WO2020070150A1 - Il2 immunoconjugates - Google Patents

Abstract

The present application relates to a conjugate for targeting an agent, such as a therapeutic or diagnostic agent, to tissues in vivo. In particular, it relates to conjugates for targeting the extracellular matrix (ECM) of tissues, particularly tumour neovasculature, and to therapeutic use of such conjugates in the treatment of a disease/disorder, such as cancer. In particular the application relates to immunocytokines for targeting interleukin-2 (IL2) to ECM components associated with neoplastic growth and/or angiogenesis.

Description

IL2 IMMUNOCONJUGATES

Related Applications

The present case is related to GB1816091.1 filed on 2 October 2018 (02.10.2018) and EP18209671.9 filed on 30 November 2018 (30.11.2018), the contents of both of which are hereby incorporated by reference in their entirety.

Field

The present invention relates to a conjugate for targeting an agent, such as a therapeutic or diagnostic agent, to tissues in vivo. In particular, it relates to conjugates for targeting the extracellular matrix (ECM) of tissues, particularly tumour neovasculature, and to therapeutic use of such conjugates in the treatment of a disease/disorder, such as cancer. In particular the invention relates to immunocytokines for targeting interleukin-2 (IL2) to ECM

components associated with neoplastic growth and/or angiogenesis.

Background

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 (Neri & Bicknell, 2005). However, genetically fusing a cytokine to an antibody or to an antibody fragment creating an“immunocytokine”, does not always result in an immunocytokine that retains the ability to target the tumor of the antibody. For example, in certain lnterleukin-7 fusions (Pasche et al. (201 1 ) J Biotechnology, 154, 84- 92) the tumor targeting was completely abrogated, while in certain GM-CSF fusions (Kaspar et al. (2007) Cancer Res, 67, 4940-4948) the tumor targeting ability was found to be dose dependent.

IL2 is a four a helix bundle cytokine produced by T helper 1 cells and plays an essential role in the activation phases of both specific and natural immune responses. IL2 promotes proliferation and differentiation of activated T and B lymphocytes and of natural killer (NK) cells and induces cytotoxic T cell (CTL) activity and NK/lymphokine-activated killer (LAK) cell antitumor cytotoxicity. IL2 has been approved for the treatment of several human cancers. Administration of recombinant IL2 (rlL2) alone or in combination with adoptively transferred lymphoid cells has been shown to result in the regression of established tumors in both animal models and patients. However, the in vivo therapeutic efficacy of IL2 is limited by its rapid clearance and, at high doses severe toxicity mainly related to a vascular leak syndrome.

To overcome the drawbacks associated with IL2 therapy, delivery of IL2 to the tumor site by means of an antibody directed against tumor-associated marker to increase local concentrations of IL2 at the tumour site, as well as reduce toxicities associated with systemic administration of IL2 has been proposed. In particular, the concentration of cytokines at the level of tumour blood vessels is an attractive therapeutic strategy as the tumour

neovasculature is more accessible to intravenously administered therapeutic agents than tumour cells, which helps avoid problems associated with the interstitial hypertension of solid tumours. In addition, angiogenesis is characteristic of most aggressive solid tumours.

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-2 to the neovasculature should allow the immunotherapy of a variety of different tumour types.

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. WO201 1/015333 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; Pini et al., 1998; W01999/058570), F8 (specific to ED-A; Villa et al., 2008; W02008/120101 ) and F16 (specific to the A1 domain of tenascin-C; Brack et al., 2006; W02006/050834). In addition, immunocytokines based on L19, F8 or F16 are currently being investigated in Phase I, Phase II and Phase III clinical trials in patients with cancer and chronic inflammatory disease such as rheumatoid arthritis and endometriosis (Sauer et al., 2009; Johannsen et al., 2010). These immunocytokines include several immunocytokines comprising IL2. L19-IL2 (W02001/062298) has been tested in a variety of therapeutic regimens and combinations for treatment of different types of cancer (W02007/1 15837, W02009/089858, WO2013/010749, WO2013/045125, WO2018/1 15377, WO2018/154517) with good results.

An F16-IL2 diabody conjugate is also being evaluated in clinical trials.

An F8-IL2 diabody conjugate has been shown to reduce tumour burden in mice

(W02008/120101 and WO2010/078945).

The immunocytokine format, as well as the format of the antibody fragment portion of the antibody, has been shown to have an impact on tumour targeting efficacy of the

immunocytokine.

For example, in W02006/119897 the L19 antibody was conjugates to Interleukin-12, a heterodimeric cytokine formed by the p35 and p40 subunits, in three different molecular formats schematically shown in Figure 3:

(i) two scFvs conjugated to either the p35 subunit or the p40 subunits held together by a disulphide bond (“dimeric format”);

(ii) a SIP antibody conjugated to two IL12 molecules (“SIP format”);

(iii) an scFv conjugated at its N-terminus to one IL12 molecule - a format previously disclosed in W02001/062298 - (“scFv format”).

The results of W02006/119897 showed that the“dimeric format” had a superior tumor targeting ability as compared to the“SIP format” and“scFv format”.

In W02013/014149 the current applicant compared the tumor targeting ability of the“dimeric format” disclosed in W02006/119897, with two new IL12 conjugates schematically shown in Figure 4:

(i) a single-chain diabody conjugated to one IL12 molecule at its N-terminus (“scDb N-terminus”)

(ii) a diabody conjugated to two IL12 molecules at its N-terminus and at its-C- terminus“format X 2”.

The results of W02013/014149 showed that the“scDb N-terminus” conjugate had superior tumor targeting ability as compared to the“dimeric format” and“format X 2”. Furthermore, in WO2018/069467 the current applicant compared the tumor targeting ability of five different molecular formats in which different antibody fragments were conjugated to lnterleukin-4 (IL4) a compact globular protein with many biological roles.

As schematically shown in Figure 5, the following conjugate formats were compared in WO2018/069467:

(i) a scDb with IL4 conjugated at its C-terminus (“scDb C-terminus”)

(ii) a scDb with IL4 conjugated at its N-terminus (“scDb N-terminus”)

(iii) a diabody with IL4 conjugated at its C-terminus (“Diabody C-terminus”)

(iv) a diabody with IL4 conjugated at its N-terminus (“Diabody N-terminus”)

(v) IL4 conjugated at its C-terminus and at its N-terminus to two scFv’s (“Crab”)

The results of WO2018/069467 showed that the“Crab” format had a superior tumor targeting ability as compared to the other four formats tested.

Summary of the invention

The present invention relates to a conjugate comprising IL2 and a single-chain diabody. More specifically, the present invention relates to a conjugate comprising IL2 and a single- chain diabody, wherein the IL2 is linked to the C-terminus of the single-chain diabody.

The invention is derived from work which compared the tumour-targeting properties of nine antibody-IL2 immunocytokines in six different formats. The formats tested are illustrated in Figure 1 , namely:

(A) a diabody comprising one of four different VH-VL domain linker sequences (“diabody”);

(B) a single-chain variable fragment (scFv) with a 12 amino acid linker between VH and VL in which L19 scFv forms a dimer (scFv₂) as disclosed in W02001/062298, Viti et al. (1999) and Borsi et al. (2002);

(C) a single-chain diabody (scDb) with IL2 conjugated at both its C-terminus and at its N-teminus (“scDb X 2”);

(D) IL2 conjugated at its C-terminus and at its N-terminus to two scFv’s (“Crab”);

(E) a scDb with IL2 conjugated to its C-terminus (“scDb C-terminus”); and

(F) a scDb with IL2 conjugated to its N-terminus (“scDb N-terminus”). The single-chain diabody (scDb) comprising IL2 conjugated to the C-terminus of the single- chain diabody was surprisingly shown to have superior tumour targeting properties compared with all of the other immunocytokine formats tested. Specifically, 24 hours after injection into tumour-bearing mice,“scDb C-terminus” conjugates comprising different scDbs reached a percentage injected dose/gram of tissue (% I D/g ) of almost 8 and about 8.5 in the tumour tissue, whereas none of the other immunocytokines tested reached 6% I D/g at the tumor site (Figures 2, 6 and 7). These results demonstrate that the“scDb C-terminus” conjugate performed better than all of the other immunocytokine formats tested, including the“Crab” format, which is surprising in light of the teachings of WO2018/069467 where the “Crab” format out-performed all of the other formats tested, including the“scDb C-terminus” format (Figure 5). When N-terminal and C-terminal fusion proteins comprising IL4 and a single-chain F8 diabody were compared in WO2018/069467, the N-terminal fusion protein outperformed the C-terminal fusion, i.e. the opposite of the results obtained here, making the present results even more unexpected (Table 1 of WO2018/069467). The conjugates tested in WO2018/069467 comprised interleukin-4 (IL4). This makes the present results even more surprising, as IL4 and IL2 are very similar in structure and molecular weight (14.9 and 15.5 kDa, respectively) and both belong to the hematopoietin family of cytokines.

It was further surprising that the“scDb C-terminus” format outperformed the“scDb X 2” format in tumour targeting, as this format also comprises a single-chain diabody but comprises two IL2 moieties, conjugated to the N- and C-terminus of the specific binding member, respectively.

A conjugate comprising a single-chain diabody and one IL2 linked e.g. to the C-terminus of the single-chain diabody therefore displays excellent tumour targeting ability.

In addition, unlike heterodimeric formats, the immunocytokines of the present invention can be expressed as a single chain polypeptide, for example as a single chain fusion protein. This format has the advantage of being easier to produce and purify since it consists of a single species and is expected to facilitate production of clinical-grade material.

The results obtained with the conjugate of the invention have significant therapeutic implications for improved targeting of IL2 to tumours. Conjugates of the invention may thus be used in the treatment of cancer. In a first aspect, the invention therefore relates to a conjugate comprising IL2 and a single- chain diabody. The IL2 is preferably linked to the C-terminus of the single-chain diabody by a peptide linker.

The conjugate preferably comprises only one IL2. Thus, where the IL2 is linked to the C- terminus of the single-chain diabody, the N-terminus of the single-chain diabody is preferably free. Preferably, the conjugate contains only one single-chain diabody.

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

Preferably, the single-chain diabody comprises an antigen binding site having the

complementarity determining regions (CDRs) of antibody L19 set forth in SEQ ID NOs 4-9. The antigen binding site may comprise VH and/or VL domains of antibody L19 set forth in SEQ ID NOs 2 and 3, respectively. The single-chain diabody preferably comprises the L19 diabody amino acid sequence set forth in SEQ ID NO: 10. More preferably the single-chain diabody comprises or consist of the L19 single-chain diabody amino acid sequence set forth in SEQ ID NO: 1 1.

