours were clearly pal from the Neurosurgery department of Basel University Hos pable (volume of ca. 200 mm3) and injected i.v. into the pital, Basel, Switzerland, and were stored at -80° C. 55 lateral tail vein with radioiodinated F16-IL2. Antibody For immunohistochemical procedures, the F16 antibody immunoreactivity after labelling was evaluated by loading a was used in biotinylated small immunoprotein (SIP) format. sample of radiolabelled F16-IL2 onto TNC-A1-Sepharose F 16 is a human monoclonal antibody specific to the extra resin, followed by radioactive counting of the flow-through domain A1 of tenascin C25. It was obtained from the ETH2 and of the eluate fractions. Immunoreactivity, defined as the phage display libraries, underwent affinity maturation, had a 60 ratio between the counts of the eluted protein and the sum of dissociation constant in the low nanomolar range, and exhib the counts of the eluted and flow-through fractions, was 84%. ited a kinetic dissociation constantkoff towards the respective Mice were sacrificed 24 hours after injection of F16-IL2 (10 cognate antigens <10-2 S-1 in real-time interaction analysis ug, 3.6 LLCi per mouse). Organs were weighed and radioac experiments performed on a BIAcore 3000 instrument (GE tivity was counted with a Packard Cobra gamma counter. Healthcare, Otelfingen). The SIP antibody format consists of 65 Radioactivity content of representative organs was expressed a covalent homodimer in which each monomeric unit com as the percentage of the injected dose per gram of tissue (% prises a schv fused to a human CH4 domain of the secretory ID/g). US 8,580,267 B2 17 18 Subcutaneous Glioma Mouse Models Sections of 10 um thickness were treated with ice-cold Subcutaneous glioblastoma bearing mice were obtained by acetone, rehydrated in PBS and blocked with PBS 10% don a s.c. flank injection of 5x 106 U87 cells in 8-week old female key serum--10% goat serum. Sections were then incubated BALB/c nude mice 66. Twelve days after tumour cell with the primary antibodies (in a PBS 12% BSA solution) rat implantation, when tumours had reached a size of 250-300 anti-mouse F4780+rabbitanti-mouse CD31, rabbitanti-asialo mm, mice were staged to maximize uniformity among the GM1+rat anti-mouse CD31, rat anti-mouse CD45+rabbit groups (n=5). anti-mouse CD31, and ratanti-human IL2+rabbitanti-mouse One group was injected i.V. (lateral tail vein) with 20 g of CD31, in order to evaluate the distribution of macrophages, F16-IL2 (corresponding to 6.6 g of IL2) in a total volume of NK cells, and leukocytes in relation with the tumour vascu 100 ul PBS solution (Phosphate Buffer Saline), one was 10 lature in every therapeutic group (placebo, F16-IL2, temoZo injected i.p. with 0.525 mg oftemozolomide (corresponding lomide, F16-II2+temozolomide). The location of the F16-IL2 to 75 mg/m) in a total volume of 150 ul saline 10% DMSO fusion protein within the tumour was examined in the F16 Solution, a third group received both the i.v. injection of 20 ug IL2 and in the F16-II2+temozolomide therapeutic groups. F16-IL2 and the i.p. of 0.525 mg temozolomide, finally, the Bound antibody was detected with fluorescent Alexa488- or control group was injected i.p. with 150 ul of saline 10% 15 Alexa594-coupled secondary antibodies (donkey anti-rator DMSO. goat anti-rabbit, BD Biosciences Pharmingen) and DAPI in a Aschedule offive total administrations was established: on PBS 12% BSA Solution. days 12, 15, 18, 21, 24 from the tumour cell injection into In each tumour section, the staining given by the primary mice (day 0). antibodies was quantified in three representative 10x micro Dosage of F16-IL2 was in line with the recommended scopic images using the Image.J Software (http://rsb.info. human dose of 18 million I.U. of recombinant IL2 (Proleukin, nih.gov/j/). Novartis) and of 22.5 million I.U. of IL2 equivalents, used in Statistical Analysis clinical trials with the immunocytokine L19-IL2. Biodistribution data are expressed as the median tSD. A 75 mg/m dose oftemozolomide was chosen in order to Tumour volumes are shown as average +SD or SE. Kaplan mimic the standard of care for newly diagnosed glioblastoma 25 Meier survival curves