IMMUNOCONJUGATES COMPRISING A SINGLE CHAIN DIABODY AND INTERLEUKIN-15 OR INTERLEUKIN-15 AND A SUSHI DOMAIN OF INTERLEUKIN-15 RECEPTOR ALPHA

Field

The present invention relates to conjugates comprising a single-chain diabody, interleukin-15 (IL15) and optionally a sushi domain of the IL15 Receptor alpha (IL15R alpha). In particular, the present invention relates to conjugates in which IL15, or IL15 and the sushi domain of IL15R alpha, is/are conjugated to the C-terminus of the single-chain diabody. The conjugate may be useful, for example, for targeting IL15 to tissues in vivo. In particular, the present invention relates to the therapeutic use of the conjugates in the treatment of a disease/disorder, such as cancer.

Background to the Invention

Cytokines are key mediators of innate and adaptive immunity. Many cytokines have been used for therapeutic purposes in patients, such as the treatment of advanced cancer, but their

administration is typically associated with severe toxicity, hampering dose escalation to

therapeutically active regimens and their development as anticancer drugs, for example. To overcome these problems, the use of‘immunocytokines’ (i.e. cytokines fused to antibodies or antibody fragments) has been proposed, with the aim of concentrating the immune system stimulating activity of the cytokines at the site of disease, whilst sparing normal tissues (Savage et al., 1993; Schrama et al., 2006; Neri et al. 2005; Dela Cruz et al., 2004; Reisfeld et al., 1997;

Kontermann et al., 2012).

Antibodies specific to splice-isoforms of fibronectin have been described as vehicles for

pharmacodelivery applications, as these antigens are virtually undetectable in the normal healthy adult (with the exception of the placenta, endometrium and some vessels in the ovaries) while being strongly expressed in the majority of solid tumors and lymphomas, as well as other diseases (Pedretti et al., 2010; Schliemann et al. 2009).

For example, antibodies F8 and L19, specific to the alternatively-spliced Extra Domain-A (ED-A) and Extra Domain-B (ED-B) of fibronectin, respectively (Villa et al., 2008, Viti et al., 1999), have been employed for the development of armed antibodies, some of which have begun clinical testing in oncology and rheumatology applications (Eigentler ef al., 2011 ; Papadia et al., 2013).

The tumor targeting properties of these antibodies have also been documented in mouse models of cancer, as well as in cancer patients.

Antibodies specific to alternatively spliced domains of tenascin C have also been described as vehicles for pharmacodelivery applications. The anti-tenascin C antibody F16 (Brack et al., Clin Cancer Res. 2006 May 15; 12(10):3200-8.) binds to the extra domain A1 of tenascin-C. This domain is virtually undetectable in normal adult tissues but is strongly expressed at sites of physiological angiogenesis and tumour angiogenesis (Brack et al. , 2006 supra). The F16 antibody has efficacy in vivo and has been successfully employed for the development of armed antibodies, in particular immunocytokines. The F16 antibody has begun clinical testing in oncology (Pasche and Neri, 2012 Drug Discov. Today. 17(1 1-12):583-90). The present applicant has also previously generated antibodies that bind to the domain D of Tenascin-C. In particular, the properties of the “P12” “p4” _anc| “□1 1” antibodies specific for Tn-D have been described in Brack et al. (Clin.

Cancer Res. (2006) 12, 3200-3208) and W02006/050834 and the properties of the“CPR01” and “CPR01.1” antibodies have been described in WO2017/097990.

Interleukin-15 (IL15) is a pro-inflammatory cytokine, which is structurally related to IL-2. The two cytokines share the same IL-2/IL15 receptor beta and gamma, which is displayed on the surface of natural killer (NK) and T cells (Alter et al., 2018; Han et al., 2011 ; Waldmann et al., 2006;

Waldmann et al., 2011). IL15 stimulates the production of other pro-inflammatory cytokines (e.g. TNFa, IL-1 , IFNy). IL15 has also been shown to stimulate the proliferation of activated B cells and Ig synthesis by these cells, as well as promoting the activation of TH1 cells, monocytes, and lymphokine activated killer cells. IL15 has further been shown to stimulate the proliferation of mast cells and T cells and to inhibit apoptosis of T cells and B cells. In addition to the mentioned functional activities, IL15 is known to play a pivotal role in the development, survival and function of NK cells (Waldmann, 2006).

When used as therapeutic protein, IL15 has been shown to stimulate anti-cancer immune responses of NK and T cells without the induction of IL-2-associated capillary leak syndrome and the expansion of T regulatory cells (Waldmann et al., 2006; Waldmann et al., 2011).

The sushi domain (SD) is the shortest region of the IL15 receptor alpha (IL15Ralpha) capable of binding IL15. It has previously been reported that a fusion protein comprising, in order, an anti fibroblast activation protein (FAP) single-chain Fv (scFv), a sushi domain of human IL15Ralpha and IL15 increased the activity of IL15 and had an enhanced effect in preventing the formation of metastatic lesions in a B16-FAP lung metastasis mouse model compared with untargeted IL15, or an equivalent conjugate lacking the sushi domain (Kermer et al. (2012), WO2012/175222). A fusion protein comprising an anti-CD206 scFv, a sushi domain of human IL15Ralpha and human IL15, has further been shown to have anti-cancer activity, as measured by reduction of tumor volume (WO2017/158436). In clinical studies (Phase I completed), a product referred to as ALT803 consisting of a novel IL15 superagonist complex (IL15 mutant IL15N72D bound to an IL15 receptor a and to an lgG1 Fc fusion protein) was assessed in patients with advanced solid tumors in combination with Nivolumab (Wrangle et a!., 2018).

It has further been shown that fusion of I L15 to a diabody specific to the ED-B isoform of fibronectin was capable of inducing tumor regression in mice models of cancer compared to a control fusion protein comprising an antibody fragment of irrelevant specificity (Kaspar et al., 2007; W02007/128563). Moreover, fusion proteins based on IL15 and antibody fragments specific to cell surface markers have shown encouraging results in mice models of cancer (Kermer et al., 2012; Alter et al., 2018). Likewise, RLI-based immunocytokines, comprising the sushi and hinge domains of IL15Ralpha and IL15 have been prepared. Testing of these molecules has shown that the biological activity of IL15 was conserved, and even increased, as compared with free IL15 in the bg context (WO2012/175222). This effect was also seen when the RLI-based immunocytokine was in IgG or scFv format.

IL15, however, remains a difficult payload to deliver, probably due to extensive N-glycosylation and potential receptor trapping in normal tissues. In addition, due to the extensive N-glycosylation of IL15, conjugates comprising this cytokine are difficult to express. For these reasons, there remains a need in the art for further immunocytokine formats based on I L15 which can be expressed at commercially relevant levels and allow targeted delivery of I L 15 to tumor tissue.

Summary of the Invention

The present inventors have surprisingly shown that immunocytokines comprising human interleukin-15 (IL15) conjugated to the C-terminus of a single-chain diabody could be produced with good yield and high quality in contrast to conjugates comprising IL15 conjugated to the N- terminus of a single-chain diabody, for which the yield was lower and accompanied by aggregate formation. The C-terminal conjugates were further shown to retain both the targeting properties of the unconjugated single-chain diabody and the biological activity of IL15. These properties were observed independently of the antigen bound by the single-chain diabody (the ED-A or ED-B of fibronectin or domain D of tenascin C) (Examples 1, 2, 8 and 9).

The present inventors have further shown that a conjugate comprising IL15 conjugated to the C- terminus of a single-chain diabody which binds the ED-A or ED-B of fibronectin was capable of selectively targeting tumor tissue in mice, with surprisingly high levels of the conjugate (—8-15%) accumulating in the tumor and only low levels of the conjugate being present in the blood or organs (Figure 7 A and B; Example 3). In contrast, only very low levels of tumor targeting (—2%), and a poor tumor to blood/organ ratio was observed with a conjugate as described in Kaspar et al.