Alternatively, the single-chain diabody may comprise an antigen binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 56-61. The antigen binding site may comprise VH and/or VL domains of antibody F8 set forth in SEQ ID NOs 54 and 55, respectively. The single-chain diabody preferably comprises the F8 diabody amino acid sequence set forth in SEQ ID NO: 72. More preferably the single-chain diabody comprises or consist of the F8 single-chain diabody amino acid sequence set forth in SEQ ID NO: 73 or 62, but most preferably SEQ ID NO: 73.

Other antibodies capable of binding to ECM proteins, for example 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 single-chain diabodies forming part of the conjugates of the invention.

Preferably, the conjugate has at 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 L19-IL2 scDb C-terminal conjugate set out in SEQ ID NO: 12. The conjugate preferably comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12.

In an alternative embodiment, the conjugate has at 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 F8-IL2 scDb C-terminal conjugate set out in SEQ ID NO: 74. The conjugate preferably comprises or consists of the amino acid sequence of F8-IL2 scDb C-terminal conjugate set forth in SEQ ID NO: 74.

The invention also provides isolated nucleic acids encoding conjugates of the invention. An isolated nucleic acid may be used to express the conjugate of the invention, for example by expression in a bacterial, yeast, insect or mammalian host cell. 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 by targeting IL2 to the neovasculature in vivo.

In another aspect the invention relates to a method of treating cancer by targeting IL2 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

Figure 1 shows the structure of the IL2 conjugates tested. A: Diabody comprising the VH and VL domains of antibody L19 linked by a 5 amino acid linker sequence (GSSGG, GGSGG, GSADG, or GSAKG) wherein the IL2 is linked to the VL domains of diabody via a 15 amino acid linker. B: scFv₂ wherein IL2 is fused at its N-terminus via a linker, to the C- terminus of the VL domain of a single-chain variable fragment (scFv) molecule comprising the VH and VL domains of L19. Dimerisation of the L19 scFv results in the formation of the scFv L19-IL2 homodimer. C: a single-chain diabody molecule comprising the VH and VL domains of L19 wherein IL2 is linked to the N- and C-terminus of the diabody molecule (scDb x2). D: IL2 linked at its N- and C-terminus to scFv molecules comprising the VH and VL domains of antibody L19 (Crab). E: Single-chain diabody comprising the VH and VL domains of antibody L19 wherein IL2 is linked to the C-terminus of the diabody (scDb C- terminus). F: Single-chain diabody comprising the VH and VL domains of antibody L19 wherein IL2 is linked to the N-terminus of the diabody (scDb N-terminus).

Figure 2 shows the results of comparative biodistribution analysis of different

immunocytokine formats comprising L19 and IL2 in a F9 teratocarcinoma mouse tumour model (n = 3 per group). The percentage of the injected dose (ID) of the immunocytokine labeled with ¹²⁵l per gram of tissue (%l D/g) in tumor, blood and organs 24 Hours after administration is shown. A: Diabody GSSGG. B: Diabody GGSGG. C: Diabody GSADG. D: Diabody GSAKG. E: scFv2. F: scDb X2. G: Crab. H: scDb C-terminus. All of the

immunocytokine formats tested showed a preferential uptake in the tumor and favourable tumor-to-organ profile, with the highest tumor uptake seen with the scDb C-terminus format (% I D/g of about 7.7).

Figure 3 shows the different immunocytokine formats tested for a conjugate comprising IL12 in W02006/1 19897. A: scFv format. B: SIP format. C: dimeric format.

Figure 4 shows the different immunocytokine formats tested for a conjugate comprising IL12 in W02013/014149. A: dimeric format. B: format x 2. C: scDb N-terminus.

Figure 5 shows the different immunocytokine formats tested for a conjugate comprising IL4 in WO2018/069467. A: scDb N-terminus. B: scDb C-terminus. C: Diabody N-terminus. D: Diabody C-terminus. E: Crab.

Figure 6 shows the results of comparative biodistribution analysis of different

immunocytokine formats comprising L19 and IL2 in a F9 teratocarcinoma mouse tumour model (n = 5 per group): scDb C-terminus, scDb N-terminus and scFv2. The percentage of the injected dose (ID) of the immunocytokine labeled with ¹²⁵l per gram of tissue (% I D/g ) in tumor, blood and organs 24 hours after administration is shown. All of the immunocytokine formats tested showed a preferential uptake in the tumor and favourable tumor-to-organ profile, with the highest tumor uptake seen with the scDb C-terminus format (%ID/g of about Figure 7 shows the results of comparative biodistribution analysis of different immunocytokine formats comprising F8 and IL2 in a F9 teratocarcinoma mouse tumour model (n = 4 per group): scDb C-terminus (A) and scDb N-terminus (B). The percentage of injected dose (ID) of the immunocytokine labeled with ¹²⁵l per gram of tissue (%l D/g) in tumor, blood and organs 24 hours after administration is shown. The two immunocytokine formats tested showed a preferential uptake in the tumor, and favourable tumor-to-organ profile, with the highest tumor uptake seen with the scDb C-terminus format (%ID/g of about 8.5).

Detailed Description

Conjugate

Conjugates of the invention comprise IL2, and single-chain diabody.

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. For example, the conjugate may be a single-chain protein comprising IL2 and a single-chain diabody. The single-chain protein may be a fusion protein, for example a single-chain fusion protein comprising IL2 and a single-chain diabody. By“single-chain fusion protein” is meant a polypeptide that is a translation product resulting from the fusion of two or more genes or nucleic acid coding sequences into one open reading frame (ORF). The fused expression products of the two genes in the ORF may be conjugated by a peptide linker encoded in- frame. Suitable peptide linkers are described herein.

The conjugate preferably comprises only one IL2. The IL2 is linked to the C-terminus of the single-chain. The linkage may be direct or may be indirect, for example via a peptide linker. Suitable linkers and ways of linking are disclosed herein. Where the IL2 is conjugated to the C-terminus of the single-chain diabody, the N-terminus of the single-chain diabody is preferably free.“Free” in this context refers to the N-terminus not being linked or otherwise conjugated to another moiety, such as IL2.

Single-chain diabody

Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen- binding site: antigen-binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804; Holliger and Winter, 1997; Holliger ef a/., 1993).

In a diabody a heavy chain variable domain (VH) is connected to a light chain variable domain (VL) on the same polypeptide chain. The VH and VL domains are connected by a peptide linker that is too short to allow pairing between the two domains (generally around 5 amino acids). This forces paring with the complementary VH and VL domains of another chain. An example of this format is shown in Figure 1A. The diabody-based IL2 conjugates tested showed lower tumour targeting that the single-chain diabody-based IL2 conjugates. The conjugate of the invention therefore preferably comprises a single-chain diabody. In a single-chain diabody two sets of VH and VL domains are connected together in sequence on the same polypeptide chain. For example, the two sets of VH and VL domains may be assembled in a single-chain sequence as follows:

(VH-VL)--(VH-VL), where the brackets indicate a set.

In the single-chain diabody format each of the VH and VL domains within a set is connected by a short or‘non-flexible’ peptide linker. This type of peptide linker sequence is not long enough to allow pairing of the VH and VL domains within the set. Generally a short or‘non flexible’ peptide linker is around 5 amino acids.

The two sets of VH and VL domains are connected as a single-chain by a long or‘flexible’ peptide linker. This type of peptide linker sequence is long enough to allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set. Generally a long or‘flexible’ linker is 15 to 20 amino acids.

Single-chain diabodies have been previously generated (Konterman & Muller, 1999).

A single-chain diabody is bivalent i.e. has two antigen-binding sites, each comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). An“antigen-binding site” describes the part of the single-chain diabody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, the single-chain diabody may only bind to a particular part of the antigen, which part is termed an epitope.

The antigen-binding sites of the single-chain diabody may be identical or different but preferably are identical. Each of the antigen-binding sites in the single-chain diabody may bind the same antigen or epitope. This can be achieved by providing two identical antigen- binding sites such as two identical VH-VL domain pairs, or by providing two different antigen- binding sites, for example comprising different VH and VL domains, which nevertheless both bind the same antigen or epitope. Alternatively, the single-chain diabody may be bispecific. By‘bispecific” we mean that each of the antigen-binding sites binds a different antigen. Optionally, two antigen-binding sites may bind two different antigens mentioned herein, e.g. two different antigens of the extracellular matrix, or two different domains of a particular antigen (e.g. fibronectin or tenascin-C).

The single-chain diabody may bind an extra-cellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis. The binding may be specific. The term "specific" may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the single-chain diabody carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

Preferably the single-chain diabody binds fibronectin. Fibronectin is an antigen subject to alternative splicing, and a number of alternative isoforms of fibronectin are known, including alternatively spliced isoforms A-FN and B-FN, comprising domains ED-A or ED-B

respectively, which are known markers of angiogenesis. The single-chain diabody may selectively bind to isoforms of fibronectin selectively expressed in the neovasculature. An antigen-binding site in the single-chain diabody may bind fibronectin isoform A-FN, e.g. it may bind domain ED-A (extra domain A). In a preferred embodiment, an antigen-binding site in the single-chain diabody binds fibronectin isoform B-FN, e.g. it may bind ED-B (extra domain B).

Fibronectin Extra Domain-A (EDA or ED-A) is also known as ED, extra type III repeat A (EIIIA) or EDI. The sequence of human ED-A has been published by Kornblihtt et al. (1984), Nucleic Acids Res. 12, 5853-5868 and Paolella et al. (1988), Nucleic Acids Res. 16, 3545- 3557. The sequence of human ED-A is also available on the SwissProt database as amino acids 1631-1720 (Fibronectin type-ill 12; extra domain 2) of the amino acid sequence deposited under accession number P02751. The sequence of mouse ED-A is available on the SwissProt database as amino acids 1721-1810 (Fibronectin type-ill 13; extra domain 2) of the amino acid sequence deposited under accession number P1 1276. The ED-A isoform of fibronectin (A-FN) contains the Extra Domain-A (ED-A). The sequence of the human A-FN can be deduced from the corresponding human fibronectin precursor sequence which is available on the SwissProt database under accession number P02751. The sequence of the mouse A-FN can be deduced from the corresponding mouse fibronectin precursor sequence which is available on the SwissProt database under accession number P1 1276. The A-FN may be the human ED-A isoform of fibronectin. The ED-A may be the Extra Domain-A of human fibronectin.

ED-A is a 90 amino acid sequence which is inserted into fibronectin (FN) by alternative splicing and is located between domain 1 1 and 12 of FN (Borsi et al., 1987). ED-A is mainly absent in the plasma form of FN but is abundant during embryogenesis, tissue remodelling, fibrosis, cardiac transplantation and solid tumour growth.

Fibronectin isoform B-FN is one of the best known markers angiogenesis (W01997/045544). An extra domain“ED-B” of 91 amino acids is found in the B-FN isoform and is identical in mouse, rat, rabbit, dog and man. B-FN accumulates around neovascular structures in aggressive tumours and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues.