were performed to display therapeutic patients 44 with immunotherapy instead of the radiation efficacy in the Subcutaneous glioma model. treatment. Radiotherapy was avoided in order to evaluate the efficacy Results of F16-IL2 and temozolomide combined together. The five administration schedule reflected the intent to mimic the daily 30 Immunohistochemistry on Human Glioblastoma Specimens temozolomide treatment in patients (75 mg/m daily for 6 and on Mouse Xenografts weeks) 44). The five-administration 75 mg/m temozolo Expression of A1 domain of tenascin-C and of the EDB mide schedule was far below the LDo for the subcutaneous domain of fibronectin was assessed in sections of glioblas glioma mouse models 67. toma Surgical specimens and of U87 gliomablastomas grafted Mice were monitored daily and tumours were measured 35 Subcutaneously in nude mice. Identical concentrations of the with a digital caliper three times per week. Tumour volume F16 and L19 antibodies were used, in full analogy to the was estimated using the formula: Volume-lengthxwidth/2. recently published comparative analysis of antigen expres Animals were sacrificed when tumours approached a volume sion in thoracic neoplasias. The F16 antibody was found to of 3,000 mm or when tumours turned necrotic and bleeding, strongly stain both experimental U87 tumours and glioblas according to the Swiss regulations and under a project license 40 tomas from patients, with patterns and intensities comparable granted by the Veterinaramt des Kantons Zürich (198/2005). to the ones of the L19 antibody, which had previously been No animals had to be sacrificed because of suffering from shown to stain blood vessels in virtually all types of high side-effects of the malignancy or from therapy-derived tox grade astrocytomas 30. icities. Biodistribution Studies with Radiolabelled F16-IL2 Immunofluorescence Assessment of Tumour-Infiltrating 45 Mice bearing subcutaneous U87 glioblastomas were Cells and of Microvascular Density in Subcutaneous Glioma injected i.v. with radioiodinated preparations of F16-IL2, in Xenografts order to study in vivo targeting performance by quantitative To evaluate the role of inflammatory cell responses in vivo, biodistribution analysis. The immunocytokine displayed a female Balb/c nude mice bearing s.c. U87 tumours (3 mice preferential accumulation in the tumour 24 h after injection per group) were treated on days 12, 15 and 18 after tumour 50 (2.3% ID/g), with a tumour-to-blood ratio of 11.5 and with cell implantation with either saline 10% DMSO (i.p.), F16 excellent tumour:organ ratios (FIG. 2). IL2 (i.v.), temozolomide (i.p.), and F16-IL2 (i.v.) plus temo Therapeutic Activity of F16-IL2 Combined with Temozolo Zolomide (i.p.). Mice were sacrificed 24 hours after the third mide in Subcutaneous Glioblastoma Models injection, and tumours were excised and Snap-frozen in OCT. Therapeutic activity of temozolomide and of F16-IL2 The immunofluorescent staining of tumour sections was per 55 (alone and in combination) was compared in nude mice bear formed using antibodies against the following antigens: ing Subcutaneous U87 tumours. Therapy was started 12 days F4/80 (rat anti-mouse F4/80, clone A3-1, AbCam, Cam after subcutaneous injection of U87 cells, when tumours had bridge, UK) for the detection of tumour-infiltrating macroph reached an average size of 300 mg. Temozolomide was ages, asialo GM1 (rabbitanti-asialo GM1, Wako Pure Chemi administered five times with i.p. injections of 0.525 mg in cal Industries Ltd, Osaka, Japan) for NK cells, CD45 (rat 60 10% DMSO solution every three days. F16-IL2 was admin anti-mouse CD45, BD Biosciences Pharmingen, Allschwil, istered i.v. at 20 ug doses. Switzerland) for leukocytes, CD31 (rabbit or rat anti-mouse Monotherapy treatment with F16-IL2 led only to a minor CD31, BD Pharmingen, Allschwil, Switzerland) for the tumour growth retardation, compared to the control group of localization of endothelial cells, and IL2 (ratanti-human IL2, mice treated with 10% DMSO, while all mice in the temozo eBioscience Inc, San Diego, USA) for the detection of the 65 lomide group exhibited a virtually complete tumour regres F16-IL2 immunocytokine