(2007), in which a diabody was employed for targeting of IL15 instead of a single-chain diabody (Figure 7C). These results are consistent with those reported in Kaspar et al. (2007), in which the L19(Db)-IL15 conjugate was shown to accumulate in tumors at a level of less than 2% ID/g in the tumor and also exhibited a poor tumor to organ/blood ratio. These results clearly demonstrate the superior tumor-targeting properties of a single-chain diabody-based conjugate format over a diabody-based format as disclosed in Kaspar et al. (2007), and further demonstrate that these superior tumor targeting properties are observed independently of the target bound by the single chain diabody.

A conjugate comprising IL15 conjugated to the C-terminus of a single-chain diabody which binds the ED-A of fibronectin was further confirmed by the inventors to have anti-tumor efficacy, as evidenced by inhibition of metastatic foci formation in mice treated with the conjugate (Example 7).

Based on these results, it is expected that conjugates comprising IL15 conjugated to the C- terminus of a single-chain diabody can be produced with good yield and quality and that I L15 will retain its biological activity in the context of the conjugate. It is further expected that such conjugates can be selectively targeted to the tumor or tumor microenvironment and will exhibit anti tumor activity, and thus will find application in the treatment of cancer, in particular in human patients. These properties of the conjugate are expected to be independent of the particular antigen bound by the single-chain diabody, which may be any antigen suitable for targeting the conjugate to the tumor or tumor microenvironment. Without being bound by theory, it is thought that a conjugate targeted to the tumor or tumor microenvironment will result in enhanced anti-tumor immune responses by e.g. stimulating the production of other pro-inflammatory cytokines and/or promoting the activation and/or proliferation of immune cells, thereby exerting an anti-tumor effect. In order to target the conjugate to the tumor or tumor microenvironment, the antigen bound by the single-chain diabody may be an antigen associated with neoplastic growth, such as an antigen (e.g. a cell-surface antigen) expressed on tumor cells and/or tumor-associated immune cells, or an extracellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis, in particular an extracellular matrix (ECM) component associated with neoplastic growth. Extracellular matrix (ECM) components associated with neoplastic growth include fibronectin, tenascin C, and cells forming part of the ECM, such as cancer-associated fibroblasts (FAP). Tumor-associated immune cells are known in the art and include tumor-infiltrating lymphocytes (TILs), as well as tumor-associated macrophages and neutrophils.

In one aspect, the present invention thus relates a conjugate comprising IL15 and a single-chain diabody, wherein the IL15 is linked to the C-terminus of the single-chain diabody. The conjugate preferably comprises only one IL15. Thus, where the IL15 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 IL15.

The present inventors have further shown that immunocytokines comprising human interleukin-15 (IL15), and a sushi domain of the IL15R alpha (sushi domain; SD), conjugated to a single-chain diabody that retain both the targeting properties of the unconjugated single-chain diabody and the biological activity of I L 15 can be prepared (Examples 4, 5, 10 and 11).

A number of conjugate formats were tested in which the IL15 and sushi domain were linked to the single-chain diabody at either the N-terminus or C-terminus of the single chain diabody and in different orientations (Figure 9). The present inventors surprisingly found that conjugates

(F8(scDb)-SD-IL15 and R6N(scDb)-SD-IL15) comprising a sushi domain of IL15R alpha conjugated to the C-terminus of a single-chain diabody and IL15 conjugated to the C-terminus of the sushi domain could be expressed at high levels and with a purity suitable for industrial production (Tables 2 and 5). The expression level of the F8(scDb)-SD-IL15 conjugate was surprisingly approximately three-fold higher than the expression levels of the same conjugate comprising an scFv as the targeting moiety (F8(scFv)-SD-IL15) and also higher than the expression levels of conjugates comprising the sushi domain and IL15 conjugated to the N- terminus of the single-chain diabody (Tables 2 and 5). The F8(scDb)-SD-IL15 conjugate also had very low levels of glycosylation (Figure 12), as well as being more stable and biologically active than conjugates where IL15 and the sushi domain of IL15R alpha were conjugated to the N- terminus of a single-chain diabody (IL15-SD-F8(scDb) and SD-IL15-F8(scDb)), or where the IL15 was conjugated to the C-terminus of a single-chain diabody and the sushi domain of IL15R alpha was conjugated to the C-terminus of the IL15 (F8(scDb)-IL15-SD) (Table 3). Specifically, the F8(scDb)-SD-IL15 conjugate was shown to have higher activity in a CTLL2 cell-proliferation assay than the other conjugate formats tested (Figure 13A). Retention of biological activity of IL15 can be affected by glycosylation (Kaspar et al., 2007) but this was not an issue with the conjugate format of the invention.

The F8(scDb)-SD-IL15 conjugate was further shown to be capable of selectively targeting tumor tissue in mice with lower levels of the conjugate accumulating in the healthy organs, demonstrating the suitability of the conjugate for treating cancer (Figure 18A). Furthermore, the tumor targeting specificity of the F8(scDb)-SD-IL15 conjugate was demonstrated to be superior to that of a conjugate comprising the F8 antibody in scFv format (F8(scFv)-SD-IL15), as described in Kermer et al. (2012). In particular, the F8(scDb)-SD-IL15 conjugate showed reduced unspecific accumulation in the healthy spleen compared with the F8(scFv)-SD-IL15 conjugate (Figure 18 A and B).

The F8(scDb)-SD-IL15 conjugate was also shown to have potent anti-tumor efficacy, as evidenced by almost complete inhibition of metastatic foci formation in mice treated with the conjugate. The anti-tumor effect of the sushi domain-containing conjugate was furthermore enhanced compared with the same conjugate without the sushi domain F8(scDb)-IL15 (Example 7). The inclusion of a sushi domain in the conjugates of the invention is therefore preferred.

In a preferred aspect, the invention thus provides a conjugate comprising interleukin-15 (IL15), a sushi domain of IL15R alpha, and a single-chain diabody, wherein the sushi domain is linked to the C-terminus of the single-chain diabody and the IL15 is linked to the C-terminus of the sushi domain.

As mentioned above, the conjugate preferably comprises only one IL15. Similarly, the conjugate preferably comprises only one sushi domain of IL15R alpha. Thus, the N-terminus of the single chain diabody is preferably free

The conjugates of the invention preferably contain only one single-chain diabody.

The antigen bound by the single-chain antibody may be any antigen suitable for targeting the conjugate to the tumor or tumor microenvironment. Preferably, the single-chain antibody binds an antigen associated with neoplastic growth. The antigen may, for example be an antigen, such as a cell-surface antigen, expressed on tumor cells and/or tumor-associated immune cells, or an extracellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis and/or tissue remodelling. Many tumor-associated antigens, including antigens expressed on the surface of tumor cells, are known in the art and can be selected as targets bound by the single chain diabody depending on the cancer to be treated. Similarly, antigens (e.g. cell-surface antigens) expressed by tumor-associated immune cells, such as tumor-infiltrating lymphocytes (TILs) or tumor-associated macrophages and neutrophils are known in the art and can be selected as targets for the single-chain diabody. ECM components associated with neoplastic growth and/or angiogenesis are also known and include fibronectin, tenascin C, as well as cells forming part of the ECM, such as cancer-associated fibroblasts (FAP), and may be selected as targets bound by the single-chain diabody.

In one embodiment, the single-chain diabody therefore binds an extra-cellular matrix component associated with neoplastic growth, angiogenesis, and/or tissue remodelling, most preferably an extra-cellular matrix component associated with neoplastic growth and/or angiogenesis, such as the Extra Domain-A (ED-A) isoform of fibronectin, the Extra Domain-B (ED-B) isoform of fibronectin, or domain D of tenascin C.

In a preferred embodiment, the single-chain diabody binds to the ED-A of fibronectin and comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 3-8. The antigen binding site may comprise VH and/or VL domains of antibody F8 set forth in SEQ ID NOs 1 and 2, respectively. The single-chain diabody preferably comprises or consists of the F8 single-chain diabody amino acid sequence set forth in SEQ ID NO: 9.

In an alternative preferred embodiment, the single-chain diabody binds to the ED-B of fibronectin and comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody L19 set forth in SEQ ID NOs 27-32. The antigen binding site may comprise VH and/or VL domains of antibody L19 set forth in SEQ ID NOs 24 and 25, respectively. The single-chain diabody preferably comprises or consists of the L19 single-chain diabody amino acid sequence set forth in SEQ ID NO: 33.