The single-chain diabody may bind tenascin-C. Tenascin-C is a large hexameric

glycoprotein of the extracellular matrix which modulates cellular adhesion. It is involved in processes such as cell proliferation and cell migration and is associated with changes in tissue architecture as occurring during morphogenesis and embryogenesis as well as under tumourigenesis or angiogenesis. Several isoforms of tenascin-C can be generated as a result of alternative splicing which may lead to the inclusion of (multiple) domains in the central part of this protein, ranging from domain A1 to domain D (Borsi L et al Int J Cancer 1992; 52:688-692, Carnemolla B et al. Eur J Biochem 1992; 205:561-567,

W02006/050834). An antigen-binding site in the single-chain diabody may bind tenascin-C domain A1.

The single-chain diabody may comprise an antigen-binding site having the complementarity determining regions (CDRs), or the VH and/or VL domains of an antibody capable of specifically binding to an antigen of interest, for example, one or more CDRs or VH and/or VL domains of an antibody capable of specifically binding to an antigen of the ECM. The antigen may be an antigen preferentially expressed by cells of a tumour or tumour neovasculature or associated with the ECM. Such antigens include fibronectin and tenascin C, as described above.

Thus, the single-chain diabody may comprise an antigen-binding site of the antibody F8, the antibody L19 or the antibody F16, which have all been shown to bind specifically to ECM antigens. The single-chain diabody may comprise an antigen-binding site having one, two, three, four, five or six CDR’s, or the VH and/or VL domains of antibody F8, L19 or F16.

L19 is a human monoclonal scFv specific alternatively spliced ED-B domain of fibronectin and has been previously described (W01999/058570; W02006/119897). F8 is a human monoclonal scFv antibody fragment specific to the alternatively spliced ED-A domain of fibronectin and has been previously described (W02008/120101 ; Villa ef a/., 2008). F16 is a human monoclonal scFv specific to the A1 domain of Tenascin C and has been previously described (W02006/050834).

An antigen-binding site may comprise one, two, three, four, five or six CDRs of antibody L19. Amino acid sequences of the CDRs of L19 are:

SEQ ID NO:4 (CDR1 VH);

SEQ ID NO:5 (CDR2 VH);

SEQ ID NO:6 (CDR3 VH);

SEQ ID NO:7 (CDR1 VL);

SEQ ID NO:8 (CDR2 VL), and/or

SEQ ID NO:9 (CDR3 VL).

SEQ ID NOs 4-6 are the amino acid sequences of the VH CDR regions (1-3, respectively) of the human monoclonal antibody L19. SEQ ID NOs 7-9 are the amino acid of the VL CDR regions (1-3, respectively) of the human monoclonal antibody L19. The amino acid sequence of the VH and VL domains of antibody L19 correspond to SEQ ID NOs 2 and 3, respectively.

An antigen-binding site may comprise one, two, three, four, five or six CDRs of antibody F8. Amino acid sequences of the CDRs of F8 are:

SEQ ID NO: 56 (CDR1 VH);

SEQ ID NO: 57 (CDR2 VH);

SEQ ID NO: 58 (CDR3 VH);

SEQ ID NO: 59 (CDR1 VL);

SEQ ID NO: 60 (CDR2 VL), and/or

SEQ ID NO: 61 (CDR3 VL). SEQ ID NOs 56-58 are the amino acid sequences of the VH CDR regions (1-3, respectively) of the human monoclonal antibody F8. SEQ ID NOs 59-61 are the amino acid of the VL CDR regions (1-3, respectively) of the human monoclonal antibody F8. The amino acid sequence of the VH and VL domains of antibody F8 correspond to SEQ ID NO: 54 and SEQ ID NO:

55, respectively.

An antigen-binding site may comprise one, two, three, four, five or six CDRs of antibody F16. Amino acid sequences of the CDRs of F16 are:

SEQ ID NO: 65 (CDR1 VH);

SEQ ID NO: 66 (CDR2 VH);

SEQ ID NO: 67 (CDR3 VH);

SEQ ID NO: 68 (CDR1 VL);

SEQ ID NO: 69 (CDR2 VL), and/or

SEQ ID NO: 70 (CDR3 VL).

SEQ ID NOs 65-67 are the amino acid sequences of the VH CDR regions (1-3, respectively) of the human monoclonal antibody F16. SEQ ID NOs 68-70 are the amino acid of the VL CDR regions (1-3, respectively) of the human monoclonal antibody F16. The amino acid sequence of the VH and VL domains of antibody F16 correspond to SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

The conjugate of the invention preferably comprises IL2 joined to a single-chain diabody, for example a single-chain diabody comprising the VH and VL domains of antibody L19, F8, or F16, preferably antibody L19.

A single-chain diabody according to the invention may have a VH domain having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the L19 VH domain amino acid sequence SEQ ID NO: 2, the F8 VH domain amino acid sequence SEQ ID NO: 54, or the F16 VH domain amino acid sequence SEQ ID NO: 63.

A single-chain diabody according to the invention may have a VL domain having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the L19 VL domain amino acid sequence SEQ ID NO: 3, the F8 amino acid sequence SEQ ID NO: 55 or the F16 amino acid sequence SEQ ID NO: 64. Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981 ) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Variants of these VH and VL domains and CDRs may also be employed in antibody molecules for use in conjugates as described herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening.

Particular variants for use as described herein may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), maybe less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, 4, 3, 2 or 1.

Alterations may be made in one or more framework regions and/or one or more CDRs. In particular, alterations may be made in VH CDR1 , VH, CDR2 and/or VH CDR3.

The single-chain diabody may comprise the sequence of the L19 diabody set forth in SEQ ID NO: 10, or sequence which has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

The amino acid sequence of the L19 single-chain diabody is found in SEQ ID NO: 11. The L19 single-chain diabody may comprise or consist the amino acid sequence of SEQ ID NO:

1 1. A single-chain diabody for use in the invention may have at least 70%, 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 the L19 single-chain diabody set forth in SEQ ID NO: 1 1 . Alternatively, the single-chain diabody may comprise the sequence of the F8 diabody set forth in SEQ ID NO: 72 or a sequence which has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 72.

The amino acid sequence of the F8 single-chain diabody may be as set forth in SEQ ID NO: 73 or 62, but preferably is as set out in SEQ ID NO: 73. The F8 single-chain diabody may therefore comprise or consist the amino acid sequence of SEQ ID NO: 73 or 62, but preferably comprise or consist the amino acid sequence of SEQ ID NO: 73. A single-chain diabody for use in the invention may have at least 70%, 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 the F8 single-chain diabody set forth in SEQ ID NO: 73 or 62, but preferably the sequence of the F8 single-chain diabody set forth in SEQ ID NO: 73.

As a further alternative, a single-chain diabody for use in the invention may have at least 70%, 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 the F16 single-chain diabody set forth in SEQ ID NO: 71.

Linkers

The single-chain diabody and IL2 may be connected to each other directly, for example through any suitable chemical bond or through a linker, for example a peptide linker, but preferably are connected by a peptide linker. The peptide linker may be a short (2-30, preferably 10-20) residue stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. The linker may be about 15 amino acids in length. An example of a suitable linker is (G₄S)₃ (SEQ ID NO:

26).

The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. For example, the single- chain diabody and IL2 may be covalently linked. For example, by peptide bonds (amide bonds). Thus, the single-chain diabody and IL2 may be produced (secreted) as a single- chain polypeptide. The single-chain diabody and IL2 may also be connected directly, for example through any suitable chemical bond, or through a linker, for example a peptide linker. Examples of individual components which may be linked within the single-chain diabody are VH and VL sequences. For example, the first and second set of VH and VL sequences of the single-chain diabody are preferably connected by a flexible peptide linker. By“flexible” is meant a linker sequence that is long enough to allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set. Generally, a long or‘flexible’ linker is at least 10 amino acids, preferably 10 to 20 amino acid. Single-chain diabodies have been previously generated and described by Kontermann, R. E., and Muller, R. (1999), J.

Immunol. Methods 226: 179-188. An example of such a linker is GSLDGAGGSAGADGG (SEQ ID NO: 25). Preferably the VH-VL sequences within each set are connected by a‘non- flexible’ linker. By a‘non-flexible’ linker is meant a peptide linker sequence that is not long enough to allow pairing of the VH and VL domains. Examples of suitable short linker sequences are GSSGG (SEQ ID NO: 21 ) and GGSGG (SEQ ID NO: 22).

Interleukin-2 (IL2)

The conjugate of the invention comprises IL2. The IL2 may be derived from any animal, e.g. human, rodent (e.g. rat, mouse), horse, cow, pig, sheep, dog, etc. Human IL2 is preferred in conjugates for administration to humans. The amino acid sequence of human IL2 is set out in SEQ ID NO: 1. The conjugate of the invention preferably comprises a single IL2 polypeptide.

IL2 in conjugates of the invention retains a biological activity of IL2, e.g. an ability to promote proliferation and differentiation of activated T and B lymphocytes and natural killer (NK) cells, induce cytotoxic T cell (CTL) activity, and/or NK/lymphokine-activated killer (LAK) cell antitumor cytotoxicity.

The IL2 is conjugated to the C-terminus of the single-chain diabody.

Methods of treatment

A conjugate according to the invention may be used in a method of treatment of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a cancer in a patient (typically a human patient) comprising administering the conjugate to the patient.

Accordingly, such aspects of the invention provide methods of treatment comprising administering a conjugate of the invention, pharmaceutical compositions comprising such a conjugate for the treatment of cancer in a patient, and a method of making a medicament or pharmaceutical composition comprising formulating the conjugate of the present invention with a physiologically acceptable carrier or excipient.

Thus, a conjugate of the invention may be for use in a method of treating cancer by targeting IL2 to the tumour neovasculature in vivo. Also contemplated is a method of treating cancer by targeting IL2 to the neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate of the invention to the patient. Also provided is the use of a conjugate of the invention for the manufacture of a medicament for the treatment of cancer.

Conditions treatable using the conjugate as described herein include cancer, other tumours and neoplastic conditions. Treatment may include prophylactic treatment.

Cancers suitable for treatment as described herein include any type of solid or non-solid cancer or malignant lymphoma and especially malignant melanoma, Merkel-cell carcinoma, renal cell cancer, leukaemia (e.g. acute myeloid leukaemia), non-small cell lung cancer (NSCLC), oligometastatic solid tumors, liver cancer, lymphoma, sarcomas, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, oesophageal cancer, pancreatic cancer, stomach cancer and cerebral cancer. Cancers may be familial or sporadic. Cancers may be metastatic or non-metastatic.

Preferably, the cancer is a cancer selected from the group of malignant melanoma, Merkel- cell carcinoma, renal cell cancer, acute myeloid leukaemia (AML), non-small cell lung cancer (NSCLC), colon carcinoma and oligometastatic solid tumors.

The cancer may express an isoform of fibronectin comprising domain ED-A or ED-B, or alternatively spliced tenascin-C comprising for example domain A1. Preferably the cancer expresses the ED-B or ED-A isoforms of fibronectin.