in the F16-IL2 and in the F16-IL2+ sion by day 30. However, by day 45, tumours started growing temozolomide therapeutic groups. again in 3/5 mice of this treatment group. By contrast, mice US 8,580,267 B2 19 20 treated with a combination of temozolomide and F16-IL2 - Continued exhibited a complete tumour eradication and remained GCG AAA. GCG CAT AAT GCT TTT GAC TAC TGG GGC CAG tumour-free for over 200 days. See FIG.3. Measurements of body weights for the four treatment GGA ACC CTG GTC ACC GTG TCG. AGA groups confirmed that no weight loss was observed in the SEQ ID NO: 2 4A1-F16 WH domain amino acid control group and in the F16-IL2 treatment group, while sequence temozolomide and the combination of temozolomide--F16 EVOLLESGGG LVOPGGSLRL SCAASGFTFS RYGASWVROA IL2 led to a transient weight loss (with a nadir of 10% and PGKGLEWWSA. ISGSGGSTYY ADSWKGRFTI SRDNSKNTLY 15% after the fifth injection, respectively). FIG.5 shows mass of the mice ingrams as recorded for the four treatment groups. 10 LOMNSLRAED TAVYYCAKAH NAFDYWGOGT LWTVSR The therapy study with Subcutaneous glioblastoma bearing SEQ ID NO : 3 4A1 - F16 WL domain nucleotide sequence mice disclosed the complete eradication of the tumour in all TCT TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG mice receiving the combined treatment of F16-IL2 and temo GCC TTG GGA CAG ACA GTC AGG ATC ACA TGC CAA. GGA Zolomide. The Kaplan-Meier survival curve is shown in FIG. 15 4. By day 35, all 10% DMSO-treated control mice were GAC AGC CTC AGA AGC TAT TAT GCA AGC TGG TAC CAG sacrificed because the tumour had approached the cut-off size of 3,000 mm. The administration of F16-IL2 alone exhibited CAG AAG CCA. GGA CAG GCC CCT GTA CTT GTC ATC TAT some therapeutic benefit, with the sacrifice of the last mouse GGT AAA AAC AAC CGG CCC. TCA. GGG ATC CCA GAC CGA at day 47 from the tumour cell inoculation. Temozolomide alone showed efficacy in determining the survival of two mice TTC TCT GGC. TCC AGC TCA. GGA AAC ACA GCT. TCC TTG over 3 months from the tumour cell injection, but the other ACC ATC ACT GGG GCT CAG GCG GAA. GAT GAG GCT GAC animals were sacrificed before day 106 (the tumour had reached the cut-off size). All mice of the combination treat TAT TAC TGT AAC TCC, TCT GTT TAT ACT ATG CCG CCC ment group (F16-IL2+TMZ) remained alive and tumour-free for over 3 months from the tumour cell injection. One of the 25 GTG GTA TTC GGC GGA. GGG ACC AAG. CTG ACC GTC CTA five mice in this treatment group had to be sacrificed at day SEQ ID NO: 4 4A1-F16 VL domain amino acid sequence 160 due to weight loss experienced since day 155, but was SSELTODPAVSVALGOTVRITCOGDSLRSYYASWYOOKPGOAPWLVIYGK found to be tumour-free at necropsy. NNRPSGIPDRFSGSSSGNTASLTITGAOAEDEADYYCNSSWYTMPPVVFG Analysis of Tumour-Infiltrating Cells and of Microvascular 30 Density in Subcutaneous Glioblastoma Xenografts GGTKLTWL Human glioblastoma (U87) xenografts were harvested after three injections and sections were analyzed by immun D NO: 5 4A1 6 W H C D R1 amino acid sequence: ofluorescence. Tumour sections were stained with antibodies anti-asialo GM1, specific for natural killer cells, anti-F4/80, 35 which recognizes macrophages, and anti-CD45, a leukocyte SEQ ID NO: 6 4A1- 6 W H C D R2 amino acid sequence: specific marker. The largest increase in the infiltration of natural killer cells, AISGSGGSTYYADSWKG macrophages and leukocytes was observed in the F16-IL2 SEQ ID NO: 7 4A1- 6 W H C D R3 amino acid sequence: plus temozolomide combination treatment group. The assess 40 ment of microvascular density by CD31 staining revealed a AHNAFDY general decrease of neo-Vessels in the temozolomide-treated groups. Analysis of the F16-IL2 fusion protein within the SEQ ID NO: 8 4A1- 6 W C D R1 amino acid sequence: tumour confirmed the expected localization of the immuno QGDSLRSYYAS cytokine in the perivascular region and in the tumour extra 45 cellular matrix. SEQ ID NO: 9 4A1- 6 W C D R2 amino acid sequence: These results demonstrate the contribution of cytotoxic effector cells to the therapeutic effect of F16-IL2 plus temo GKNNRPS Zolomide combination therapy. SEQ ID NO: 10 4A1-F16 VL CDR3 amino acid sequence: List of Sequences: 50 NSSWYTMPPVV