In a further preferred embodiment, the single-chain diabody binds to domain D of tenascin C and comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody R6N set forth in SEQ ID NOs 67-72. The antigen binding site may comprise VH and/or VL domains of antibody R6N set forth in SEQ ID NOs 73 and 74, respectively. The single-chain diabody preferably comprises or consists of the R6N single-chain diabody amino acid sequence set forth in SEQ ID NO: 75.

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 the single-chain diabody-IL15 fusion set forth in SEQ ID NO: 14 (F8(scDb)-IL15)). Yet more preferably, the conjugate comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14.

In an alternative preferred 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 the single-chain diabody-IL15 fusion set forth in SEQ ID NO: 23 (L19(scDb)-IL15)). Yet more preferably, the conjugate comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. In a further preferred 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 the single-chain diabody-IL15 fusion set forth in SEQ ID NO: 76 (R6N(scDb)-IL15). Yet more preferably, the conjugate comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76.

In a more preferred 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 the single-chain diabody-sushi-domain-IL15 fusion

F8(scDb)-SD-IL15 set forth in SEQ ID NO: 41. Yet even more preferably, the conjugate preferably comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41.

In an alternative more preferred embodiment, the conjugate has at least 70% sequence identity, e.g. one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity, to the amino acid sequence of the single-chain diabody-sushi-domain-IL15 fusion L19(scDb)-SD- IL15 set forth in SEQ ID NO: 64. Yet more preferably, the conjugate comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64.

In a further more preferred embodiment, the conjugate has at least 70% sequence identity, e.g. one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity, to the amino acid sequence of the single-chain diabody-sushi-domain-IL15 fusion R6N(scDb)-SD- IL15 set forth in SEQ ID NO: 78. Yet more preferably, the conjugate may comprise or consists of the amino acid sequence set forth in SEQ ID NO: 78.

Other antibodies capable of binding to an antigen, such as a cell-surface antigen, expressed on tumor cells and/or tumor-associated immune cells, or an extracellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis and/or tissue remodelling are known, or may be prepared by those skilled in the art, 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.

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 of the invention for use in a method of treating cancer, e.g. by targeting IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in vivo.

In another aspect the invention relates to a method of treating cancer, e.g. by targeting IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate of the invention to the patient.

In a further aspect, the present invention relates to conjugate of the invention for use in a method of delivering IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in a patient.

The present invention also provides a method of delivering IL15 to the tumor or tumor

microenvironment, such as the tumor neovasculature in a patient comprising administering to the patient a conjugate of the invention.

Brief Description of the Figures

Figure 1 shows a schematic representation of a single-chain diabody conjugated at its C-terminus to IL15

Figure 2 shows the biochemical properties of the purified F8(scDb)-IL15 conjugate. Figure 2A shows the size exclusion chromatography profile of the F8(scDb)-IL15 conjugate. Figure 2B shows the results of an SDS-PAGE analysis of the F8(scDb)-IL15 conjugate under reducing (R) and non-reducing (NR) conditions. Figure 2C shows the ESI-MS profile of the F8(scDb)-IL15 conjugate.

Figure 3 shows the biochemical properties of the purified L19(scDb)-IL15 conjugate. Figure 3A shows the size exclusion chromatography profile of the L19(scDb)-IL15 conjugate. Figure 3B shows the results of an SDS-PAGE analysis of the L19(scDb)-IL15 conjugate under reducing (R) and non-reducing (NR) conditions. Figure 4 shows the biochemical properties of the purified F8(Db)-IL15 conjugate. Figure 4A shows the size exclusion chromatography profile of the F8(Db)-IL15 conjugate. Figure 4B shows the results of an SDS-PAGE analysis of the F8(Db)-IL15 conjugate under reducing (R) and non reducing (NR) conditions.

Figure 5 shows binding of the purified F8(scDb)-IL15 (A) and F8(Db)-IL15 (B) conjugates to ED-A and the binding of the purified L19(scDb)-IL15 (C) conjugate to ED-B as measured by BiaCORE.

Figure 6 shows the induction of T cell proliferation by the purified F8(scDb)-IL15 conjugate (A) and by the purified L19(scDb)-IL15 conjugate (B) whereas Figure 6C shows that the purified IL15- F8(scDb) conjugate was not able to induce T cell proliferation.

Figure 7 shows the biodistribution of the purified conjugates in the tumor, blood and organs in tumor-bearing mice: F8(scDb)-IL15 (A), L19(scDb)-IL15 (B), and F8(Db)-IL15 (C).

Figure 8 shows the biochemical properties of the purified IL15-F8(scDb) conjugate. Figure 8A shows the size exclusion chromatography profile of the IL15-F8(scDb) conjugate. Figure 8B shows the results of an SDS-PAGE analysis of the IL15-F8(scDb) conjugate under reducing (R) and non-reducing (NR) conditions.

Figure 9 shows schematic representations of the different conjugates comprising a single-chain diabody, IL15 and a sushi domain of IL15R alpha. Figure 9A shows a schematic representation of the F8 single-chain diabody conjugated at its C-terminus to the sushi domain (SD) and IL15 (F8(scDb)-SD-IL15). Figure 9B shows a schematic representation of the F8 single-chain diabody conjugated at its C-terminus to IL15 and the SD (F8(scDb)-IL15-SD). Figure 9C shows a schematic representation of the F8 single-chain diabody conjugated at its N-terminus to the SD and IL15 (IL15-SD-F8(scDb)). Figure 9D shows a schematic representation of the F8 single-chain diabody conjugated at its N-terminus to IL15 and the SD (SD-IL15-F8(scDb)).

Figure 10 shows the results of SDS-PAGE analysis (NR: non-reducing conditions, R: reducing conditions) of conjugates F8(scDb)-SD-IL15 (A), F8(scDb)-IL15-SD (B), IL15-SD-F8(scDb) (C), SD-IL15-F8(scDb) (D), F8(scFv)-SD-IL15 (E), and L19(scDb)-SD-IL15 (F).

Figure 11 shows the results of FPLC profile analysis of conjugates F8(scDb)-SD-IL15 (A),

F8(scDb)-IL15-SD (B), SD-IL15-F8(scDb) (C) and F8(scFv)-SD-IL15 (D). Figure 12 shows the results of ESI-MS analysis of conjugates F8(scDb)-SD-IL15 (A), F8(scDb)- IL15-SD (B), IL15-SD-F8(scDb) (C), and SD-IL15-F8(scDb) (D).

Figure 13 shows the ability of the purified conjugates to stimulate the proliferation of CTLL2 cells: F8(scDb)-SD-IL15 (A), IL15-SD-F8(scDb) (B), and SD-IL15-F8(scDb) (C).

Figure 14 shows the binding activity of the purified F8(scDb)-SD-IL15 conjugate to ED-A measured by BiaCORE.

Figure 15 shows the capability of F8(scDb)-SD-IL15 (F8-F8-SD-IL15) to target ED-A on CT26 tumor sections in immunofluorescence analysis.

Figure 16 shows the capability of F8(scDb)-SD-IL15 (F8-F8-SD-IL15) to target ED-A on CT26- CAIX tumor sections in immunofluorescence analysis.

Figure 17 shows the capability of F8(scDb)-SD-IL15 (F8-F8-SD-IL15) to target ED-A on CT26- CAIX tumor sections 24 hours after intravenous administration in immunofluorescence analysis.

Figure 18 shows the biodistribution of purified F8(scDb)-SD-IL15 (A) in tumor bearing mice compared with F8(scFv)-SD-IL15 (B), and the control conjugate KSF(scDb)-SD-IL15 (C).

Figure 19 shows the number of metastatic foci counted on the lungs of mice (A) and body weight changes of mice (B) treated with F8(scDb)-IL15 (F8F8-IL15), F8(scDb)-SD-IL15 (F8F8-SD-IL15) and PBS as a negative control.

Figure 20 shows the biochemical properties of the purified R6N(scDb)-IL15 conjugate. Figure 20A shows the size exclusion chromatography profile of the R6N(scDb)-IL15 conjugate. Figure 20B shows the results of an SDS-PAGE analysis of the R6N(scDb)-IL15 conjugate under reducing (R) and non-reducing (NR) conditions. Figure 20C shows the induction of T cell proliferation by the purified R6N(scDb)-IL15 conjugate.