Expression of the ED-B and of ED-A isoforms of fibronectin has been reported in a number of different cancers.

Pharmaceutical compositions

A further aspect of the invention relates to a pharmaceutical composition comprising at least one conjugate of the invention and optionally a pharmaceutically acceptable excipient. Pharmaceutical compositions of the invention typically comprise a therapeutically effective amount of a conjugate according to the invention and optionally auxiliary substances such as pharmaceutically acceptable excipient(s). Said pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. A carrier or excipient may be a liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art and include, for example, stabilisers, antioxidants, pH- regulating substances, controlled-release excipients. The pharmaceutical preparation of the invention may be adapted, for example, for parenteral use and may be administered to the patient in the form of solutions or the like.

Compositions comprising the conjugate of the invention may be administered to a patient. Administration is preferably in a“therapeutically effective amount", this being sufficient to show benefit to the patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors. Treatments may be repeated at daily, twice-weekly, weekly, or monthly intervals at the discretion of the physician

Conjugates of the invention may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream and/or directly into the site to be treated, e.g. tumour or tumour vasculature. The precise dose and its frequency of administration will depend upon a number of factors, the route of treatment, the size and location of the area to be treated (e.g. tumour).

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or

polyethylene glycol may be included

For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Kits

Another aspect of the invention provides a therapeutic kit for use in the treatment of cancer comprising a conjugate of the invention. The components of a kit are preferably sterile and in sealed vials or other containers. A kit may further comprise instructions for use of the components in a method described herein. The components of the kit may be comprised or packaged in a container, for example a bag, box, jar, tin or blister pack.

Nucleic acids, vectors, host cells and methods of production

Also provided is an isolated nucleic acid molecule encoding a conjugate according to the invention. Nucleic acid molecules may comprise DNA and/or RNA and may be partially or wholly synthetic.

Further provided are constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise such nucleic acids. Suitable vectors can be chosen or

constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids e.g. phagemid, or viral e.g. 'phage, as appropriate. For further details, see, for example, Sambrook & Russell (2001 ) Molecular Cloning: a Laboratory Manual: 3rd edition, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. (1999) 4^th eds., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons.

A recombinant host cell that comprises one or more constructs as described above is also provided. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.

A conjugate according to the present invention may be produced using such a recombinant host cell. The production method may comprise expressing a nucleic acid or construct as described above. Expression may conveniently be achieved by culturing the recombinant host cell under appropriate conditions for production of the conjugate. Following production, the conjugate may be isolated and/or purified using any suitable technique, and then used as appropriate. The conjugate may be formulated into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. The expression of antibodies, including conjugates thereof, in prokaryotic cells is well established in the art. For a review, see for example Pluckthun (1991 ),

Bio/Technology 9: 545-551. A common bacterial host is E.coli.

Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of conjugates for example Chadd et al. (2001 ), Current Opinion in Biotechnology 12: 188-194); Andersen et al. (2002) Current Opinion in Biotechnology 13: 1 17; Larrick & Thomas (2001 ) Current Opinion in Biotechnology 12:41 1-418. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.

A method comprising introducing a nucleic acid or construct disclosed herein into a host cell is also described. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The nucleic acid or construct may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote

recombination with the genome, in accordance with standard techniques.

Further aspects and embodiments of the invention will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification. All documents mentioned in this specification are incorporated herein by reference in their entirety.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term“comprising” replaced by the term“consisting of or “consisting essentially of”, unless the context dictates otherwise.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Examples

Example 1 - Cloning of L19-IL2 immunocytokines

The below examples describe the production and characterisation of nine L19-IL2 immunocytokines which differ in format and/or sequence, and compare these formats with regard to their ability to specifically target tumours in a mouse cancer model, thereby demonstrating the superiority of the single-chain diabody format as claimed herein for in vivo targeting.

7.1 Cloning of four L19 diabody-IL2 conjugates comprising different VH-VL domain linker sequences

1.1.1 Cloning of the L19 diabody-IL2 conjugate with a GSSGG VH-VL domain linker sequence

DNA fragments encoding the L19 diabody comprising different VH-VL domain linker sequences were prepared: GSSGG (composed of two polar serines and neutral glycines), GGSGG (composed of the polar amino acid serine and neutral glycines), GSADG

(negatively charged under physiological conditions of pH 7.4) and GSAKG (positively charged under physiological conditions of pH 7.4). Differently charged linker sequences were tested to determine which linker would exhibit the best performance.

The DNA fragment encoding the L19 diabody comprising the GSSGG VH-VL domain linker sequence was cloned by PCR-amplification of the L19 gene using primers HindlllSIP (SEQ ID NO: 50) and L19Linker (SEQ ID NO: 51 ). The DNA sequence encoding the IL2 was cloned from the IL2 gene using primers LinkerlL2 (SEQ ID NO: 52) and IL2stopNotl (SEQ ID NO: 53). The two DNA fragments (L19 antibody and IL2) were assembled by means of PCR, amplified using primers HindlllSIP and IL2stopNotl, double digested with Hindi ll/Notl and cloned into a pcDNA 3.1 (+) vector. The nucleic acid sequence of the L19 diabody-IL2 polypeptide is set out in SEQ ID NO: 85. The amino acid sequence of the L19 diabody-IL2 polypeptide with the GSSGG VH-VL domain linker is shown is SEQ ID NO: 16. A schematic representation of the L19 diabody-IL2 conjugate is shown in Figure 1A.

1 .1 .2 Cloning of diabody-IL2 conjugates with different VH-VL domain linker sequences

Three further diabody-IL2 conjugates were prepared by inserting linker sequences GGSGG, GSADG and GSAKG between the heavy and light chain variable domains of the L19 diabody- IL2 conjugate prepared in 1.1.1 . The L19 diabody-IL2 conjugates were prepared by means of PCR assembly of a fragment“A” (encoding the L19 heavy chain variable domain), and a fragment“B” (encoding the L19 light chain variable domain and the IL2 payload). Fragments “A” and“B” were amplified from the L19 diabody-IL2 molecule prepared in 1.1 .1 using the primers listed in the Tables 1 and 2 below.

Table 2: Primers used for PCR amplification of“B” fragments of L19 diabody-IL2.

The“A” and“B” fragments were then PCR-assembled, PCR-amplified and double digested with Hindlll/Notl-HF and cloned into the double digested vector pcDNA 3.1 (+).

The resulting plasmids were amplified and used for cell transfection. The amino acid sequences of the L19 diabody-IL2 polypeptides with the GGSGG, GSADG and GSAKG linker sequences are set out in SEQ ID NOs 17 to 19. A schematic representation of the L19 diabody-IL2 conjugates is shown in Figure 1A.

1.2 Cloning of scFv L 19-1 L2 (^,scFv¾)

The scFv L19-IL2 (SCFV2) is an immunocytokine, consisting of human IL2 fused at its N- terminus, via a 17 amino acid linker, to the C-terminus of antibody L19 in scFv format (see Figure 1 B). The production and purification of scFv L19-IL2 was performed as described in W001/062298. Dimerisation via the L19 scFv results in the formation of scFv L19-IL2 homodimers. The amino acid sequence of the scFv L19-IL2 polypeptide is set out in SEQ ID NO: 15. A schematic representation of the scFv L19-IL2 conjugate is shown in Figure 1 B.

7.3 Cloning of the L19 scDb with IL2 conjugated to its C- and N-termini (“scDb X 2”)

The L19 scDb X2 immunocytokine (IL2-L19L19-IL2) coding sequence was generated using the cloned the scDb C-terminus (L19L19-IL2) and the scDb N-terminus (IL2-L19L19) as starting material. The nucleic acid sequences of the scDb C-terminus and the scDb N- terminus are set out in SEQ ID NOS: 86 and 87 respectively.

The vector pcDNA 3.1 (+) containing the sequence Nhel (restriction site)-l L2-L19-Hindll I (restriction site)-L19 was digested by Nhel/Hindl II in order to obtain the IL2-L19 DNA fragment. At the same time, the vector pcDNA 3.1 (+) containing the sequence Nhel (restriction site)-L19- Hindi 11 (restriction site)-L19-IL2 was digested by Nhel/Hindlll, in order to remove the first L19 moiety and replace it with Nhel (restriction site)-l L2-L19-Hindl 11 (restriction site). By doing so, the full length scDb X2 (IL2-L19L19-IL2) was obtained.

The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of above scDb X2 polypeptide is set out in SEQ ID NO: 20. A schematic representation of the L19 scDb X2 conjugate is shown in Figure 1C.

1.4 Cloning of IL2 conjugated at its C-terminus and at its N-terminus to two scFv’s (“Crab")

The“Crab” immunocytokine L19-IL2-L19 coding sequence was generated using L19-IL2 as template. The nucleic acid sequence of the L19-IL2 is set out in SEQ ID NO: 85.

A first DNA fragment was amplified using primers Hindl_ead> (SEQ ID NO: 76) and

DP47G4S2.5< (SEQ ID NO: 47). A second DNA fragment was amplified using primers G4S2.5VL> (SEQ ID NO: 48) and IL2G4S3Bam< (SEQ ID NO: 43). The two intermediate fragments were PCR-assembled, PCR-amplified using primers Hindl_ead> and

IL2G4S3Bam<, double digested with Hindlll/BamHI and cloned into a pcDNA 3.1 (+) vector (resulting in the vector pcDNA 3.1 (+) containing Hindi 11 (restriction site)-L19-IL2-BamHI (restriction site)).

A third DNA fragment was amplified using primers BamG4S3L19> (SEQ ID NO: 44) and DP47G4S2.5<. A fourth DNA fragment was amplified using primers G4S2.5VL> and L19StopNot< (SEQ ID NO: 45). The two intermediate fragments were PCR-assembled, PCR-amplified using primers BamG4S3L19> and L19StopNot<, double digested with BamHI/Notl HF and cloned into the previously generated pcDNA 3.1 (+) vector containing the sequence Hindi 11 (restriction site)-L19-IL2-BamHI (restriction site), resulting in the full length L19-IL2-L19“Crab” molecule. The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of L19-IL2-L19 polypeptide is set out in SEQ ID NO: 14. A schematic representation of the L19“Crab” conjugate is shown in Figure 1D.

1.5 Cloning of the single-chain diabody (scDb) C-terminal fusion protein (L19L19-1 L2)

The scDb C-terminal fusion protein coding sequence was generated using L19-IL2 as template. The nucleic acid sequence of the L19-IL2 is set out in SEQ ID NO: 85.

The L19 gene was PCR amplified using primers LnkDP47> (SEQ ID NO: 39) and L19G4S3< (SEQ ID NO: 42). The IL2 gene was amplified using primers G4S3IL2> (SEQ ID NO: 41 ) and IL2StopNot< (SEQ ID NO: 46). The two intermediate fragments were PCR-assembled, PCR-amplified using primers Hindl_nk> (SEQ ID NO: 40) and IL2StopNot<, double digested with Hindlll/Notl-HF and cloned into a pcDNA 3.1 (+) vector (resulting into a pcDNA 3.1 vector containing the sequence Hindi 11 (restriction site)-L19-IL2-Notl (restriction site)).