SEO ID NO: 1. 4A1-F16 WH domain nucleotide SEQ ID NO: 11 hIL2 precursor sequence sequence MYRMOLLSCI ALSLALVTNS APTSSSTKKT OLOLEHLLLD GAG. GTG. CAG CTG TTG GAG TCT GGG (GGA. GGC TTG GTA 55 LOMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLOCLE CAG CCT GGG GGG TCC CTG AGA CTC. TCC TGT, GCA GCC EELKPLEEWL, NLAOSKNFHL RPRDLISNIN WIVLELKGSE TCT GGA TTC ACC TTT AGC CGG TAT GGT GCG. AGC TGG TTFMCEYADE TATIVEFLNR WITFCOSIIS TLT GTC CGC CAG GCT CCA. GGG AAG GGG CTG GAG TGG GTC 60 TCA GCT ATT AGT GGT AGT GGT GGT AGC ACA. TAC TAC SEQ ID NO: 12 Peptide linker amino acid sequence:

GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC. TCC AGA GGGGSGGGGSGGGG

GAC AAT TCC AAG AAC ACG. CTG TAT. CTG CAA ATG AAC SEQ ID NO: 13 Peptide linker amino acid sequence:

AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT 65 GGGGSGGGGSGGGGS US 8,580,267 B2 21 22 - Continued - Continued SEO ID NO: 4 Peptide linker amino acid sequence: GCG AAA. GCG CAT AAT GCT TTT GAC TAC TGG GGC CAG SSSSGSSSSGS SSSG GGA ACC CTG GTC ACC GTG TCG. AGA SEO ID NO: 5 Peptide linker amino acid sequence: GSGSAGSGSAG SGSA References SEO ID NO: 6 Peptide linker amino acid sequence: 1 Bosslet Cancer Res. 1998 GGSGGGGSGGGGSGG 10 2 Adams G. P. Weiner L. M. Nat Biotechnol. 23:1147-1157 2005 SEO ID NO: 7 Improved F16 VH domain amino acid sequence 3 Carter P.J. Nat Rev Immunol. 6:343-357 2006 EVOLLESGGG LVOPGGSLRL SCAASGFTFS RYGMSWVROA 4 Schrama D, Reisfeld RA. Becker J.C. Nat Rev Drug Discov. 5:147-159 2006 PGKGLEWWSA SGSGGSTYY ADSWKGRFTI SRDNSKNTLY 15 5 Neri D, Bicknell R. Nat Rev Cancer. 5:436-446 2005 LOMNSLRAED TAVYYCAKAH NAFDYWGOGT LWTVSR 6 Thorpe P E. Clin Cancer Res. 10:415-427 2004 7 Schliemann C, Neri D. Biochim Biophys Acta. 1776:175 SEO ID NO: 8 Improved F16 VH CDR1 amino acid 192 2007 8 Riva Petal. Int J Cancer, 51:7-13 1992 sequence: RYGMS 9 Riva Petal. Cancer Res 55:5952S-5956S 1995 SEO ID NO: 9 Human IL-2 amino acid sequence 10 Paganelli Getal Eurj Nucl Med 21:314-321 1994 mature sequ ecce 11 Reardon DA et al. J. Clin Oncol 20:1389–1397 2002 APTSSSTKKT OLO EHILLD LOMILNGINN YKNPKLTRML 12 Bigner DD et al. J Clin Oncol 16:2202-2212 1998 TELKHLOCLE EELKPLEEWL, NLAOSKNFHL 13 Halin C, et al. Nat Biotechnol. 20:264-269 2002 25 14 Carnemolla B, et al. Blood. 99:1659-1665 2002 RPRDLISNIN WIW ELKGSE TTFMCEYADE TATIVEFLNR 15 Borsi L, et al. Blood. 102:4384-4392 2003 16 Ebbinghaus C, et al. IntJ Cancer. 116:304-313 2005 WITFCOSIIS TLT 17 Kaspar M, Trachsel E, Neri D. Cancer Res.67:4940-4948 SEO ID NO: Peptide linker amino acid sequence 2007 30 18 Tijink B Met al., J. Nucl Med. 47: 1127-1135 2006 EFSSSSGSSSSGSSSSG 19 Birchler et al. Nat Biotechnol. 17:984-988 1999 SEO ID NO: 21 F16 - IL2 Immunocytokine amino acid 20 Halin C, et al. Cancer Res.63:3202-3210 2003 sequence 21 Menrad A, Menssen HD. 9:491-500 2005 EVOLLESGGG LVO PGGSLRL SCAASGFTFS RYGMSWVROA 22 Marlind et al. Clin Cancer Res. 14(20):6515-6524 2008 35 23 Viti, F., et al., Cancer Res, 1999. 59(2): p. 347-52 PGKGLEWWSA ISGSGGSTYY ADSWKGRFTI SRDNSKNTLY 24 Nilsson, F., et al., Cancer Res, 2001. 