News Column

Researchers Submit Patent Application, "Il-12 Immunoconjugate", for Approval

July 8, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventor Wulhfard, Sarah (Baden, CH), filed on July 24, 2012, was made available online on June 26, 2014 (see also Philogen S.p.A).

The patent's assignee is Philogen S.p.A.

News editors obtained the following quote from the background information supplied by the inventors: "Cytokines are key mediators of innate and adaptive immunity. Many cytokines have been used for therapeutic purposes in patients with advanced cancer, but their administration is typically associated with severe toxicity, hampering dose escalation to therapeutically active regimens and their development as anticancer drugs. To overcome these problems, the use of Immunocytokines' (i.e. cytokines fused to antibodies or antibody fragments) has been proposed, with the aim to concentrate the immune-system stimulating activity at the site of disease while sparing normal tissues.sup.1-5.

"The heterodimeric cytokine interleukin-12 (IL-12) is a key mediator of innate and cellular immunity with potent antitumour and antimetastatic activity.sup.6-8. It consists of a p35 and a p40 subunit covalently linked by a disulphide bridge.

"Secretion of the isolated p35 subunit has never been detected; in contrast, the cells that produce the biologically active IL-12 heterodimer secrete p40 in free form in a 10-100-fold excess over the IL-12 heterodimer; depending on the stimulus.sup.9. A biological function of free p40 has never been observed and its physiological significance is still debated. Disulphide linked homodimers of p40 are produced in the mouse; murine p40 homodimers, in contrast to the free p40, have the ability to block IL-12 functions in vitro and in vivo.sup.10. The existence of human p40 homodimers has been demonstrated up to now only in p40 transfected cell lines and the physiological relevance of human p40 homodimers is still debated.sup.11,12.

"IL-12 acts primarily on T and NK cells. The most important functions of IL-12 are the priming of the T helper 1 (Th1) immune responses and IFN-.gamma. secretion by NK cells.sup.13.

"IL-12 generates the Th1 response in three modalities: (i) it promotes the differentiation of naive T cells, during initial encounter with an antigen, into a population of Th1-cells capable of producing large amounts of IFN-.gamma. following activation.sup.14, (ii) it serves as a costimulus required for maximum secretion of IFN-.gamma. by differentiated Th1 cells responding to a specific antigen.sup.15, and (iii) it stimulates the development of IFN-.gamma. producing Th1 cells from populations of resting memory T cells interacting with an antigen to which they have been previously exposed.sup.16.

"IL-12 strongly inhibits neo-vascularisation and IFN-.gamma. seems to play a critical role as a mediator of the anti-angiogenic effects of IL-12.sup.17. Interferon gamma-induced protein 10 (IP-10) is known to be a potent inhibitor of angiogenesis.sup.18,19.

"As with many other cytokines, however, the administration of recombinant human IL-12 is associated with severe toxicity, hampering its development as an anticancer drug. Clinical trials in patients with cancer have revealed promising therapeutic activities, but have also shown that recombinant human IL-12 is extremely toxic to humans, with a maximal tolerated dose of 0.5 .mu.g/kg of body weight.sup.20,21.

"The toxic side effects of toxins, particularly cytokines such as such as IL-12 have made it difficult to administer an effective dose and to reach high concentrations at the site of a tumour.

"Previously, researchers have attempted to overcome these drawbacks by targeting delivery of IL-12 to the tumour environment and in particular to tumour blood vessels (tumour vascular targeting). Tumour vascular targeting aims at disrupting the tumour vasculature, reducing blood flow to deprive the tumour of oxygen and nutrients, causing tumour cell death.

"A targeted delivery of IL-12 to the tumour environment is expected to increase the therapeutic index of the cytokine. The concentration of cytokines, and in particular IL-12, at the level of tumour blood vessels is an attractive therapeutic strategy for a number of reasons.

"First, the tumour neovasculature is more accessible to intravenously administered therapeutic agents than are tumour cells, which helps avoid problems associated with the interstitial hypertension of solid tumours.sup.22.

"Second, angiogenesis is characteristic of most aggressive solid tumours.sup.23. Angiogenesis describes the growth of new blood vessels from existing blood vessels. Tumours can induce angiogenesis through secretion of various growth factors (e.g. Vascular Endothelial Growth Factor). Tumour angiogenesis allows tumours to grow beyond a few millimetres in diameter and is also a prerequisite for tumour metastasis. New blood vessels formed as the result of angiogenesis form the neovasculature of the tumour or the tumour metastases. Targeting IL-12 to the neovasculature should allow the immunotherapy of a variety of different tumour types.