A second L19 gene was PCR amplified using primers Nhel_ead> (SEQ ID NO: 35) and L19Hind< (SEQ ID NO: 38), double digested by Nhel/Hindlll and inserted into the previously generated pcDNA 3.1 (1 ) vector containing the sequence Hindi 11 (restriction site)-L19-I L2- Notl (restriction site), resulting into the full length L19L19-IL2.

The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of L19L19-IL2 polypeptide is set out in SEQ ID NO: 12. A schematic

representation of the L19 scDb C-terminal fusion protein is shown in Figure 1E.

1.6 Cloning of the single-chain diabody (scDb) N-terminal fusion protein (IL2-L 19L19)

The scDb N-terminal fusion protein (IL2-L19L19) coding sequence has been generated using L19-IL2 as template. The nucleic acid sequence of the L19-IL2 is set out in SEQ ID NO: 85.

The IL2 gene was amplified using primers LeadlL2> (SEQ ID NO: 49) and IL2G4S3< (SEQ ID NO: 36). The L19 gene was PCR amplified from G4S3L19> (SEQ ID NO: 37) and L19Hind< (SEQ ID NO: 38). The two intermediate fragments were PCR-assembled, PCR- amplified using primers Nhel_ead> (SEQ ID NO: 35) and L19Hind<, double digested with Nhel/Hindlll and cloned into a pcDNA 3.1 (+) vector (resulting into a pcDNA 3.1 (+) vector containing the sequence Nhel (restriction site)-l L2-L19-Hindl 11 (restriction site)).

A second L19 gene was PCR amplified a first time using primers LnkDP47> (SEQ ID NO: 39) and L19StopNot< (SEQ ID NO: 45) and a second time using primers Hindl_nk> (SEQ ID NO: 40) and L19StopNot<. The DNA fragment was subsequently double digested using Hindi ll/Not-HF and inserted into the previously generated pcDNA 3.1 (+) vector containing the sequence Nhel (restriction site)-l L2-L19-Hindl 11 (restriction site), resulting in the full length IL2-L19L19.

The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of the scDb N-terminal fusion protein is set out in SEQ ID NO: 13. A schematic representation of the L19 scDb N-terminal fusion protein is shown in Figure 1 F. Example 2 - Expression and purification of L19-IL2 immunocytokines

2.1 Cell culture and transfection

Transfected CHO-S cells (Chinese Hamster Ovary) were cultured in suspension in

PowerCHO-2CD medium, supplemented with Ultraglutamine-1 , HT-supplement and an antibiotic-antimycotic.

2.2 Expression and purification of L19-1 L2 immunocytokines

The nine L19-IL2 immunocytokines described in Example 1 above were expressed using transient gene expression in CHO-S cells. For 1 ml. of production 4 x 10⁶ CHO-S cells in suspension were centrifuged and resuspended in 1 ml. ProCH04 medium. 0.625 mg of plasmid DNAs followed by 2.5 mg polyethylene imine (PEI; 1 mg/ml_ solution in water at pH 7.0) per million cells were then added to the cells and gently mixed. The transfected cell cultures were incubated in a shaker incubator at 31 °C for 6 days. The L19-IL2

immunocytokines were purified from the cell culture medium by protein A affinity

chromatography and then dialyzed against PBS.

Example 3 - characterization of the L19-IL2 immunocytokines

3.1 SDS-PAGE and size-exclusion chromatography

The purified immunocytokines prepared as described in Example 2 were characterized by SDS-PAGE and size-exclusion chromatography. SDS-PAGE was performed with 10% gels under reducing and non-reducing conditions. Purified clones were analyzed by size- exclusion chromatography on a Superdex 200 increase 10/300 GL column on an AKTA FPLC.

SDS-PAGE characterization confirmed the expected molecular weight of the fusion proteins: around 80 kDa for the clones bearing a modified linker (based on non-covalent homodimeric diabody, Figure 1 A), 66 kDa for the scDb fusion proteins (Figures 1 E and 1 F) and the crab format (Figure 1 D) and 80 kDa for the fusion protein bearing one IL2 at both ends (scDb X 2, Figure 1 C). The proteins exhibited a main peak of the expected elution volume in gel- filtration.

3.2 Affinity measurements

Affinity measurements were performed by surface plasmon resonance using a BIAcore X100 instrument using a fibronectin 7B89 domain coated CM5 chip. Samples were injected as serial-dilutions, in a concentration range from 1 mM to 250nM. Regeneration of the chip was performed using 10 mM HCI. The BIAcore analysis confirmed the ability of the L19 antibody in the nine L19-IL2 immunocytokines to recognize the fibronectin 7B89 domain.

Example 4 - Immunofluorescence and biodistribution experiments

4.1 Tumor implantation

The murine F9 teratocarcinoma tumor cell line was used to generate the syngeneic tumor model. F9 cells were cultured on 0.1 % gelatin-coated tissue culture flasks in DMEM medium supplemented with 10% FCS. F9 tumor cells (12 ^c 10⁶ cells resuspended in 150 mI_ of HBSS buffer) were then implanted subcutaneously in the right flank of 129/SvEv mice (females, six to eight weeks-old).

4.2 Biodistribution studies

The L19-IL2 immunocytokines (100 mg) were radioiodinated with ¹²⁵l and Chloramine T hydrate and purified on a PD10 column. Radiolabeled immunocytokines were injected into the lateral tail vein of immunocompetent (129/Sv) mice bearing subcutaneously implanted F9 murine teratocarcinomas. The injected dose per mouse varied between 12 and 15 mg. Mice were sacrificed 24 hours after injection. Organ samples were weighed and radioactivity was counted using a Packard Cobra gamma counter. The protein uptake in the different organs was calculated and expressed as the percentage of the injected dose per gram of tissue (%l D/g).

4.3 Immunofluorescence experiments

Immunofluorescence was performed on frozen murine F9 teratocarcinoma sections (8 mGh). The tumor sections were fixed using ice-cold acetone (5 min) and blocked with 20% fetal bovine serum in PBS for 45 min. The L19-IL2 immunocytokines were added to the tumour sections at a concentration of 5 mg/mL in a 2% BSA/PBS solution and incubated for 1 h at room temperature. Anti-human interleukin-2 (final dilution 1 : 150) was used as the secondary antibody to detect the L19-IL2 immunocytokines. The secondary antibody was added to the tumour sections in a 2% BSA/PBS solution and incubated at room temperature for 1 h.

Donkey anti-rat Alexa 488 antibody (final dilution 1 :500) was used as the tertiary antibody. Nuclei were counterstained with DAPI. Slides were analyzed with an Axioskop2 mot plus microscope. All of the L19-IL2 immunocytokines showed specific binding to the vasculature compared to the negative control. Example 5 - Comparative biodistribution experiments between the L19 scDb N-terminal fusion protein and L19 scDb C-terminal fusion protein

5.1 Tumor implantation

F9 tumor cells (25 ^c 10⁶ cells resuspended in 200 mI_ of HBSS buffer) were implanted subcutaneously in the right flank of 129/SvEv mice (females, six to eight weeks-old).

5.2 Radiolabeling and tumor targeting

The purified immunocytokines (150 mg) were radioiodinated with ¹²⁵l and Chloramine T hydrate and purified on a PD10 column. Radiolabeled immunocytokines were injected into the lateral tail vein of immunocompetent (129/Sv) mice bearing subcutaneously implanted F9 murine teratocarcinoma. Injected dose per mouse varied between 14 and 17 mg. Mice were sacrificed 24 hours after injection. Organ samples were weighed, and radioactivity was counted using a Packard Cobra gamma counter. The protein uptake in the different organs was calculated and expressed as the percentage of the injected dose per gram of tissue (%l D/g).

Conclusion

Six novel immunocytokine formats comprising the VH and VL domains of the L19 antibody and an IL2 payload were generated. The L19-IL2 immunocytokines were designed to comprise different linker sequences between the VH and VL domains in diabody format (neutral, positively charged and negatively charged) (Example 1.1 ), or to have different immunocytokine formats (e.g. number of IL2 payloads per molecule) (Examples 1.2 to 1.6). The different L19-IL2 immunocytokines showed only minor differences when characterised in vitro.

Surprisingly, in vivo biodistribution studies performed in F9 tumour-bearing mice using radioiodinated immunocytokine preparations showed that one immunocytokine format (scDb C-terminus [L19L19IL2]) had significantly superior tumour targeting properties, as reflected by a higher percentage of the injected dose of the immunocytokine being taken up by the tumour, compared with the other immunocytokine formats tested. Specifically, the scDb C- terminal fusion protein showed an accumulation of about 7.7% I D/g in the tumour and a favourable tumor-to-organ profile, while the other immunocytokines tested only reached values of around 5% ID/g in the tumour at a maximum (Figure 2). A further in vivo biodistribution study performed in F9 tumour-bearing mice using

radioiodinated immunocytokine preparations further showed that the scDb C-terminus

[L19L19IL2] immunocytokine format also had significantly superior tumour targeting properties compared with the scDb N-terminus [IL2L19L19] immunocytokine format, as well as the scFv L19-IL2 homodimer [SCFV2] immunocytokine format, as previously shown.

Specifically, the scDb C-terminal fusion protein [L19L19IL2] showed an accumulation of about 7.8% ID/g in the tumour and a favourable tumor-to-organ profile, while the scDb N- terminal fusion protein [IL2L19L19] could only reach values around 3.9% ID/g and the scFv L19-IL2 homodimer around 4.6% ID/g (Figure 6).

Example 6 - Cloning of F8-IL2 immunocytokines

The below examples describe the production and characterisation of two F8-IL2

immunocytokines which differ in format, and compare these formats with regard to their ability to specifically target tumours in a mouse cancer model, thereby demonstrating the superiority of the single-chain diabody C-terminal format as claimed herein for in vivo targeting.

6.1 Cloning of the single-chain diabody (scDb) C-terminal fusion protein (F8F8-IL2)

The scDb C-terminal fusion protein (F8F8-IL2) coding sequence was generated using F8F8 and L19-IL2 as templates.

The first F8 gene (also known as F8.2) was PCR amplified using primers

Bam_GSLD_F8.2_fw> and F8.2_G4S3_bw<. The IL2 gene was amplified using primers G4S3_IL2_fw> and IL2_Stop_Not_bw<. The two intermediate fragments were PCR- assembled, PCR-amplified using primers Bam_GSLD_F8.2_fw> and IL2_Stop_Not_bw<, double digested with BamHI-HF/Notl-HF and cloned into a pcDNA 3.1 (+) vector.