61(2): p. 711-6 LOMNSLRAED TAWYYCAKAH NAFDYWGOGT LWTVSRGSSG 25 Brack et al. Clin Cancer Res; 12:3200-8 2006 26 Burnet N G et al Br. Cancer 92:241-245 2005 GSSELTODPA WSWALGOTVR ITCOGDSLRS YYASWYOOKP 27 CBTRUS, Statistical Reports: Primary Brain Tumours in KNNRPSGIPD 40 the United States: 1995-1999 and 1997-2000 RFSGSSSGNT ASLTITGAOA 28 Ferlay Jet al: Globocan 2000: Cancer incidence, mortality EDEADYYCNS SWYTMPPWWF GGGTKLTWLE FSSSSGSSSS and prevalence worldwide. IARC Press, Lyon 2000 29 Castellani IJ C 1994 GSSSSGAPTS SSTKKTOLQL EHLLLDLOMI LNGINNYKNP 30 Castellani Pet al. American Journal of Pathology: 161: KLTRMLTFKF YMPKKATELK HLOCLEEELK PLEEWLNLAQ 45 1695-17OO 2002 31 Santimaria, 2003, Clin Cancer Res SKNFHLRPRD LISNINWIWL ELKGSETTFM CEYADETATI 32 Leins et al. Cancer 98(11):2430-2439 2003 WEFLNRWITF COSIISTLT 33 Bartolomei et al. Q J Nucl Med 48:220-228 2004 34 Darkes et al. Am. J. Cancer 1(1):55-90 2002 SEO ID NO: 22 Peptide linker amino acid sequence: 50 35 Kleihues P. Cavenee W K: WHO, Pathology & Genetics, Tumours of the Nervous System. IARC Press, Lyon 2000 GSSGG 36 Ohgaki Het al. Cancer Res 64, 6892-6899 2004 SEO ID NO: 23 Improved F16 VH domain nucleotide 37 Schliemann et al. Blood November 2008 sequence 38 Dunne Jet al. J Immunol. 167:3129-3138 2001 GAG. GTG CAG CTG TTG GAG TCT GGG (GGA. GGC TTG GTA 55 39 Albertsson PA et al., Trends Immunol. 24:603-609 2003 CAG CCT GGG GGG TCC CTG. AGA CTC. TCC TGT, GCA GCC 40 Altschul et al. J. Mol. Biol. 215: 405-410 1990 41 Pearson and Lipman PNAS USA 85: 2444-2448 1988 TCT GGA TTC ACC TTT AGC CGG TAT GGT ATG AGC TGG 42 Smith and Waterman J. Mol Biol. 147: 195-197 1981 43 Nucl. Acids Res. 253389-3402 1997 GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC 60 44 Stuppetal The New England Journal of Medicine, 352: TCA GCT ATT AGT GGT AGT GGT GGT AGC ACA. TAC TAC 987-96 2005 45 Holliger & Hudson, Nature Biotechnology 23(9): 1126 GCA GAC TCC AAG GGC CGG TTC ACC ATC. TCC AGA 1136 2005 GAC AAT TCC AAG AAC ACG CTG TAT. CTG CAA ATG AAC 46 Ward, E. S. et al., Nature 341, 544-546 (1989) 65 47 McCafferty et al (1990) Nature, 348, 552-554 AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT 48 Holt et al (2003) Trends in Biotechnology 21, 484-490 49 Bird et al, Science, 242, 423-426, 1988 US 8,580,267 B2 23 24 50 Huston et al., PNAS USA, 85,5879-5883, 1988 59 Staerz U. D. and Bevan M. J. 1986 PNAS 83 51 Holliger, P. et al. Proc. Natl. Acad. Sci. USA 90 6444 60 Suresh M. R. et al., 1986 Method Enzymol. 121:210-228 6448, 1993 61 Merchand et al., 1998 Nature Biotech. 16:677-681 52 Reiter, Y. etal, Nature Biotech, 14, 1239-1245, 1996 62 Ridgeway, J. B. B. etal, Protein Eng., 9, 616-621, 1996 53 Hu, S. et al, Cancer Res., 56, 3055-3061, 1996 54 Quiet al., Nat. Biotechnol. 25:921-929 2007 63 Harlow and Lane, Antibodies: A Laboratory Manual, Cold 55 Holliger and Bohlen 1999 Cancer and metastasis rev. 18: Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 411-41 726, 1988 56 Holliger, P. and Winter G. Current Opinion Biotechnol 4, 64 Köhler and Milstein, Nature, 256:495-497, 1975 446-449 1993 10 65 Borsi Let al. IntJ Cancer; 102:75-85 2002 57 Glennie MJ et al., 1987.J. Immunol. 139, 2367-2375 66 Bello et a Clinical Cancer Research, 10: 4527-37 2004 58 Repp R. et al., 1995 J. Hemat. 377-382 67 Friedman et al. Clinical Cancer Research 6:2585-97 2000