Figure 21 shows the biochemical properties of the purified IL15-R6N(scDb) conjugate. Figure 21 A shows the size exclusion chromatography profile of the IL15-R6N(scDb) conjugate. Figure 21 B shows the results of an SDS-PAGE analysis of the IL15-R6N(scDb) conjugate under reducing (R) and non-reducing (NR) conditions. Figure 21 C shows the induction of T cell proliferation by the purified IL15-R6N(scDb) conjugate. Figure 22 shows the results of FPLC profile analysis of conjugates R6N(scDb)-SD-IL15 (A) and R6N(scDb)-IL15-SD (B).

Figure 23 shows the results of SDS-PAGE analysis (NR: non-reducing conditions, R: reducing conditions) of conjugates R6N(scDb)-SD-IL15 (A) and R6N(scDb)-IL15-SD (B).

Detailed Description

Conjugate

Conjugates of the invention comprise IL15, and a single-chain diabody. In some preferred embodiments, the conjugates of the invention further comprises a sushi domain of IL15R alpha (sushi domain).

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 IL15 and a single-chain diabody. Alternatively, the conjugate may be a single-chain protein comprising IL15, a sushi domain, and a single-chain diabody. The single-chain protein may be a fusion protein, for example a single-chain fusion protein comprising IL15, a single-chain diabody and optionally a sushi domain. 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 IL15. Similarly, the conjugate preferably comprises only one sushi domain.

Where the conjugate does not comprise a sushi domain, the IL15 is linked to the C-terminus of the single-chain diabody.

Where the conjugate comprises a sushi domain, the sushi domain is linked to the C-terminus of the single-chain diabody and the IL15 is linked to the C-terminus of the sushi domain.

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 I L15 or a sushi domain. The ED-A and the ED-B Domain of Fibronectin

Fibronectin (FN) is a multimodular glycoprotein found abundantly in the extracellular matrix (ECM) of various connective tissues. FN regulates a wide spectrum of cellular and developmental functions, including cell adhesion, migration, growth, proliferation and wound healing.

FN is secreted from cells as a dimer consisting of two -250 kDa subunits covalently linked by a pair of disulfide bonds near their C-termini. Each monomer of FN consists of three types of homologous repeat subunits termed FNI, FNII and FNIII domains, with binding affinity for various ECM proteins. FN contains 12 FNI, 2 FNII and 15-17 FNIII domains. Based on solubility and tissue distribution, FN occurs in two principal forms, the soluble plasma FN (pFN) circulating in the blood, and the cellular FN (cFN), which polymerizes into insoluble fibers in the ECM of connective tissues.

In the plasma, the pFN dimer does not polymerize and adopts a compacted conformation. cFN on the other hand is synthesized by various cell types including fibroblasts, smooth muscle cells and endothelial cells.

Even though coded by a single gene, FN exists in multiple isoforms as a result of alternative splicing of the precursor mRNA.

Splicing occurs at three sites, including the complete 90 amino acid domain ED-A or EIIIA, located between 11 FNIII and 12FNIII, the complete 91 amino acid ED-B or EIIIB domain located between the 7FNIII and 8FNIII domain, and various portions of the 120 amino acid V (variable) or IIICS (connecting segment) domain present between domains 14FNIII and 15FNIII.

All possible combinations of ED-A and ED-B (A+B+, A+B-, A-B+ and A-B-) exist in cFN. Thus, cFN represents a heterogeneous group of isoforms resulting from developmental^ regulated, species-specific and cell-specific splicing patterns, giving rise to about 20 different isoforms of cFN in humans. Contrary to cFN, pFN is almost always devoid of the ED-A and ED-B alternatively spliced domains.

ED-A and ED-B containing splice variants of FN are sometimes referred to as oncofetal variants because they are expressed typically during embryonic development and are re-expressed in adults during cancer.

The presence of ED-A and ED-B domains in adulthood is very restricted in normal tissue, but prominent in highly remodeling ECM for example during wound healing, atherosclerosis, liver and pulmonary fibrosis, and in vascular tissue and stroma of many cancer types. The most established mechanism of delivering drugs to the site of tumor is by using antibodies, which are raised against antigens that are highly and specifically expressed in tumors. As mentioned above, both ED-A and ED-B represent such antigens as they are highly expressed in the tumor neovasculature but are not abundantly found in the adult tissues.

Over the years, the current applicant has developed a number of anti-cancer agents, including targeted cytokines (“immunocytokines”) based on the anti-ED-A antibody“F8” and on the anti-ED- B“L19”.

Reference to the work on the anti-ED-A“F8” antibody and conjugates thereof can be found in W02008/120101 , W02009/013619, W02009/056268, WO2010/078945, WO2010/078950, WO2011/015333, W02012/041451 , W02013/014149, WO2014/055073, WO2014/173570, W02014/174105, WO2015/114166, W02016/180715, WO2017/009469, WO2018/069467, WO2018/087172 and WO2018/224550,

Reference to the work on the anti-ED-B“L19” antibody and conjugates thereof can be found in W02001/062298, W02003/076469, W02005/023318, W02006/119897, W02007/115837, W02007/128563, W02009/089858, WO2013/010749, WO2013/045125, WO2017/178562,

WO2018/011404, WO2018/154517, WO2019/154896 and WO2019/185792.

The Domain D of Tenascin C

Tenascin-C is a glycoprotein of the extracellular matrix. It comprises several fibronectin type 3 homology repeats that can be either included or omitted in the primary transcript by alternative splicing, leading to small and large isoforms that have distinct biological functions. Whereas the small isoform is expressed in several tissues, the large isoform of tenascin-C exhibits a restricted pattern of expression. It is virtually undetectable in healthy adult tissues but is expressed during embryogenesis and is expressed in adult tissues undergoing tissue remodeling including neoplasia.

Traditionally, the literature has referred to the large isoform of tenascin-C for tenascin molecules, which would putatively comprise all alternatively spliced domains A1 , A2, A3, A4, B, AD, C, D, and to the small isoform of tenascin-C whenever these domains were absent.

Antibodies that bind to the domain D of Tenascin-C have been generated by the applicant of the present application: for example“P12”,“F4” and“D11” antibodies (Brack et a!., Clin. Cancer Res. (2006) 12, 3200-3208 and in W02006/050834),“CPR01” and“CPR01.1” (WO2017/097990), as well as the R6N scDb described herein. Single-chain diabodies

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 et aL, 1993).

The conjugate of the invention 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.

An example of this format is shown in Figure 1 and Figures 9A-D.

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. 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. Suitable linkers are disclosed herein.

Single-chain diabodies have been previously generated (Kontermann & Muller, 1999). A bispecific single-chain diabody has been used to target adenovirus to endothelial cells (Nettelbeck et a!., 2001).

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).

The single-chain diabody may bind any antigen suitable for targeting the conjugate to the tumor or tumor microenvironment. Preferably, the single-chain antibody binds an antigen associated with neoplastic growth, e.g. an antigen, such as a cell-surface antigen, expressed on tumor cells and/or tumor-associated immune cells, or an extracellular 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.

In one embodiment, the specific binding member binds ECM components associated with neoplastic growth and/or angiogenesis, such as fibronectin or tenascin C.

In a preferred embodiment, 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 B-FN, e.g. it may bind domain ED-B (extra domain B). In an alternative embodiment, an antigen-binding site in the single-chain diabody binds fibronectin isoform A-FN, e.g. it may bind ED-A (Extra Domain-A).

In an alternative preferred embodiment, the single-chain diabody binds domain D of Tenascin C. Tenascin C is an antigen subject to alternative splicing, and a number of alternative isoforms of tenascin C are known, including alternatively spliced domain D. The single-chain diabody may selectively bind to domain D of Tenascin C selectively expressed in the extracellular matrix. An antigen-binding site in the single-chain diabody may bind domain D of Tenascin C.

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 expressed on tumor cells and/or tumor- associated immune cells, or an extracellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis. The antigen may be an antigen preferentially expressed by cells of a tumor or tumor 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 R6N, 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 R6N.