A second F8 gene (also known as F8.1 ) was PCR amplified using primers Hindlll_Lead_fw> and F8.1_GSLD_bam_bw< double digested by Hindlll-HF/ BamHI-HF and inserted into the previously generated vector resulting into the full length F8F8-IL2.

The sequence of the PCR primers used to clone F8F8-IL2 are listed in Table 3.

The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of the F8F8-IL2 polypeptide is set out in SEQ ID NO: 74. A schematic

representation of the F8 scDb C-terminal fusion protein is shown in Figure 1E. 6.2 Cloning of the single-chain diabody (scDb) N-terminal fusion protein (IL2-F8F8)

The scDb N-terminal fusion protein (IL2-F8F8) coding sequence was generated using L19- I L2 and F8F8 as templates.

The I L2 gene was amplified using primers Hindl ll_Lead_fw> and I L2-G4S3_bw<. The first F8 gene (also known as F8.1 ) was PCR amplified from G4S3_F8.1_fw> and

F8.1_GSLD_Bam_bw<. The two intermediate fragments were PCR-assembled, PCR- amplified using primers Hindl l l_Lead_fw > and F8.1_GSLD_Bam_bw<, double digested with Hindl ll/BamHI-HF and cloned into a pcDNA 3.1 (+) vector.

A second F8 gene (also known as F8.2) was PCR amplified using primers

Bam_GSLD_F8.2_fw> and F8.2_stop_not_bw<. The DNA fragment was subsequently double digested by BamHI-HF/Not-HF and inserted into the previously generated vector resulting into the full length I L2-F8F8.

The sequences of the PCR primers used to clone I L2-F8F8 are listed in Table 3.

The resulting plasmids were amplified and used for cell transfection. The amino acid sequence of the scDb N-terminal fusion protein is set out in SEQ I D NO: 75. A schematic representation of the F8 scDb N-terminal fusion protein is shown in Figure 1 F.

Table 3: Primers used for PCR amplification of the F8 scDb C-terminal fusion protein and the F8 scDb N-terminal fusion protein

Example 7 - Expression and purification of F8-IL2 immunocytokines

7.1 Cell culture and transfection

Transfected CHO-S cells (Chinese Hamster Ovary) were cultured in suspension in PowerCHO-2CD medium, supplemented with Ultraglutamine-1 , HT-supplement and Antibiotic-Antimycotic.

7.2 Expression and purification of F8-IL2 immunocytokines

The two F8-IL2 immunocytokines described in Example 6 above were expressed using transient gene expression in CHO-S cells. For 1 ml. of production 4 x 10⁶ CHO-S cells in suspension were centrifuged and resuspended in 1 ml. ProCH04. 0.625 mg of plasmid DNAs followed by 2.5 mg polyethylene imine (PEI; 1 mg/ml_ solution in water at pH 7.0) per million cells were then added to the cells and gently mixed. The transfected cultures were incubated in a shaker incubator at 31 °C for 6 days. The F8-IL2 immunocytokines were purified from the cell culture medium by protein A affinity chromatography and then dialyzed against PBS.

Example 8 - characterization of the F8-IL2 immunocytokines by SDS-PAGE

The purified immunocytokines prepared as described in Example 7 were characterized by SDS-PAGE. SDS-PAGE was performed with 4% - 12% Bis - Tris gels under reducing and non-reducing conditions. 2pg of purified fusion proteins were analysed.

SDS-PAGE characterization confirmed the expected molecular weight of the scDb fusion proteins (Figures 1 E and 1 F) of around 66 kDa. Example 9 - Comparative biodistribution experiments between the F8 scDb N-terminal fusion protein and F8 scDb C-terminal fusion protein

9.1 Tumor implantation

The murine tumor cell line F9 teratocarcinoma was used to generate the syngeneic tumor model. F9 cells were cultured on 0.1% gelatin-coated tissue culture flasks in DMEM medium supplemented with 10% FCS.

F9 tumor cells (10 ^c 10⁶ cells resuspended in 200 mI_ of HBSS buffer) were implanted subcutaneously in the right flank of 129/SvEv mice (females, six to eight weeks-old).

9.2 Radiolabeling and tumor targeting

Purified fusion protein samples (100 mg) were radioiodinated with ¹²⁵l and Chloramine T hydrate and purified on a PD10 column. Radiolabeled immunocytokines were injected into the lateral tail vein of immunocompetent (129/Sv) mice bearing subcutaneously implanted F9 murine teratocarcinomas. The injected dose per mouse was about 15 mg. Mice were sacrificed 24 hours after injection. Organ samples were weighed, and radioactivity was counted using a Packard Cobra gamma counter. The protein uptake in the different organs was calculated and expressed as the percentage of the injected dose per gram of tissue (%l D/g).

Conclusion

Example 5 demonstrated that the scDb C-terminus [L19L19-IL2] immunocytokine format had significantly superior tumour targeting properties compared with the scDb N-terminus [IL2- L19L19] immunocytokine format. The superior tumour targeting properties of the scDb C- terminal immunocytokine format were thought to be due to the position of the IL2 payload rather than the nature of the scDb which was common to both formats tested. To

demonstrate that it is indeed the immunocytokine format which results in the superior tumour targeting, a further set of scDbs comprising the IL2 payload at either the N- or C-terminus were generated using the F8 scDb (Examples 6.1 to 6.2).

The results again showed that the scDb C-terminus format [F8F8-IL2] had significantly superior tumour targeting properties, as reflected by a higher percentage of the injected dose of the immunocytokine being taken up by the tumour, compared with the scDb N- terminus format [IL2-F8F8] in in vivo biodistribution studies performed in F9 tumour-bearing mice. Specifically, the scDb C-terminal fusion protein showed an accumulation of about 8.5 % ID/g in the tumour and a favourable tumor-to-organ profile (Figure 7A), while the scDb N- terminal fusion protein only reached values of around 5.6% ID/g in the tumour at a maximum (Figure 7B). These results confirm that the scDb C-terminus format is responsible for the superior tumour targeting properties observed. Based on these results it is expected that other scDbs targeting an extracellular matrix component associated with neoplastic growth and/or angiogenesis, and comprising IL2 conjugated to their C-terminus would show similarly superior tumour targeting properties to the scDb C-terminal conjugates tested.

Sequence listing

SEQ ID NO: 1 : lnterleukin-2 (IL2)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS

IISTLT

SEQ ID NO: 2: L19 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

SEQ ID NO: 3: L19 VL

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDR

FSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

SEQ ID NO: 4: L19 VH CDR1

SFSMS

SEQ ID NO: 5: L19 VH CDR2

SISGSSGTTYYADSVKG

SEQ ID NO: 6: L19 VH CDR3

PFPYFDY

SEQ ID NO: 7: L19 VL CDR1

RASQSVSSSFLA

SEQ ID NO: 8: L19 VL CDR2

YASSRAT

SEQ ID NO: 9: L19 VL CDR3

QQTGRIPPT SEQ ID NO: 10: L19 Diabody

L19 VH - LINKER - L19 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

SGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

SEQ ID NO: 11 : L19 scDb

L19 VH - LINKER - L19 VL - LINKER - L19 VH - LINKER - L19 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

SGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKGSLDGAGGSAGA

DGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSS

GTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVS

SGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSR

ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

SEQ ID NO: 12: L19-IL2 (scDb C-Terminus)

L19 VH - LINKER - L19 VL - LINKER - L19 VH - LINKER - L19 VL - (G4S)3 Linker -

HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

SGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKGSLDGAGGSAGA

DGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSS

GTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVS

SGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK GGGGSGGG GSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN

RWITFCQSIISTLT SEQ ID NO: 13: IL2-L19 (scDb N-terminus)

HulL2 - (G4S)3 Linker - L19 VH - LINKER - L19 VL - Linker - L19 VH - LINKER - L19

VL

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS

I ISTLT GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVR

QAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

PFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWY

QQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTF

GQGTKVEIKGSLDGAGGSAGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMS

WVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY

YCAKPFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFL

AWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIP

PTFGQGTKVEIK

SEQ ID NO: 14: L19-IL2 (Crab)

L19 VH - LINKER - L19 VL - (G4S)3 Linker- HulL2 - (G4S)3 Linker- L19 VH - LINKER

- L19 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGG

GGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRL

LIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKGG

GGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK

KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET

ATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCA

ASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ

MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGT

LSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTD

FTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

SEQ ID NO: 15: L19-IL2 (scFv₂)

L19 VH - LINKER - L19 VL - Linker - HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGD

GSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYY ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSS

SGSSSSGSSSSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA

TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI

VEFLNRWITFCQSIISTLT

SEQ ID NO: 16: L19-IL2 (Diabody linker 1 : GSSGG)

L19 VH - LINKER - L19 VL - (S4G)3 Linker - HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

SGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSS

SGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF

CQSIISTLT

SEQ ID NO: 17: L19-IL2 (Diabody linker 2: GGSGG)

L19 VH - LINKER - L19 VL - (S4G)3 Linker - HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGG

SGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSS

SGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF

CQSIISTLT

SEQ ID. NO: 18: L19-IL2 (Diabody linker 3: GSADG)

L19 VH - LINKER - L19 VL - (S4G)3 Linker - HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

APGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSS

SGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF

CQSIISTLT

SEQ ID NO: 19: L19-IL2 (Diabody linker 4: GSAKG)

L19 VH - LINKER - L19 VL - (S4G)3 Linker - HulL2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGS

AKGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGI

PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSS

SGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF

CQSIISTLT

SEQ ID NO: 20: L19-IL2 (scDb X 2)

HulL2- (G4S)3 Linker - L19 VH - LINKER - L19 VL - LINKER - L19 VH - LINKER - L19 VL - (G4S)3 Linker- HulL2

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS

I ISTLT GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVR

QAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

PFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWY

QQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTF

GQGTKVEIKGSLDGAGGSAGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMS

WVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY

YCAKPFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFL

AWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIP

PTFGQGTKVEIKGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP

KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKG

SETTFMCEYADETATIVEFLNRWITFCQSI ISTLT

SEQ ID NO: 21 : Linker between VH and VL in diabody and scDb

GSSGG

SEQ ID NO: 22: Linker between VH and VL in diabody and scDb

GGSGG

SEQ ID NO: 23: Linker between VH and VL in diabody

GSADG

SEQ ID NO: 24: Linker between VH and VL in diabody

GSAKG SEQ ID NO: 25: Linker between Diabody in scDb

GSLDGAGGSAGADGG

SEQ ID NO: 26: Linker between VL or VH and IL2

GGGGSGGGGSGGGGS

SEQ ID NO: 27 Clones GGSGG, GSADG, and GSAKG Fragment A Forward Primer T AAT AC G ACT C ACT ATAG G G