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 27

<21 Oc SEO I D NO 1 <211 LENGT H: 348 <212> TYPE : DNA <213> ORGANISM: Homo sapiens

< 4 OOs SEQUE NCE: 1

gaggtg Cagc tgttggagtic tgggggaggc ttgg tacagc Ctggggggt C cctgagactic 60

toctdt gCag cctictoggatt cacctittagc cgg tatggtg cgagctgggit cogc.caggct 12O

CC aggg aagg ggctggagtg ggtct cagct attagtggta gtggtggtag cacatact ac 18O

gcagac tgaaggg.ccg gttcaccatc to cagaga.ca attic caagaa cacgctgtat 24 O

ctgcaa. atga acagcctgag agc.cgaggac acggc.cgitat attact.gtgc gaaag.cgcat 3 OO

aatgct tittg actactgggg cc agggalacc Ctggtcaccg tgtc.gaga 348

<21 Oc SEO I D NO 2 <211 LENGT H: 116 <212> TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUE NCE: 2

Glu Wall Glin Lieu. Lieu Gl u Ser Gly Gly Gly Luell Val Glin Pro Gly Gly 1. 5 15

Ser Le u Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 25 3 O

Gly Al a Ser Trp Val Air g Glin Ala Pro Gly Gly Lieu. Glu Trp Val 35 4 O 45

Ser Al a Ile Ser Gly Se r Gly Gly Ser Thir Tyr Ala Asp Ser Val SO 55 60

Lys Gl y Arg Phe Thir II e Ser Arg Asp Asn Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O

Lieu Gl in Met Asn. Ser Lue u Arg Ala Glu Asp Thir Ala Val Tyr Tyr Cys 85 90 95

Ala Lys Ala His Asn Al a Phe Asp Tyr Trp Gly Gln Gly Thr Lieu Val 1OO 105 110

Thir Wa l Ser Arg 115

SEO I D NO 3 LENGT H: 324 TYPE : DNA ORGANISM: Homo sapiens

< 4 OOs SEQUE NCE: 3 US 8,580,267 B2 25 26 - Continued tottctgagc tgacticagga CCCtgctgtg tctgtggcct tgggacagac agt caggat C 6 O acatgccaag gagacagc ct Cagaa.gct at tatgcaa.gct ggtaccagca gaa.gc.cagga 12 O

Caggcc cctg tacttgtcat citatgtaaa aacaa.ccggc Cct cagggat CCC agaccga 18O ttct ctdgct Ccagct cagg aaacacagct tcc ttgacca t cactggggc t caggcggaa 24 O gatgaggctg act attactg taact cost ct gtttatacta ggt attcggc 3OO ggagggacca agctgaccgt. CCta 324

<210s, SEQ ID NO 4 &211s LENGTH: 108 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4

Ser Ser Glu Lieu. Thr Glin Asp Pro Ala Wal Ser Wall Ala Luell Gly Glin 1. 1O 15

Thir Wall Arg Ile Thr Cys Glin Gly Asp Ser Lieu. Arg Ser Tyr Tyr Ala 25

Ser Trp Tyr Gln Gln Lys Pro Gly Glin Ala Pro Wall Lell Wall Ile Tyr 35 4 O 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser SO 55 6 O

Ser Ser Gly Asn. Thir Ala Ser Lieu. Thir Ilie Thir Gly Ala Glin Ala Glu 65 70 7s

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ser Wall Thir Met Pro Pro 85 90 95

Wall Wall Phe Gly Gly Gly Thr Lys Lieu. Thir Wall Lell 105

<210s, SEQ ID NO 5 &211s LENGTH: 5 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 Arg Tyr Gly Ala Ser 1.

SEQ ID NO 6 LENGTH: 17 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 6 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1. 5 15 Gly

<210s, SEQ ID NO 7 &211s LENGTH: 7 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Ala His Asn Ala Phe Asp Tyr 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 11 212. TYPE : PRT US 8,580,267 B2 27 28 - Continued <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Glin Gly Asp Ser Lieu. Arg Ser Tyr Tyr Ala Ser 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 9 Gly Lys Asn. Asn Arg Pro Ser 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 10 Asn Ser Ser Val Tyr Thr Met Pro Pro Val Val 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 153 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 Met Tyr Arg Met Glin Lieu Lleu Ser Cys Ile Ala Lieu. Ser Lieu Ala Lieu. 1. 5 1O 15 Val Thr Asn Ser Ala Pro Thir Ser Ser Ser Thr Lys Llys Thr Glin Leu 2O 25 3O Glin Lieu. Glu. His Lieu Lleu Lieu. Asp Lieu Gln Met Ile Lieu. Asn Gly Ile 35 4 O 45 Asn Asn Tyr Lys Asn Pro Llys Lieu. Thr Arg Met Lieu. Thr Phe Llys Phe SO 55 6 O Tyr Met Pro Llys Lys Ala Thr Glu Lieu Lys His Lieu. Glin Cys Lieu. Glu 65 70 7s 8O Glu Glu Lieu Lys Pro Lieu. Glu Glu Val Lieu. Asn Lieu Ala Glin Ser Lys 85 90 95 Asn Phe His Lieu. Arg Pro Arg Asp Lieu. Ile Ser Asn. Ile Asin Val Ile 1OO 105 11 O Val Lieu. Glu Lieu Lys Gly Ser Glu Thir Thr Phe Met Cys Glu Tyr Ala 115 12 O 125 Asp Glu Thir Ala Thr Ile Val Glu Phe Lieu. Asn Arg Trp Ile Thr Phe 13 O 135 14 O Cys Glin Ser Ile Ile Ser Thr Lieu. Thr 145 150

<210s, SEQ ID NO 12 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 12 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1. 5 1O US 8,580,267 B2 29 30 - Continued

<210s, SEQ ID NO 13 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1. 5 1O 15

<210s, SEQ ID NO 14 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 14 Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly 1. 5 1O 15

<210s, SEQ ID NO 15 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 15 Gly Ser Gly Ser Ala Gly Ser Gly Ser Ala Gly Ser Gly Ser Ala 1. 5 1O 15