The amino acid sequences of the VH and VL of F8 are:

SEQ ID NO: 1 (VH)

SEQ ID NO: 2 (VL)

The amino acid sequences of the CDRs of F8 are:

SEQ ID NO: 3 (CDR1 VH);

SEQ ID NO: 4 (CDR2 VH);

SEQ ID NO: 5 (CDR3 VH);

SEQ ID NO: 6 (CDR1 VL);

SEQ ID NO: 7 (CDR2 VL), and

SEQ ID NO: 8 (CDR3 VL).

The amino acid sequences of the VH and VL of L19 are:

SEQ ID NO: 24 (VH)

SEQ ID NO: 25 (VL)

The amino acid sequences of the CDRs of L19 are:

SEQ ID NO: 27 (CDR1 VH);

SEQ ID NO: 28 (CDR2 VH);

SEQ ID NO: 29 (CDR3 VH); SEQ ID NO: 30 (CDR1 VL);

SEQ ID NO: 31 (CDR2 VL), and

SEQ ID NO: 32 (CDR3 VL).

The amino acid sequences of the VH and VL of R6N are:

SEQ ID NO: 73 (VH)

SEQ ID NO: 74 (VL)

The amino acid sequences of the CDRs of R6N are:

SEQ ID NO: 67 (CDR1 VH);

SEQ ID NO: 68 (CDR2 VH);

SEQ ID NO: 69 (CDR3 VH);

SEQ ID NO: 70 (CDR1 VL);

SEQ ID NO: 71 (CDR2 VL), and

SEQ ID NO: 72 (CDR3 VL).

In one aspect, the conjugate of the invention comprises I L15 joined to the C-terminus of a single chain diabody, for example a single-chain diabody comprising the VH and VL domains of antibody L19, F8 or R6N.

In a preferred aspect, the conjugate of the invention comprises IL15 and a sushi domain of IL15 Receptor alpha (IL15R alpha), wherein the sushi domain is joined to the C-terminus of a single chain diabody, for example a single-chain diabody comprising the VH and VL domains of antibody L19, F8 or R6N, and the IL15 is joined to the C-terminus of the sushi domain.

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 F8 VH domain amino acid sequence set forth in SEQ ID NO: 1 , the L19 VH domain amino acid sequence set forth in SEQ ID NO: 24, or the R6N VH domain amino acid sequence set forth in SEQ ID NO: 73.

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 F8 VL domain amino acid sequence set forth in SEQ ID NO: 2, the L19 VL domain amino acid sequence set forth in SEQ ID NO: 25, or the R6N VL domain amino acid sequence set forth in SEQ ID NO: 74. 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: 403-410), 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 (Altschul et ai, 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 of the invention. 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 set forth in SEQ ID NO: 9 (F8 scDb), 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: 9.

Alternatively, the single-chain diabody may comprise the sequence set forth in SEQ ID NO: 33 (L19 scDb), 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: 33.

As a further alterative, the single-chain diabody may comprise the sequence set forth in SEQ ID NO: 75 (R6N scDb), 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: 75. Interleukin-15

The conjugate of the invention comprises IL15. The IL15 may be derived from any animal, e.g. human, rodent (e.g. rat, mouse), horse, cow, pig, sheep, dog, etc. Human IL15 is preferred in conjugates for administration to humans. The amino acid sequence of human IL15 is set out in SEQ ID NO: 13. The conjugate of the invention preferably comprises a single IL15 polypeptide.

Typically, the IL15 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 shown in SEQ ID NO: 13.

IL15 in conjugates of the invention retains a biological activity of IL15, e.g. an ability to promote production of proinflam matory cytokines TNFa, IL-1 , and/or IFNy, the proliferation and Ig synthesis of activated B cells, the activation of TH1 cells, monocytes, and/or lymphokine activated killer cells, the proliferation of mast cells and/or T cells, and/or to inhibit the apoptosis of T cells and/or B cells. In particular, retention of biological activity of IL15 in conjugates of the invention may be tested by determining the ability of the conjugate to stimulate the proliferation of CTLL-2 cells.

The IL15 is conjugated to the C-terminus of the single-chain diabody.

Where the conjugate does not comprise a sushi domain, the IL15 is conjugated to the C-terminus of the single-chain diabody either directly, or preferably via a peptide linker. Suitable peptide linkers are described herein. In a preferred embodiment, the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 14 (F8(scDb)-IL15), SEQ ID NO: 23 (L19(scDb)-IL15), or SEQ ID NO: 76 (R6N(scDb)-IL15).

Where the conjugate comprises a sushi domain, the sushi domain is linked to the C-terminus of the single-chain diabody, and the IL15 is linked to the C-terminus of the sushi domain. Linkage may be direct but preferably is via a peptide linker, such as a peptide linker as described herein. In a preferred embodiment, the conjugate comprises or consists of the sequence set forth in SEQ ID NO: 41 (F8(scDb)-SD-IL15), SEQ ID NO: 64 (L19(scDb)-SD-IL15), or SEQ ID NO: 78 (R6N(scDb)- SD-IL15).

IL15 is a heavily glycosylated protein. It is generally preferable to avoid glycosylation, as glycosylation may interfere with conjugate production, including batch consistency, and result in more rapid clearance of the conjugate from the patient’s body. Preferably, a conjugate of the present invention, and in particular the IL15 present in a conjugate of the present invention, is not glycosylated. Thus, the IL15 included in a conjugate of the invention may comprise or consist of the sequence shown in SEQ ID NO: 13, except that the residue at position 79 of SEQ ID NO: 13 is a glutamine, serine, or alanine residue rather than an asparagine residue. In a preferred embodiment, the residue at position 79 of SEQ ID NO: 13 is a glutamine rather than an

asparagine. Thus preferably, the mutant IL15 included in a conjugate of the invention comprises or consists of the sequence set forth in SEQ ID NO: 38.

Sushi Domain of Interleukin-15 Receptor Alpha

The Sushi Domain (SD) is the shortest region of the IL15 Receptor alpha capable of binding IL15.

In addition, the sushi domain has been shown to stabilize the conjugate and enhance the activity of IL15. It has been previously shown that a fusion protein with IL15 and a portion of IL15R alpha was able to increase the activity of IL15 (Kermeret al., 2012). In clinical studies (Phase I completed) a product named ALT803 consisting of a novel IL15 superagonist complex (an IL15 mutant

IL15N72D bound to an IL15 receptor alpha and to an lgG1 Fc fusion protein) was assessed in patients with advanced solid tumors in combination with Nivolumab (Wrangle et al., 2018).

Where the conjugate comprises a sushi domain, the sushi domain is linked to the C-terminus of the single-chain diabody, either directly, or preferably via a peptide linker. Suitable peptide linkers are described herein.

The amino acid sequence of human lnterleukin-15R alpha is set forth in SEQ ID NO: 39. The sushi domain may be a fragment of IL15R alpha comprising the sushi domain IL15R alpha. The amino acid sequence of the sushi domain of human IL15R alpha is set forth in SEQ ID NO: 40. The sushi domain 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 shown in SEQ ID NO: 40.

Linkers

In the conjugates of the invention, the single-chain diabody and IL15, single-chain diabody and sushi domain, and/or sushi domain and IL15, 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.

In a preferred embodiment, the linker connecting the single-chain diabody and IL15 in a conjugate not comprising a sushi domain may be 12 to 15 amino acids in length, e.g. 12 or 15 amino acids in length. Examples of suitable linker sequences are set forth in SEQ ID NOs 10 and 36. In a conjugate comprising a sushi domain, the linker connecting the single-chain diabody and sushi domain is preferably 12 to 15, for example 14 to 15 amino acids in length. The linker may be 14 amino acids or 15 amino acids in length. Examples of suitable linkers are set forth in SEQ ID NOs 36 and 88. The linker connecting the sushi domain and IL15 in such conjugates is preferably 12 to 20 amino acids in length, for example 12 amino acids or 20 amino acids in length. Examples of a suitable linker sequences are set forth in SEQ ID NOs 10 and 37.