"Third, IL-12 shows an anti-angiogenic activity conferred by its downstream mediator, IP-10.sup.17,24.

"The alternatively spliced extra domains A (ED-A) and B (ED-B) of fibronectin and the A1 domain of tenascin-C represent three of the best-characterised markers of angiogenesis and have been reported to be expressed around the neo-vasculature and in the stroma of virtually all types of aggressive solid tumours. Furthermore, even non-solid cancers, such as leukaemia, may be amenable to treatment by targeting antigens of the neovasculature. WO2011/015333 described treating leukaemia, including acute myeloid leukaemia, by targeting the bone marrow neovasculature.

"Three human monoclonal antibodies specific to these targets have been developed and moved to clinical trials: L19 (specific to ED-B).sup.25, F8 (specific to ED-A).sup.26 and F16 (specific to the A1 domain of tenascin-C).sup.27.

"In addition, several antibody derivatives, based on the modification of L19, F8 or F16 with cytokines or iodine radionuclides, are currently being investigated in Phase I and Phase II clinical trials in patients with cancer and with rheumatoid arthritis.sup.28,29. These biopharmaceuticals are called L19-.sup.124I, L.sup.19-.sup.131I, L19-IL2, L19-TNF, F8-IL10, F16-.sup.124I, F16-.sup.131I, F16-IL2, indicating the modular nature of these derivatives, in which the antibody moiety is used to deliver a payload at the site of disease.

"In WO2008/120101 an I.sup.125-labelled F8 diabody was shown to selectively target I.sup.125 to tumours in mice.

"An F8-IL2 diabody conjugate has been shown to reduce tumour burden in mice (WO2008/120101, WO2010/078945).

"Researchers have attempted to improve targeting of IL-12 to the vasculature using antibody-IL-12 conjugates. Halin et al. sequentially fused the p40 and p35 domains of the heterodimeric IL-12 using a (Ser.sub.4Gly).sub.3 linker and appended at the N-terminal end of the antibody fragment scFv(L19). This immunocytokine showed an increased therapeutic activity of IL12; however, only a modest tumour targeting was observed.sup.30.

"Gafner et a(successfully cloned and tested a heterodimeric fusion protein in which the disulphide-linked p35 and p40 subunits were fused to scFv(L19).sup.31 to produce the fusion protein p40-scFv(L19)/scFv(L19)-p35 (see also WO2006/119897). This heterodimeric fusion protein showed an excellent tumour-targeting performance in biodistribution studies and enhanced therapeutic activity compared to the Halin format."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "The present invention relates to a conjugate comprising a therapeutic or diagnostic agent portion, such as a cytokine, e.g. IL-12, and single chain targeting portion comprising two antigen binding sites, such as a single chain diabody.

"More specifically, the present invention relates to a conjugate comprising linked interleukin 12 (IL-12) subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites.

"One exemplary embodiment of the new format is a single chain protein comprising linked interleukin 12 (IL-12) subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites. The single chain protein may be a single chain fusion protein comprising linked IL-12 subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites.

"The invention is derived from work which compared the tumour-targeting abilities of three antibody-IL-12 immunocytokine formats. Surprisingly, a new format was discovered, which improves tumour targeting ability compared with known formats. The new format also has the further advantages of easier production and purification.

"As shown in the Examples, a single chain fusion protein comprising the p40 and p35 subunits of IL-12 linked to a single chain F8 diabody (p40p35F8F8), demonstrates improved tumour targeting in vivo compared with the scFv-IL-12-scFv immunocytokine format described by Gafner et al WO2006/119897. In contrast, an F8-IL-12 diabody (p40p35F8).times.2, does not show any tumour uptake. These formats are illustrated in FIG. 2.

"Thus, surprisingly, a single chain bivalent immunocytokine displays a better biodistribution profile compared with previously known formats. This is remarkable since the heterodimeric format described by Gafner et al. (WO2006/119897) already showed very good biodistribution, and it was unexpected that a new format could retain or even further improve this targeting profile.

"A conjugate comprising the p40 and p35 subunits of IL-12 linked to a single chain targeting portion comprising two antigen binding sites displays excellent tumour targeting ability.

"In addition, unlike the Gafner et al. heterodimeric format, the immunocytokine of the present invention can be expressed as a single chain polypeptide, for example as a single chain protein comprising linked IL-12 subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites. This format has the advantage of being easier to produce and purify since it consists of one single species. This facilitates production of clinical-grade material. Further, expression of a single chain immunocytokine avoids homodimerization of the p35 subunit, which can be associated with separate expression of the p35 and p40 subunits. Purification of a heterodimeric immunocytokine is facilitated by the use of peptidic tags, but these must be removed for clinical grade material. The immunocytokine of the present invention offers a simpler route to purification and production, while retaining and even improving on the biodistribution profile of previous products.