<210s, SEQ ID NO 16 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 16 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1. 5 1O 15

<210s, SEQ ID NO 17 &211s LENGTH: 116 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Improved F16 VH domain amino acid sequence

<4 OOs, SEQUENCE: 17 Glu Val Glin Lieu. Lieu. Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 2O 25 3O Gly Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val SO 55 6 O US 8,580,267 B2 31 - Continued Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala His Asn Ala Phe Asp Tyr Trp Gly Glin Gly. Thir Lieu Val 1OO 105 11 O Thr Val Ser Arg 115

<210s, SEQ ID NO 18 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Improved F16 VH CDR1 domain amino acid sequence

<4 OOs, SEQUENCE: 18 Arg Tyr Gly Met Ser 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 133 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19 Ala Pro Thir Ser Ser Ser Thr Lys Llys Thr Gln Leu Gln Leu Glu. His 1. 5 1O 15 Lieu. Lieu. Lieu. Asp Lieu Gln Met Ile Lieu. Asn Gly Ile Asn. Asn Tyr Lys 2O 25 3O Asn Pro Llys Lieu. Thr Arg Met Lieu. Thr Phe Llys Phe Tyr Met Pro Llys 35 4 O 45 Lys Ala Thr Glu Lieu Lys His Lieu. Glin Cys Lieu. Glu Glu Glu Lieu Lys SO 55 6 O Pro Lieu. Glu Glu Val Lieu. Asn Lieu Ala Glin Ser Lys Asn. Phe His Lieu. 65 70 7s 8O Arg Pro Arg Asp Lieu. Ile Ser Asn. Ile Asin Val Ile Val Lieu. Glu Lieu 85 90 95 Lys Gly Ser Glu Thir Thr Phe Met Cys Glu Tyr Ala Asp Glu. Thir Ala 1OO 105 11 O Thir Ile Val Glu Phe Lieu. Asn Arg Trp Ile Thr Phe Cys Glin Ser Ile 115 12 O 125

Ile Ser Thir Lieu. Thir 13 O

<210s, SEQ ID NO 2 O &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 2O Glu Phe Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser 1. 5 1O 15 Gly

<210s, SEQ ID NO 21 &211s LENGTH: 379 US 8,580,267 B2 33 34 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: F16-IL2 Immunocytokine amino acid sequence

<4 OOs, SEQUENCE: 21 Glu Val Glin Lieu. Lieu. Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 2O 25 3O Gly Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala His Asn Ala Phe Asp Tyr Trp Gly Glin Gly. Thir Lieu Val 1OO 105 11 O Thr Val Ser Arg Gly Ser Ser Gly Gly Ser Ser Glu Lieu. Thr Glin Asp 115 12 O 125 Pro Ala Val Ser Val Ala Lieu. Gly Glin Thr Val Arg Ile Thr Cys Glin 13 O 135 14 O Gly Asp Ser Lieu. Arg Ser Tyr Tyr Ala Ser Trp Tyr Glin Glin Llys Pro 145 150 155 160 Gly Glin Ala Pro Val Lieu Val Ile Tyr Gly Lys Asn. Asn Arg Pro Ser 1.65 17O 17s Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser 18O 185 19 O Lieu. Thir Ile Thr Gly Ala Glin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 195 2OO 2O5 Asn Ser Ser Val Tyr Thr Met Pro Pro Val Val Phe Gly Gly Gly Thr 21 O 215 22O Lys Lieu. Thr Val Lieu. Glu Phe Ser Ser Ser Ser Gly Ser Ser Ser Ser 225 23 O 235 24 O Gly Ser Ser Ser Ser Gly Ala Pro Thr Ser Ser Ser Thr Lys Llys Thr 245 250 255 Glin Lieu. Glin Lieu. Glu. His Lieu. Lieu. Lieu. Asp Lieu Gln Met Ile Lieu. Asn 26 O 265 27 O Gly Ile Asn Asn Tyr Lys Asn Pro Llys Lieu. Thir Arg Met Lieu. Thr Phe 27s 28O 285 Llys Phe Tyr Met Pro Llys Lys Ala Thr Glu Lieu Lys His Lieu. Glin Cys 29 O 295 3 OO Lieu. Glu Glu Glu Lieu Lys Pro Lieu. Glu Glu Val Lieu. Asn Lieu Ala Glin 3. OS 310 315 32O Ser Lys Asn. Phe His Lieu. Arg Pro Arg Asp Lieu. Ile Ser Asn. Ile Asn 3.25 330 335 Val Ile Val Lieu. Glu Lieu Lys Gly Ser Glu Thir Thr Phe Met Cys Glu 34 O 345 35. O Tyr Ala Asp Glu Thir Ala Thir Ile Val Glu Phe Lieu. Asn Arg Trp Ile 355 360 365 Thr Phe Cys Glin Ser Ile Ile Ser Thr Lieu. Thr 37 O 375 US 8,580,267 B2 35 36 - Continued