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 acids, e.g. 15 to 20 amino acids. Single-chain diabodies have been previously generated and described by Kontermann, R. E., and Muller, R. (1999), J. Immunol. Methods 226: 179-188. Examples of such linkers are set forth in SEQ ID NOs 11 and 81.

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. The non-flexible linker may be about 5 amino acids in length. Examples of non- flexible linker sequences are set forth in SEQ ID NOs 12 and 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 IL15, or single-chain diabody, sushi domain and IL15, may be covalently linked. For example, by peptide bonds (amide bonds).

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, or 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. Also contemplated is a method of treating cancer 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. Further provided is a conjugate of the invention for use in a method of delivering IL15 to a tumor in a patient, as well as a method of delivering IL15 to a tumor in a patient comprising administering to the patient a conjugate of the invention.

A conjugate of the invention may be for use in a method of treating cancer by targeting IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in vivo. Also contemplated is a method of treating cancer by targeting IL15 to the tumor or tumor microenvironment, such as the neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate of the invention to the patient. Further provided is a conjugate of the invention for use in a method of delivering IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in a patient, as well as a method of delivering IL15 to the tumor or tumor microenvironment, such as the tumor neovasculature in a patient comprising administering to the patient a conjugate of the invention.

In addition, or alternatively, the conjugate of the invention may be for use in a method of treating cancer by targeting IL15 to an extracellular matrix component associated with neoplastic growth and/or angiogenesis in a patient. Also contemplated is a method of treating cancer by targeting IL15 to an extracellular matrix component associated with neoplastic growth and/or angiogenesis in a patient, the method comprising administering a therapeutically effective amount of a conjugate of the invention to the patient. Further provided is a conjugate of the invention for use in a method of delivering IL15 to an extracellular matrix component associated with neoplastic growth and/or angiogenesis in a patient, as well as a method of delivering IL15 to an extracellular matrix component associated with neoplastic growth and/or angiogenesis in a patient comprising administering to the patient a conjugate of the invention.

Conditions treatable using the conjugate of the invention include cancer, other tumors and neoplastic conditions. Treatment may include prophylactic treatment. The cancer may comprise an extracellular matrix component associated with neoplastic growth and/or angiogenesis, such as an isoform of fibronectin comprising domain ED-A and/or ED-B, the domain D of tenascin C.

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. tumor or tumor 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.

tumor).

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. A pharmaceutical composition comprising a conjugate of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Other treatments may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics, as well as a second anti-cancer therapy.

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 of the invention. 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: 117; Larrick & Thomas (2001) Current Opinion in Biotechnology 12:411-418. 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 and production of conjugates comprising a scDb or Db specific for the ED-A or ED-B of fibronectin and IL15

1.1 Cloning procedure for F8(scDb)-IL 15

The genes encoding for F8 and human IL15 were PCR amplified, PCR assembled and cloned into a mammalian expression vector pcDNA3.1 (+) using Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify F8 (VH-VL) with:

“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEC ID NO: 15) and“L19-SSG Ba”

(CGGAAGAGCT ACT ACCCG AT G AGG AAG ATTT G ATTTCCACCTT GGTCC) (SEC ID NO: 16).

The resulting fragment was PCR amplified with

“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEC ID NO: 15) and“Link-BamHI ba”

(GCCGCTGGACGAGGATCCGGAAGAGCTACTACCCGATGAGGAAGA) (SEC ID NO: 17) to add the restriction site for BamHI. The PCR product was digested with Nhel and BamHI and ligated into pCDNA 3.1 vector digested with same enzymes. IL15 was PCR amplified from a synthetic gene with“GDGS-IL15 fwd”

(TCTTCAGGCGGCTCTGGCGGAGCTTCCAACTGGGTGAATGTAATAAG) (SEQ ID NO: 18) and “Notl-STOP-IL15 ba” (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

With the aim to obtain the second F8 (VH-VL) primers“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “GDGS-IL15-kappa ba”

(CCGCCAGAGCCGCCTGAAGAGCCGTCACCTTTGATTTCCACCTTGGTCC) (SEQ ID NO: 21) were used.

The two fragments were PCR assembled with primers“BamHI-L19VH fo” and“Notl-STOP-IL15 ba”. The final product was digested with BamHI and Notl and ligated into pCDNA 3.1 F8 (VH-VL) vector digested with the same enzymes. The amino acid sequence of the F8(scDb)-IL15 conjugate is shown in SEQ ID NO: 14.

1.2 Cloning procedure for L19(scDb)-IL15

The genes encoding L19 and human IL15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify the first L19(VH-VL) adding a Nhel Leader sequence, Nhel restriction site and a part of the (S4G)3 linker. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“L19-SSG Ba”

(CGGAAGAGCT ACT ACCCGAT GAGGAAGATTT GATTTCCACCTT GGTCC) (SEQ ID NO: 16).

A second PCR was performed to amplify the first L19(VH-VL) adding a BamHI restriction site. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Link-BamHI ba”

(GCCGCTGGACGAGGATCCGGAAGAGCTACTACCCGATGAGGAAGA) (SEQ ID NO: 17).

The PCR product was digested with Nhel and BamHI restriction enzymes and ligated in the expression vector digested with the same enzymes.

A third PCR was performed to amplify the second L19(VH-VL) adding a part of the (S4G)3 linker, a BamHI restriction site and a part of the GDGSSGGSGGAS linker. Primers used were:“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “GDGS-IL15-kappa ba 2”

(GGACCAAGGTGGAAATCAAAGGTGACGGCTCTTCAGGCGGCTCTGGCGG) (SEQ ID NO: 34).

A fourth PCR was performed to amplify hi L15 adding: a part of GDGSSGGSGGAS linker and Notl restriction site. Primers used were:“GDGS-IL15 fwd”

(TCTTCAGGCGGCTCTGGCGGAGCTTCCAACTGGGTGAATGTAATAAG) (SEQ ID NO: 18) and “Notl-STOP-IL15 ba” (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A fifth PCR was performed to assemble the third and the fourth PCR products. Primers used were: “BamHI-L19VH fo” (CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19). The final PCR product was digested with BamHI and Notl restriction enzymes and ligated in the pCDNA 3.1 expression vector containing the first L19(VH-VL) digested with the same enzymes. The amino acid sequence of the L19(scDb)-IL15 conjugate is shown in SEQ ID NO: 23.

1.3 Cloning procedure for F8(Db)-IL 15

The genes encoding F8 and human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using Nhel/Notl restriction enzymes.

Primers were designed to amplify F8(VH-VL) adding a Nhel Leader sequence, a Nhel restriction site and a part of the GDGSSGGSGGAS linker. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“GDGS-IL15-kappa ba”

(CCGCCAGAGCCGCCTGAAGAGCCGTCACCTTTGATTTCCACCTTGGTCC) (SEQ ID NO: 21).

A second PCR was performed to amplify hi L15 adding a part of the GDGSSGGSGGAS linker and a Notl restriction site. Primers used were:“GDGS-IL15 fwd”

(TCTTCAGGCGGCTCTGGCGGAGCTTCCAACTGGGTGAATGTAATAAG) (SEQ ID NO: 18) and “Notl-STOP-IL15 ba” (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A third PCR was performed to assemble the first and the second PCR products. Primers used were: Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

The final PCR product was digested with Nhel and Notl restriction enzymes and ligated in the pcDNA3.1 (+) expression vector digested with the same enzymes. The amino acid sequence of the F8(Db)-IL15 conjugate is shown in SEQ ID NO: 35.

1.4 Cloning procedure for IL 15-F8(scDb)

The genes encoding F8 and human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify the first F8(VH-VL) adding: a part of the GDGSSGGSGGAS linker, a part of the (SSSSG)3 linker and the BamHI restriction site. Primers used were“24-RC GAS-F8 Vh Fo”

(GGCTCTTCAGGCGGCTCTGGCGGAGCTTCCGAGGTGCAGCTGTTGGAGTCTG) (SEQ ID NO:

65) and“Link-BamHI ba”

(GCCGCTGGACGAGGATCCGGAAGAGCTACTACCCGATGAGGAAGA) (SEQ ID NO: 17).