"These results have significant therapeutic implications for improved targeting of IL-12 to tumours and to other sites of pathological angiogenesis. Conjugates of the invention may be used in the treatment of cancer or treatment of pathological angiogenesis. The wider implications also include a variety of other applications involving targeting of substances in vivo, including diagnostic methods as well as the prevention and treatment of diseases and other pathological conditions.

"In a first aspect, the invention relates to a conjugate comprising linked interleukin 12 (IL-12) subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites.

"The conjugate may be or may comprise a single chain protein. When the conjugate is a single chain protein, the entire protein can be expressed as a single polypeptide or fusion protein. For example, the conjugate may be a single chain protein comprising IL-12 subunits p40 and p35 and a single chain targeting portion comprising two antigen binding sites. Alternatively, the conjugate may comprise a heterodimeric agent (e.g. IL-12) linked to the single chain targeting portion. One subunit of the heterodimeric agent may be linked by a peptide bond or peptide linker to the single chain targeting portion, and thus expressed as a fusion protein, then assembled with the other subunit. For example, the conjugate may comprise heterodimeric IL-12 p40 and p35 subunits, and a single chain targeting portion linked to one of the subunits (e.g. p35), optionally by a peptide linker.

"The linkage may be at the N or C end of the targeting portion. Suitable ways of linking are disclosed herein. Preferably the p35 subunit is linked to the single chain targeting portion.

"Preferably the conjugate contains only one IL-12. Preferably the conjugate contains only one of each p35 and p40 subunit. Preferably the conjugate contains only one targeting portion. Preferably the targeting portion is bivalent, having only two antigen binding sites. The conjugate may be an immunocytokine, wherein one or preferably both of the antigen binding sites is provided by an antibody molecule. Preferably the targeting portion is a single chain diabody.

"Preferably the targeting portion is linked to the C terminus of the p35 subunit. The conjugate may therefore have the format [p40]-[p35]-[targeting portion]. Preferably the p40 subunit has a free N terminus, as this arrangement has been shown to provide improved tumour targeting in vivo.

"Preferably the targeting portion binds an extra-cellular matrix component associated with neoplastic growth and/or angiogenesis. For example, the targeting portion may bind fibronectin (e.g. domain ED-A or ED-B) or tenascin-C (e.g. domain A1).

"The targeting portion may comprise an antigen binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 9-14. The antigen binding site may comprise VH and/or VL domains of antibody F8 set forth in SEQ ID NOs 23 and 24, respectively. The targeting portion may comprise or consist of the F8 single chain diabody amino acid sequence set forth in SEQ ID NO: 31.

"Other antibodies capable of binding to ECM proteins such as fibronectin, for example L19 (specific to ED-B), or F16 (specific to the A1 domain of tenascin-C) are known, and fragments of these antibodies, for example their CDRs, VH and/or VL domains, may be used in targeting portions in the present invention.

"Preferably the conjugate has a molecular weight of less than 150 kDa, more preferably 140, 130, 120 kDa or less. Preferably the conjugate has a molecular weight of between 100 and 150 kDa, preferably between 100 and 120 kDa.

"The conjugate may have least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity, to the amino acid sequence of p40p35F8F8 (SEQ ID NO: 8).

"The conjugate may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 8. The conjugate may be encoded by the nucleotide sequence consisting of or comprising SEQ ID NO: 1.

"The invention also provides isolated nucleic acids encoding conjugates of the invention. Examples of encoding nucleic acid sequences are disclosed herein. An isolated nucleic acid and may be used to express the fusion protein of the invention, for example by expression in a bacterial, yeast, insect or mammalian host cell. A preferred host cell is E. coli. The encoded nucleic acid will generally be provided in the form of a recombinant vector for expression. Host cells in vitro comprising such vectors are part of the invention, as is their use for expressing the fusion proteins, which may subsequently be purified from cell culture and optionally formulated into a pharmaceutical composition.

"A conjugate or immunocytokine of the invention may be provided for example in a pharmaceutical composition, and may be employed for medical use as described herein, either alone or in combination with one or more further therapeutic agents.

"In another aspect the invention relates to a conjugate as herein described for use in a method of treating cancer or inhibiting angiogenesis by targeting IL-12 to the neovasculature in vivo.