<210s, SEQ ID NO 22 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic sequence: Peptide linker amino acid sequence

<4 OOs, SEQUENCE: 22 Gly Ser Ser Gly Gly 1. 5

<210s, SEQ ID NO 23 &211s LENGTH: 348 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic sequence: Improved F16 VH domain nucleotide sequence

<4 OOs, SEQUENCE: 23 gaggtgcagc tigttggagtic tgggggaggc ttggtacagc CtggggggtC cctgagactic 6 O t cct gtgcag cct ctdgatt cacctittagc cgg tatggta tgagctgggt cc.gc.caggct 12 O cCagggalagg gctggagtg ggt ct cagct attagtggta gtggtgg tag CaCatactac 18O gCagactic.cg taagggc.cg gttcaccatc tccagaga.ca att coaagaa cacgctgitat 24 O

Ctgcaaatga acagoctgag agc.cgaggac acggc.cgitat attactgtgc gaaag.cgcat 3OO aatgcttittg act actgggg cCagggalacc Ctggit caccg tgtc.gaga 348

<210s, SEQ ID NO 24 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic sequence: Linker motif

<4 OOs, SEQUENCE: 24 Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 25 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic sequence: Linker motif

<4 OOs, SEQUENCE: 25 Ser Ser Ser Ser Gly 1. 5

<210s, SEQ ID NO 26 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic sequence: Linker motif

<4 OOs, SEQUENCE: 26 Gly Ser Gly Ser Ala 1. 5

<210s, SEQ ID NO 27 &211s LENGTH: 5 212. TYPE: PRT US 8,580,267 B2 37 38 - Continued <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic sequence: Linker motif

<4 OOs, SEQUENCE: 27 Gly Gly Ser Gly Gly 1. 5

The invention claimed is: 6. A method according to claim 2, wherein the immunocy 1. A method of treating glioblastoma in an individual, tokine comprises IL2 conjugated to an ScFV antibody mol comprising administering a chemotherapeutic agent and an ecule. immunocytokine to the individual, wherein the chemothera 7. A method according to claim3, wherein the immunocy peutic agent is temozolomide and wherein the immunocytok 15 ine comprises interieukin-2 (IL2) conjugated to an antibody tokine comprises IL2 conjugated to an ScFV antibody mol molecule that binds domain A1 of tenasin-C large isoform, ecule. wherein the antibody molecule comprises an antibody-anti 8. A pharmaceutical composition for the treatment of glio gen binding site comprising a VH domain and a VL domain, blastoma comprising effective amounts oftemozolomide and the VH domain comprising: interleukin 2 conjugated to an antibody molecule that binds a VHCDR1 of SEQID NO: 18 or a VHCDR1 of SEQID domain A1 of tenascin-C isoform, wherein the antibody mol NO:5; ecule comprises an antibody-antigenbinding site comprising a VHCDR2 of SEQID NO: 6; a VH domain and a VL domain, the VH domain comprising: and a VHCDR3 of SEQID NO: 7; a VHCDR1 of SEQID NO: 18 or a VHCDR1 of SEQID and the VL domain comprising: 25 NO:5; a VLCDR1 of SEQID NO:8: a VHCDR2 of SEQID NO: 6; a VLCDR2 of SEQID NO:9; and a VLCDR3 of SEQID and a VHCDR3 of SEQID NO: 7; NO:10. and the VL domain comprising: 2. A method according to claim 1 wherein the antibody a VLCDR1 of SEQID NO: 8: molecule comprises an antibody-antigen binding site com 30 a VLCDR2 of SEQID NO:9; and a VLCDR3 of SEQID prising the 4A1-F16 VH domain SEQ ID NO. 17 and the NO:10 in a pharmaceutically acceptable carrier. 4A1-F16 VL domain SEQID NO:4. 9. The pharmaceutical composition of claim 8 wherein the 3. A method according to claim 1, wherein the antibody immunocytokine comprises IL2 conjugated to an Schv anti molecule comprises an antibody-antigen binding site com body molecule. prising the 4A1-F16VH domain SEQID NO:2 and the 4A1 35 10. The method of claim 1, comprising administering the F16 VH domain SEC ID NO:4. immunocytokine Lathe individual by intravenous injection. 4. A method according to claim 1, wherein the immunocy 11. The pharmaceutical composition of claim 8, wherein tokine comprises IL2 conjugated to an Schv antibody mol the composition is formulated for intravenous injection of the ecule. interleukin 2 conjugated to the antibody molecule. 5. A method according to claim 1, wherein treating the 40 12. The pharmaceutical composition of claim 11, wherein glioblastoma comprises retarding growth of the glioblas the composition is formulated for separate administration of toma, reducing size of the glioblastoma or inhibiting the temozolomide and the antibody molecule. regrowth of the glioblastoma in the individual. k k k k k