A second PCR was performed to amplify hlL15 by adding the Leader Sequence, the Nhel restriction site and a part of GDGSSGGSGGAS linker. Primers used were“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“25-RC I L15-GAS Ba”

(CTCCGCCAGAGCCGCCT GAAGAGCCGTCACCAGAAGT GTT GAT GAACATTT G) (SEQ ID NO:

66).

A third PCR was performed to assemble the first and the second PCR products. Primers used were Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“25-RC I L15-GAS Ba”

(CTCCGCCAGAGCCGCCT GAAGAGCCGTCACCAGAAGT GTT GAT GAACATTT G) (SEQ ID NO: 66). The final PCR product was digested with Nhel and BamHI restriction enzymes and ligated in the expression vector, containing the second F8(VH-VL), digested with the same enzymes. The amino acid sequence of the IL15-F8(scDb) conjugate is shown in SEQ ID NO: 22.

1.5 Production of conjugates

The conjugates were expressed using transient gene expression (TGE) in CHO-S cells. For 1 mL of production 4 ^c 10 ⁶ CHO-S cells in suspension were centrifuged and resuspended in 1 mL of a suitable medium. 0.625-0.9 mg of plasmid DNA 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. Proteins were purified from the cell culture medium by protein A affinity chromatography and then dialyzed against PBS or Tris 50mM, NaCI 100mM buffer. The yield obtained for each of the conjugates is shown in Table 1.

Table 1

Example 2 - In vitro characterization of conjugates comprising a scDb specific for the ED-A or ED- B of fibronectin and IL15

2.1 Protein characterization

SDS-PAGE of the F8(scDb)-IL15, IL15-F8(scDb), L19(scDb)-IL15 and F8(Db)-IL15 conjugates was performed with 4-12% Bis-Tris gel under reducing and non-reducing conditions.

The F8(scDb)-IL15, IL15-F8(scDb), L19(scDb)-IL15 and F8(Db)-IL15 conjugates were analyzed by size-exclusion chromatography using a Superdex 200 increase 10/300 GL column on an AKTA FPLC.

For ESI-MS analysis, samples of F8(scDb)-IL15 were diluted to about 0.1 mg/ml_ and LC-MS was performed on a Waters Xevo G2XS Qtof instrument (ESI-ToF-MS) coupled to a Waters Acquity UPLC H-Class System using a 2.1 ^c 50 mm Acquity BEH300 C4 1.7 pm column.

2.2 Affinity measurement

Affinity measurements of F8(scDb)-IL15 and F8(Db)-IL15 were performed by surface plasmon resonance using a BIAcore X100 instrument and an ED-A coated CM5 chip whereas affinity measurements of L19(scDb)-IL15 were performed by surface plasmon resonance using a BIAcore X100 instrument and an ED-B coated CM5 chip. The F8(scDb)-IL15 sample was injected at a concentration of 100nM whereas the L19(scDb)-IL15 and F8(Db)-IL15 samples were injected as serial-dilutions, in a range concentration from 62.5nM to 1000nM. Regeneration of the chip was performed by HCI 10 mM.

2.3 Biological activity

The biological activity of IL15 in the F8(scDb)-IL15, IL15-F8(scDb) and L19(scDb)-IL15 conjugates was determined by assessing the ability of the conjugates to stimulate the proliferation of CTLL-2 cells. Cells were seeded in 96-well plates in the culture medium supplemented with varying concentrations of the F8(scDb)-IL15, IL15-F8(scDb) or L19(scDb)-IL15 conjugates. After incubation at 37°C for 72 hours, cell proliferation was determined with Cell Titer Aqueous One Solution.

2.4 Results and Conclusions

The conjugates F8(scDb)-IL15, the L19(scDb)-IL15 and F8(Db)-IL15 were produced with excellent quality as evidenced by the single peak in gel filtration (Figure 2A, 3A and 4A) whereas IL15- F8(scDb) was much difficult to produce due to the presence of aggregates (Figure 8A). The two bands seen for each conjugate in the SDS-PAGE analysis were shown to be due to the heavy glycosylation of IL15 (Figure 2B, 3B, 4B and 8B). The presence of N-linked glycans was confirmed by MS analysis of the F8(scDb)-IL15 conjugate (Figure 2C). BIAcore analysis confirmed that the F8(scDb)-IL15 and F8(Db)-IL15 conjugates were able to recognize and bind ED-A (Figure 5A and 5B) and that the L19(scDb)-IL15 conjugate was able to recognize and bind ED-B (Figure 5C). The activity assay based on CTLL2 cells showed that the F8(scDb)-IL15 and L19(scDb)-IL15 conjugates were further able to induce T cell proliferation (Figure 6A and 6B) whereas IL15- F8(scDb) was not able to induce T cell proliferation (Figure 6C), thereby demonstrating that a conjugate format where the IL15 moiety is conjugated to the C-terminus of the scDb or Db is superior to formats where the IL15 is conjugated to the N-terminus.

Example 3 - In vivo characterization of conjugates comprising a scDb or Db specific for the ED-A or ED-B of fibronectin and IL15

3. 1 Biodistribution studies

The ability of the F8(scDb)-IL15, L19(scDb)-IL15 and F8(Db)-IL15 conjugates to target ED-A in vivo was assessed by quantitative biodistribution analysis.

Purified samples (100 mg) of the conjugates were radioiodinated with ¹²⁵ l and Chloramine T hydrate and purified on a PD10 column (GE Healthcare). Radiolabeled conjugates were injected into the lateral tail vein of 129SvEv mice bearing subcutaneously implanted F9 lesions. Injected dose per mouse varied between 4 and 10 mg. Mice were sacrificed 24 hours after injection. Organ samples were weighed and radioactivity was counted using a Packard Cobra gamma counter. The conjugate uptake in the organs and tumour was calculated and expressed as the percentage of the injected dose per gram of tissue (%ID/g ± standard error). Data were corrected for tumor growth.

3.2 Results and Conclusions

The biodistribution data showed a surprisingly high uptake of the F8(scDb)-IL15 and L19(scDb)- IL15 conjugates in the tumor of around 15% percent and 8% of the injected dose per gram

(%ID/g), respectively, with a good tumor to organ/blood ratio (Figure 7A and B). In contrast, the F8(Db)-IL15 conjugate showed very low tumor uptake of about 2% and a poor tumor to

organ/blood ratio (Figure 7C). This is similar to the results reported Kaspar et al. (2007), in which a L19(Db)-IL15 conjugate was shown to accumulate in tumors at a level of less than 2% ID/g in the tumor and also exhibited a poor tumor to organ/blood ratio when biodistribution of the conjugate was tested using the same mouse strain, tumor model and a comparable conjugate dose. The results in Figure 7 thus clearly demonstrate the superiority of the tumor-targeting properties of the single-chain diabody-based conjugate format of the invention over a diabody-based conjugate format disclosed in Kaspar et al. (2007), and further demonstrate that these superior tumor targeting properties are observed independently of the target bound by the single-chain diabody. Based on these results, it is therefore expected that other conjugates comprising IL15 conjugated to the C-terminus of a single-chain diabody which binds an antigen capable of targeting the conjugate to the tumor or tumor microenvironment will exhibit similarly superior tumor-targeting properties as the conjugates exemplified herein.

Example 4 - Cloning and production of conjugates comprising a scDb or scFv specific for the ED-A or ED-B of fibronectin, a sushi domain and IL15

4.1 Cloning procedure for F8(scDb)-SD-IL 15

The genes encoding F8, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using

Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify IL15 from a synthetic gene adding a part of the (G3S, G4S)2LQ linker:“RC-9 Fo” (CGGATCGGGAGGCGGGGGTAGTCTGCAAAACTGGGTGAATGTAATAAG) (SEQ ID NO: 46) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A second PCR was performed to amplify the previous fragment adding and finishing the (G3S, G4S)2LQ linker:“RC-10 Fo” (GAGGTAGCGGCGGTGGGGGAAGTGGTGGCGGATCGGGAGGCGGGGGTAG) (SEQ ID NO: 47) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A third PCR product was prepared to obtain the sushi domain fragment on the synthetic gene using“RC-1 1 Fo”

(CAGGCGGAGGTGGCTCTGGCGGCGGTGGGTCAATCACGTGCCCTCCCCCCATG) (SEQ ID NO: 48), an“RC-12 Ba” (CTTCCCCCACCGCCGCT ACCTCCCCCTCT AAT GCATTT GAG ACT GG) (SEQ ID NO: 49).