SEQ ID NO: 28 Clone GGSGG Fragment A Backward Primer

C AC CGCCTGATCCCC C ACT C GAG AC G GT G AC C AG G GT

SEQ ID NO: 29 Clone GSADG Fragment A Backward Primer

CGT CT GCT GACCCACT CGAGACGGT GACCAGGGTT CCC

SEQ ID NO: 30 Clone GSAKG Fragment A Backward Primer

ACCTTTT GCT GACCCACT CGAGACGGT GACCAGGGTT CCC

SEQ ID NO: 31 Clone GGSGG Fragment B Forward Primer

TCTCGAGTGGGGGATCAGGCGGTGAAATTGTGTTGACGCAG

SEQ ID NO: 32 Clone GGSGG, GSADG, and GSAKG Fragment B Backward Primer T AG AAG G C AC AGT C GAG G

SEQ ID NO: 33 Clone GSADG Fragment B Forward Primer

CCGTCTCGAGTGGGTCAGCAGACGGTGAAATTGTGTTGACGCAGTCT

SEQ ID NO: 34 Clone GSAKG Fragment B Forward Primer

CCGTCT CGAGTGGGT CAGCAAAAGGT GAAATT GT GTT GACGCAGT CT

SEQ ID NO: 35 Primer NheLead>

CTAGCTAGCTAGGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCT

A

SEQ ID NO: 36 Primer IL2G4S3<

CCGCCAGAACCCCCTCCGCCTGACCCGCCTCCACCAGTCAGTGTTGAGATGATGCTTT

G SEQ ID NO: 37 Primer G4S3L19>

GGTCAGGCGGAGGGGGTTCTGGCGGTGGCGGATCGGAGGTGCAGCTGTTGGAGTCT

GGG

SEQ ID NO: 38 Primer L19Hind<

GAAGCTT CCTTT GATTT CCACCTT GGT CCCTT G

SEQ ID NO: 39 Primer LnkDP47>

ATGGAGCAGGTGGCAGTGCAGGAGCGGACGGGGGTGAGGTGCAGCTGTTGGAGTCT

GGG

SEQ ID NO: 40 Primer HindLnk>

C AAG CTTG GAT G GAG C AG GTG G C AGT G C AG GAG

SEQ ID NO: 41 Primer G4S3IL2>

GGTCAGGCGGAGGGGGTTCTGGCGGTGGCGGATCGGCACCTACTTCAAGTTCTACAA

AG

SEQ ID NO: 42 Primer L19G4S3<

CCGCCAGAACCCCCTCCGCCTGACCCGCCTCCACCTTTGATTTCCACCTTGGTCCCTT

G

SEQ ID NO: 43 Primer IL2G4S3Bam<

CGCGGATCCCCCTCCGCCTGACCCGCCTCCACCAGTCAGTGTTGAGATGATGCTTTG

SEQ ID NO: 44 Primer BamG4S3L19>

CGCGGATCCGGCGGTGGCGGATCGGAGGTGCAGCTGTTGGAGTCTGGG

SEQ ID NO: 45 Primer L19StopNot<

TTTT C CTTTT GCGGCCGCT C ATT ATTT GATTT C C AC CTTGGTCCCTTG

SEQ ID NO: 46 Primer IL2StopNot<

TTTT C CTTTT GCGGCCGCT C ATT AAGT C AGT GTT GAG AT G ATG CTTT G

SEQ ID NO: 47 Primer DP47G4S2.5<

CCCT GACCCT CCGCCACCAGAGCCCCCACCT CCACT CGAGACGGT GACCAGGGTT CC SEQ ID NO: 48 Primer G4S2.5VL>

GGGCTCTGGTGGCGGAGGGTCAGGGGGAGGCGGTGAAATTGTGTTGACGCAGTCTCC

A

SEQ ID NO: 49 Primer LeadlL2>

CTGTTCCTCGTCGCTGTGGCTACAGGTGTGCACTCGGCACCTACTTCAAGTTCTACAAA

SEQ ID NO: 50 Primer HindlllSIP

CCCAAGCTTGTCGACCATGGGCTGGAGCC

SEQ ID NO: 51 Primer L19Linker

GAG C C G G AAG AG CT ACT ACCCGATGAG G AAG ATTT G ATTT C C AC CTTGGTCCCTTG

SEQ ID NO: 52 Primer LinkerlL2

TCGGGTAGTAGCTCTTCCGGCT CAT C GTC C AG C G G C G C AC CT ACTT C AAGTT CT AC A

SEQ ID NO: 53 Primer IL2stopNotl

TTTT C CTTTT GCGGCCGCT C ATT AAGT C AGT GTT GAG AT GAT

SEQ ID NO: 54: F8 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS

SEQ ID NO: 55: F8 VL

EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPD

RFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 56: F8 VH CDR1

LFT

SEQ ID NO: 57: F8 VH CDR2

SGSGGS

SEQ ID NO: 58: F8 VH CDR3

STHLYL SEQ ID NO: 59: F8 VL CDR1

MPF

SEQ ID NO: 60: F8 VL CDR2

GASS RAT

SEQ ID NO: 61 : F8 VL CDR3

MRGRPP

SEQ ID NO: 62: F8 scDb

F8 VH - LINKER - F8 VL - LINKER - F8 VH - LINKER - F8 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSG

SSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRAT

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKGSLDGAGGS

AGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAIS

GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQG

TLVTVSSGSSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLI

YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 63: F16 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR

SEQ ID NO: 64: F16 VL

SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF

SGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPWFGGGTKLTVLG

SEQ ID NO: 65: F16 VH CDR1

RYGMS

SEQ ID NO: 66: F16 VH CDR2

AISGSGGSTYYADSVKG

SEQ ID NO: 67: F16 VH CDR3

AHNAFDY SEQ ID NO: 68: F16 VL CDR1

QGDSLRSYYAS

SEQ ID NO: 69: F16 VL CDR2

GKNNRPS

SEQ ID NO: 70: F16 VL CDR3

NSSVYTMPPVV

SEQ ID NO: 71 : F16 scDb

F16 VH - LINKER - F16 VL - LINKER - F16 VH - LINKER - F16 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISGSGGST

YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSRG

SSGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSG

IPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPWFGGGTKLTVLGGSLDGAG

GSAGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVS

AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQ

GTLVTVSRGSSGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVI

YGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPWFGGGTKLTVL

G

SEQ ID NO: 72: F8 Diabody

F8 VH - LINKER - F8 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSG

GSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRAT

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 73: F8 scDb

F8 VH - LINKER - F8 VL - LINKER - F8 VH - LINKER - F8 VL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSG

GSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRAT

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKGSLDGAGGS AGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAIS

GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQG

TLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLI

YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 74: F8-IL2 (scDb C-Terminus)

F8 VH - LINKER - F8 VL - LINKER - F8 VH - LINKER - F8 VL - (G4S)3 Linker- HulL2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSG

GSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRAT

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKGSLDGAGGS

AGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAIS

GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQG

TLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK GG GGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET

ATIVEFLNRWITFCQSIISTLT

SEQ ID NO: 75: IL2-F8 (scDb N-terminus)

HulL2 - (G4S)3 Linker- F8 VH - LINKER - F8 VL - Linker - F8 VH - LINKER - F8 VL

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEEL

KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS

I ISTLT GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVR

QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

KSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFL

AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGR

PPTFGQGTKVEIKGSLDGAGGSAGADGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSL

FTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED

TAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQ

SVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY

CQQMRGRPPTFGQGTKVEIK SEQ ID NO: 76 Primer Hindlll_Lead_fw> (also called Hindl_ead>)

CCCAAGCTTCCACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCTAC

A

SEQ ID NO: 77 Primer IL2-G4S3_bw<

CCGCCAGAACCCCCTCCTCCGGACCCGCCTCCACCAGTCAGTGTTGAGATGATGCTTT

G

SEQ ID NO: 78 Primer G4S3_F8.1_fw>

GGTCCGGAGGAGGGGGTTCTGGCGGTGGCGGATCGGAGGTGCAGCTGTTGGAGTCT

GGG

SEQ ID NO: 79 Primer F8.1_GSLD_Bam_bw<

CCCGGAT CCACCTGCT CCAT CCAACGAACCTTT GATTT CCACCTT GGT CCCTT G

SEQ ID NO: 80 Primer Bam_GSLD_F8.2_fw>

CCCGGATCCGCAGGAGCGGACGGGGGTGAAGTCCAGTTGCTGGAATCTGGC

SEQ ID NO: 81 Primer F8.2_stop_not_bw<

TTTT C CTTTT GCGGCCGCCTACTT GAT CTC G AC CTTT GTTC C CTG SEQ ID NO: 82 Primer F8.2_G4S3_bw<

CCGCCAGAACCCCCT CCT CCGGACCCGCCT CCACCCTT GAT CT CGACCTTT GTT CCCT G

SEQ ID NO: 83 Primer G4S3_IL2_fw>

GGTCCGGAGGAGGGGGTTCTGGCGGTGGCGGATCGGCACCTACTTCAAGTTCTACAA

AG

SEQ ID NO: 84 Primer IL2_Stop_Not_bw<

TTTT C CTTTT GCGGCCGCCT AAGT C AGT GTT GAG AT G ATG CTTT G

SEQ ID NO: 85: L19-IL2 nucleotide sequence

L19 VH - LINKER- L19 VL- (G4S)3 Linker- HulL2

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACC ACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAG AACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCGTATATTAC TGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT CGAGTGGGTCCAGTGGCGGTGAAATT GT GTT GACGCAGT CT CCAGGCACCCT GT CTTT GTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAG CTTTTT AGCCTGGT AC CAG CAG AAAC CTG G C CAG G CAC CCAGGCTCCT CAT CT ATT AT G CAT C CAG CAG G G C CACT G G CAT C C CAG AC AG GTT CAGT G G CAGT GGGTCTGG G AC AG ACTT CACT CT CAC CAT CAG CAG ACT G GAG C CT GAAG ATTTT G CAGT GT ATT ACTGT CAG CAG AC G G GTC GT ATT C C G C C G AC GTT C G G C C AAG G G AC C AAG GT G G AAAT CAAAtcttcc

CAC CT ACTT CAAGTT CT ACAAAGAAAACA

C AG CT AC AACT G G AG C ATTT ACTG CTG G ATTT AC AG AT G ATTTT G AAT G G AATT AAT AAT T AC AAG AAT C C C AAACT CAC CAG G ATG CT C AC ATTT AAGTTTT AC AT G C C C AAG AAG G C CAC AG AACT G AAAC AT CTT CAGT G C CT AG AAG AAG AACT C AAAC CTCTG GAG G AAGT G C T AAATTT AG CT C AAAG C AAAAACTTT C ACTT AAG AC C CAG G G ACTT AAT CAG C AAT AT C A AC GT AAT AGTT CTG G AACT AAAG G G ATCT G AAAC AAC ATT CAT GTGT G AAT ATG CTG AT GAG AC AG C AAC C ATT GT AG AATTT CT G AAC AG AT G GATT AC CTTTT GT C AAAG CAT CAT C T CAAC ACT GACT