"In another aspect the invention relates to a method of treating cancer or inhibiting angiogenesis by targeting IL-12 to the neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate as herein described to the patient.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1a shows a schematic representation of the F8-IL12 single chain diabody fusion protein (p40p35F8F8) (SEQ ID NO: 8), an exemplary embodiment of the present invention. In this embodiment, the IL-12 p40 and p35 subunits were fused using a linker sequence (peptide/amino acid linker) and connected via a linker to two sets of F8 antibody fragments (two VH-VL sets). Each VH and VL within the set is connected by a linker between the variable heavy (F8 VH) and variable light (F8-VL) chains. The linkers within each set are not long enough to allow pairing between the VH and VL domains. Each VL-VL set is connected by a linker which is long enough to allow pairing between the VH and VL domains of the first set with the complementary VH and VL domains of the second set. The amino acid linkers are shown as black rectangles.

"FIG. 1b shows the amino acid sequence of the F8-IL12 fusion protein (p40p35F8F8). The sequence reads in the direction N-C of FIG. 1a. Each of the p40, p35, VH and VL subunits are joined by linker sequences, which are shown in grey. The two F8VH sequences are underlined. Each F8VH sequence is followed by a VL sequence. The VH and VL complementarity determining regions (CDR's), CDR1 VH, CDR2 VH, CDR3 VH and CDR1 VL, CDR2 VL and CDR3 VL are shown in boxes within the VH and VL sequences. The amino acid sequences of the CDR's are also indicated separately (SEQ ID NOs 9-14, respectively). The amino acid sequences of the F8-VH and F8-VL domains (SEQ ID NOs 15 and 16, respectively); the IL-12 p40 and p35 domains (SEQ ID NOs 17 and 18, respectively) and the peptide linkers(SEQ ID NOs 19-22) are also indicated separately below.

"FIG. 2 shows (A) the structure of the scFv-p30:p40-scFv heterodimeric immunocytokine format described by Gafner et al. ('old format'); (B) a single chain p40p35F8F8 fusion protein according to the invention; and (C) F8 diabody (p40p35F8).times.2.

"FIG. 3a shows the cloning strategy of p40p35F8F8

"FIG. 3b shows the cloning strategy of (p40p35F8).times.2

"FIG. 4 shows the results of a Biacore analysis of p40p35F8F8 to calculate the apparent KD (binding affinity constant) of the protein to the antigen ED-A. Each line on the graph represents an independent repeat of the p40p35F8F8 protein. The top line indicates a KD of 0.15 mg/ml, the middle line indicates a KD of 0.062 mg/ml, the bottom line indicates a KD of 0.031 mg/ml.

"FIG. 5 shows gel filtration profiles of the IL-12 single chain diabody format and the IL-12 diabody format compared with the old format. A) shows a preparative and analytical profile of the F8hIL12 format (old format). B) and C) show preparative and analytical profiles of the two new formats. B) shows the p40p35F8F8 single chain diabody format, C) shows the (p40p35F8).times.2 diabody format.

"FIG. 6 shows SDS page in reducing and non-reducing conditions for the p40p35F8F8 protein (lanes 1 and 2) (p40p35F8).times.2 protein (lanes 3 and 4). Lanes 1 and 3 show the protein under non reducing conditions, lanes 2 and 4 shows the protein under reducing conditions. The calculated molecular mass of p40p35F8F8 is 110 kDa, the calculated molecular mass of the dimeric (p40p35F8).times.2 is 170 kD.

"FIG. 7 shows a comparison of the in vivo targeting performance of the old heterodimeric format (scFv-IL-12-scFv), the new p40p35F8F8 format and the new (p40p35F8).times.2 format in a mouse tumour model. The results are presented in the order: old format (black bar), p40p35F8F8 (light grey), (p40p35F8).times.2 (dark grey)."

For additional information on this patent application, see: Wulhfard, Sarah. Il-12 Immunoconjugate. Filed July 24, 2012 and posted June 26, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3689&p=74&f=G&l=50&d=PG01&S1=20140619.PD.&OS=PD/20140619&RS=PD/20140619

Keywords for this news article include: Antibodies, Cytokines, Th1 Cells, Immunology, Amino Acids, Fibronectins, Blood Proteins, Immunoproteins, Interleukin-12, Nanotechnology, Philogen S.p.A, Immunoglobulins, Serum Globulins, Immunoconjugates, Emerging Technologies, Membrane Glycoproteins, Helper-Inducer T-Lymphocytes, Extracellular Matrix Proteins.

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Source: Life Science Weekly


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