SEQ ID NO: 86: L19-IL2 (scDb C-Terminus) nucleotide sequence

L19 VH - LINKER - L19 VL - LINKER - L19 VH - LINKER - L19 VL- (G4S)3 Linker- HulL2

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGC

CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACC

ACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAG

AACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCGTATATTAC

TGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT

CGAGTGGGTCCAGTGGCGGTGAAATT GT GTT GACGCAGT CT CCAGGCACCCT GT CTTT

GTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAG

CTTTTT AGCCTGGT AC C AG C AG AAAC CTG G C C AG G C AC C C AG GCTCCTCATCT ATT AT G

CAT C C AG C AG G G C C ACT G G CAT C C C AG AC AG GTT C AGT G G C AGT GGGTCTGG G AC AG

ACTT CACT CT CAC CAT CAG CAG ACT GG AGC CT GAAG ATTTT G CAGT GT ATT ACT GT CAG

CAGACGGGTCGTATTCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAGGA

AGCTTGGATGGAGCAGGTGGCAGTGCAGGAGCGGACGGGGGTGAGGTGCAGCTGTT

GGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG

CCTCTGGATTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACATACTACGCAGA

CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT

GCAAATGAACAGCCTGAGAGCCGAAGACACGGCCGTATATTACTGTGCGAAACCGTT

TCCGTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGGGTCCAG

JGGCGGJGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTTTTAGCCTGGT AC C AG C AG AAAC CTG G C C AG G C AC C C AG G CTC CT CAT CT ATT AT G CAT C C AG C AG G G C CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC AT CAG CAGACT G GAG CCT GAAGATTTT G CAGT GT ATT ACT GT CAG CAGACG GGT CGTAT TCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA GGTGGAGGCGGGTCAGG CGGA GGGGGTTCTGGCGGTGGCGGA TCGGCACCT ACTT CAAGTT CT ACAAAGAAAACA C AG CT AC AACT G G AG C ATTT ACTG CTG G ATTT AC AG AT G ATTTT GAAT G G AATT AAT AAT T AC AAG AAT C C C AAACT C AC CAG G ATG CT C AC ATTT AAGTTTT AC AT G C C C AAG AAG G C CACAGAACT GAAACAT CTT CAGTGCCTAGAAGAAGAACT CAAACCT CTGGAGGAAGT GC T AAATTT AG CT C AAAG C AAAAACTTT C ACTT AAG AC C CAG G G ACTT AAT CAG C AAT AT C A ACGT AAT AGTT CT G GAACT AAAG G GAT CT G AAACAACATT CAT GTGT GAAT AT G CT GAT GAG AC AG CAACCATT GT AGAATTT CT GAACAGAT GGATT ACCTTTT GT CAAAGCAT CAT C T CAACACT GACT

SEQ I D NO: 87: I L2-L19 (scDb N-terminus) nucleotide sequence

Hul L2 - (G4S)3 Linker - L19 VH - LINKER - L19 VL- LINKER - L19 VH - LINKER -L19 VL

G C AC CTACTT C AAGTT CT ACAAAG AAAAC AC AG CT AC AACT G GAG C ATTT ACT G CTG G A

TTT AC AG AT G ATTTT G AAT G G AATT AAT AATT AC AAG AAT C C C AAACT C AC C AG G ATG CT

C AC ATTT AAGTTTT ACATGCCCAAGAAGGCCACAGAACT GAAACAT CTT CAGT GCCT AG

AAG AAG AACT C AAAC CTCTG GAG G AAGT G CTAAATTT AG CT C AAAG C AAAAACTTT C AC

TT AAG AC C C AG G G ACTT AAT C AG C AAT AT C AAC GT AAT AGTT CTG G AACT AAAG G G ATC

T G AAACAACATT CAT GTGT GAAT AT G CT GAT G AG ACAG CAACCATT GT AGAATTT CT G AA

CAGATGGATT ACCTTTT GT CAAAGCAT CAT CT CAACACT GACT GGTGGAGGCGGGTCA

GGCGGAGGGGGTTCTGGCGGTGGCGGATCGGAGGJGCAGCJGJJGGAGJCJGGGGG

AGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAC

CTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG

GGTCTCATCTATTAGTGGTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGGG

CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAG

CCTGAGAGCCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGAC

T ACTGGGGCCAGGGAACCCTGGT CACCGTCT CGAGTGGGTCCAGTGGCGGTGAAATT

GTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT C CTG C AG G G C C AGT C AG AGT GTT AG C AG C AG CTTTTT AG C CTG GT AC C AG C AG AAAC C

TGGCCAGGCACCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACTGGCATCCCA

G AC AG GTT C AGT G G C AGT GGGTCTGG G AC AG ACTT C ACT CT C AC CAT C AG C AG ACT G G

AGCCT GAAGATTTTGCAGT GT ATT ACT GT CAGCAGACGGGT CGT ATT CCGCCGACGTT C

GGCCAAGGGACCAAGGTGGAAATCAAAGGAAGCTTGGATGGAGCAGGTGGCAGTGC

AGGAGCGGACGGGGGTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGC

CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTTTTC

GATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTA

GTGGTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCT

CCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAAG

ACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAGGG

AACCCTGGTCACCGTCTCGAGTGGGTCCAGTGGCGGTGAAATT GT GTT GACGCAGT CT

C C AG G C AC C CTGT CTTT GTCTC C AG G G G AAAG AG C C AC CCTCTCCTG C AG G G C C AGT C

AGAGTGTTAGCAGCAGCTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCACCCAG

G CTC CT CAT CT ATT AT G CAT C C AG C AG G G C C ACT G G CAT C C C AG AC AG GTT C AGT G G C

AGTGGGTCTGG G AC AG ACTT C ACT CT C AC CAT C AG C AG ACT G GAG C CT G AAG ATTTT G

CAGT GT ATT ACT GT CAGCAGACGGGT CGT ATT CCGCCGACGTT CGGCCAAGGGACCAA

GGTGGAAATCAAA

References

All documents mentioned in this specification are incorporated herein by reference in their entirety.

Borsi et al. (1987), J. Cell. Biol., 104, 595-600

Borsi L et al Int J Cancer 1992; 52:688-692

Borsi et al. (2002) Int. J. Cancer: 102, 75-85

Brack, S.S., Silacci, M., Birchler, M. & Neri, D. Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clin Cancer Res 12, 3200-3208 (2006).

Carnemolla B et al. Eur J Biochem 1992; 205:561-567

Holliger and Winter. Diabodies: small bispecific antibody fragments. Cancer Immunol Immunother (1997) 45: 128-130.

Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993.

Johannsen, M., et al. The tumour-targeting human L19-IL2 immunocytokine: preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma. Eur J Cancer 46, 2926-2935 (2010).

Kaspar et al. (2007) Cancer Res, 67, 4940-4948

Kontermann, R. E., and Muller, R. (1999). Intracellular and cell surface display of single- chain diabodies. J. Immunol. Methods 226: 179-188.

Kornblihtt et al. (1984), Nucleic Acids Res. 12, 5853-5868

Neri, D. & Bicknell, R. Tumour vascular targeting. Nat Rev Cancer 5, 436-446 (2005).

Pasche et al. (201 1 ) J Biotechnology, 154, 84-92

Pini, A., et al. Design and use of a phage display library. Human antibodies with

subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J Biol Chem 273, 21769-21776 (1998).

Paolella et al. (1988), Nucleic Acids Res. 16, 3545-3557.

Sauer, S., et al. Expression of the oncofetal ED-B containing fibronectin isoform in hematologic tumors enables ED-B targeted 1311-L19SIP radioimmunotherapy in Hodgkin lymphoma patients. Blood (2009).

Villa, A., et al. A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. Int J Cancer 122, 2405-2413 (2008).

Viti et al. (1999) Cancer Res. 59(2): 347-52.

Claims

Claims

1. A conjugate comprising interleukin-2 (IL2) and a single-chain diabody, wherein the single-chain diabody binds an extracellular matrix component associated with neoplastic growth and/or angiogenesis, wherein the IL2 is linked to the C-terminus of the single-chain diabody.

2. The conjugate according to claim 1 , wherein the extracellular matrix component associated with neoplastic growth and/or angiogenesis is fibronectin.

3. The conjugate according to claim 2, wherein the fibronectin is the Extra Domain-B (ED-B) of fibronectin.

4. The conjugate according to any one of the preceding claims, wherein the single- chain diabody comprises an antigen-binding site having the complementarity determining regions (CDRs) of antibody L19 set forth in SEQ ID NOs 4 to 9.

5. The conjugate according to claim 4, wherein the single-chain diabody comprises the VH and VL domains of antibody L19 set forth in SEQ ID NOs 2 and 3.

6. The conjugate according to claim 5, wherein the single-chain diabody comprises the amino acid sequence set forth in SEQ ID NO: 11.

7. The conjugate according to any one of the preceding claims, wherein the IL2 is human IL2.

8. The conjugate according to claim 7, wherein the IL2 comprises the sequence set forth in SEQ ID NO: 1.

9. The conjugate according to claim 8, wherein the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 12.

10. The conjugate according to claim 2, wherein the fibronectin is the Extra Domain-A (ED-A) of fibronectin.

1 1. The conjugate according to claim 10, wherein the single-chain diabody comprises an antigen-binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 56 to 61.

12. The conjugate according to claim 1 1 , wherein the single-chain diabody comprises the VH and VL domains of antibody F8 set forth in SEQ ID NOs 54 and 55.

13. The conjugate according to claim 12, wherein the single-chain diabody comprises the amino acid sequence set forth in SEQ ID NO: 73 or 62.

14. The conjugate according to any one of claims 10 to 13, wherein the IL2 is human IL2.

15. The conjugate according to claim 14, wherein the IL2 comprises the sequence set forth in SEQ ID NO: 1.

16. The conjugate according to claim 15, wherein the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 74.

17. The conjugate according to any one of the preceding claims which is a single-chain fusion protein.

18. A nucleic acid molecule encoding a conjugate according to any one of claims 1 to 17.

19. An expression vector comprising the nucleic acid of claim 18.

20. A host cell comprising the nucleic acid molecule of claim 18 or vector of claim 19.

21. A method of producing a conjugate according to any one of claims 1 to 17, the method comprising culturing the host cell of claim 20 under conditions for expression of the conjugate.

22. The method of claim 21 further comprising isolating and/or purifying the conjugate.

23. The conjugate according to any of claims 1 to 17 for use in a method of treating cancer in a patient by targeting IL2 to the neovasculature in vivo.

24. The conjugate according to any one of claims 1 to 17 for use in a method of delivering IL2 to the tumour neovasculature in a patient.

25. A method of treating cancer by targeting IL2 to the neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate according to any of claims 1 to 17 to the patient.

26. A method of delivering IL2 to the tumour neovasculature in a patient comprising administering to the patient a conjugate according to any one of claims 1 to 17.