To amplify the second F8“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “GGGGS3-VL-kappa ba”

(CAGAGCCACCTCCGCCTGAACCGCCTCCACCTTTGATTTCCACCTTGGTCC) (SEQ ID NO:

50) were used.

The last three fragments described above were then assembled by PCR, digested with Bam HI and Notl and cloned into the pCDNA 3.1 vector digested with the same enzymes already carrying the first F8. The amino acid sequence of the F8(scDb)-SD-IL15 conjugate is shown in SEQ ID NO: 41.

4.2 Cloning procedure for F8(scDb)-IL 15-SD

The genes encoding F8, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using

Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify IL15 from an in-house plasmid adding a part of the (G4S) ₃ linker with“RC-20 Fo”

(TTCAGGCGGAGGTGGCTCTGGCGGCGGTGGGTCAAACTGGGTGAATGTAATAAG) (SEQ I D NO: 62) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A second PCR was performed to amplify the second F8 (VH and VL) with“BamHI-L19VH fo” (CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “GGGGS3-VL-kappa ba”

(CAGAGCCACCTCCGCCTGAACCGCCTCCACCTTTGATTTCCACCTTGGTCC) (SEQ ID NO: 50). The two PCR fragments described above (one carrying the second F8 and the other the IL15-SD) were PCR assembled with:“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “Notl-STOP-IL15 ba” (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

The PCR product was digested with BamHI and Notl and cloned into the F8(Db) pCDNA 3.1 vector digested with the same enzymes already carrying the first F8. The amino acid sequence of the F8(scDb)-IL15-SD conjugate is shown in SEQ ID NO: 42.

4.3 Cloning procedure for IL 15-SD-F8(scDb)

The genes encoding for F8, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using

Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify IL15 from an in-house plasmid adding a part of the (G3S, G4S)2LQ linker with“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“13-RC Ba”

(CACCACTTCCCCCACCGCCGCT ACCTCCCCCAGAAGT GTT GAT GAACATTT G) (SEQ ID NO: 51). A PCR was performed on the previous fragment with the aim to finish the (G3S, G4S)2LQ with “nhelleader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“14-RC Ba”

(TTGCAGACTACCCCCGCCTCCCGATCCGCCACCACTTCCCCCACCGCCGCTAC) (SEQ ID NO: 52).

Another PCR amplification was conducted to amplify the sushi domain with:“15-RC Fo”

(CGGGAGGCGGGGGTAGTCTGCAAATCACGTGCCCTCCCCCCATGTC) (SEQ ID NO: 53) and “16-RC Ba”

(CGCCGCCAGAGCCACCTCCGCCT GAACCGCCTCCACCTCT AAT GCATTT GAGACTGG) (SEQ ID NO: 54).

A PCR was performed to amplify the first F8 (VH and VL) with“17-RC Fo”

(CGGAGGTGGCTCTGGCGGCGGTGGGTCAGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 55) and“L19-SSG Ba” (CGGAAG AGCT ACT ACCCGAT G AGG AAGATTT GATTTCCACCTT GGTCC) (SEQ ID NO: 16). The second F8 was PCR amplified with:“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “Not Stop DPK Ba<”

(T AGTTT AGCGGCCGCATTCTT ATTCACT ATTT GATTTCCACCTTGGTCCCTTGGCC) (SEQ I D NO: 56).

All of the above fragments were PCR assembled and the final products were digested and inserted into the pCDNA 3.1 vector. The amino acid sequence of the IL15-SD-F8(scDb) conjugate is shown in SEQ ID NO: 43.

4.4 Cloning procedure for SD-IL15-F8(scDb)

The genes encoding F8, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using

Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify SD and IL15 from an in-house plasmid linker with“18-RC Fo” (CTGTTCCTCGTCGCTGTGGCTACAGGTGTGCACTCGATCACGTGCCCTCCCCCC) (SEQ I D NO: 57) and“19-RC Ba”

(CCGCCAGAGCCACCTCCGCCT GAACCGCCTCCACCAGAAGT GTT GAT GAACATTT G) (SEQ ID NO: 58).

A PCR was performed to amplify the first F8 (VH and VL) with“17-RC Fo”

(CGGAGGTGGCTCTGGCGGCGGTGGGTCAGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 55) and“L19-SSG Ba” (CGGAAG AGCT ACT ACCCGAT G AGG AAGATTT GATTTCCACCTT GGTCC) (SEQ ID NO: 16).

A PCR assembly was done to obtain SD-IL15-F8 (VH and VL) with the two previous PCRs with the primers:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Link-BamHI ba”

(GCCGCTGGACGAGGATCCGGAAGAGCTACTACCCGATGAGGAAGA) (SEQ ID NO: 17).

The product was digested with Nhel and BamHI and ligated into the pCDNA3.1 vector containing the second F8 (VH and VL) digested with the same enzymes. The amino acid sequence of the SD- IL15-F8(scDb) conjugate is shown in SEQ ID NO: 44. 4.5 Cloning procedure for F8(scFv)-SD-IL15

To clone the F8(scFv)-SD-IL15 conjugates (SEQ ID NO: 59), the genes encoding F8, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector pcDNA3.1 (+) using Nhel/Notl restriction enzymes.

Primers were designed to amplify F8 scFv (VH-VL) adding a Nhel leader sequence, Nhel restriction site and a part of the (S4G)3 linker. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“30-RC GGGG -SD F8 Ba”

(CCACCGCTACCTCCCCCACCACTGCCACCGCCCCCTTTGATTTCCACCTTGGTCC) (SEQ I D NO: 60).

A second PCR was performed to amplify human IL15 and the sushi domain of IL15R alpha adding a part of the (S4G)3 linker and a Notl restriction site. Primers used were:“28-RC New GGGG -SD link Fo” (GTGGTGGGGGAGGTAGCGGTGGAGGGGGCTCCATCACGTGCCCTCCCCCCATG) (SEQ ID NO: 61) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A third PCR was performed to assemble the first and the second PCR products. Primers used were primers“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19). The final PCR product was digested with Nhel and Notl restriction enzymes and cloned into the pCDNA 3.1 expression vector digested with the same enzymes. The amino acid sequence of the F8(scFv)- SD-IL15 conjugate is shown in SEQ ID NO: 59.

4.6 Cloning procedure for L 19(scDb)-SD-IL 15

The genes encoding L19, human IL15 and the sushi domain of IL15R alpha were PCR amplified, PCR assembled and cloned into the mammalian expression vector using Nhel/BamHI/Notl restriction enzymes.

Primers were designed to amplify the first L19(VH-VL) adding: Nhel Leader sequence, Nhel restriction site and a part of the (S4G)3 linker. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“L19-SSG Ba”

(CGGAAGAGCT ACT ACCCG AT G AGG AAG ATTT G ATTTCCACCTT GGTCC) (SEQ ID NO: 16). A second PCR was performed to amplify the first L19(VH-VL) adding: BamHI restriction site.

Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Link-BamHI ba”

(GCCGCTGGACGAGGATCCGGAAGAGCTACTACCCGATGAGGAAGA) (SEQ ID NO: 17).

The PCR product was digested with Nhel and BamHI restriction enzymes and ligated in the expression vector digested with the same enzymes.

A third PCR was performed to amplify the second L19(VH-VL) adding: a part of the (S4G)3 linker, BamHI restriction site and a part of (G4S)3 linker.

Primers used were:“BamHI-L19VH fo”

(CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and “GGGGS3-VL-kappa ba”

(CAGAGCCACCTCCGCCTGAACCGCCTCCACCTTTGATTTCCACCTTGGTCC) (SEQ ID NO: 50).

A fourth PCR was performed to amplify hlL15 and the sushi domain of IL15R alpha adding: a part of (G4S)3 linker and Notl restriction site. Primers used were:“GGGGS3-SD fwd”

(TTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAATCACGTGCCCTCCCCCCAT) (SEQ ID NO: 63) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A fifth PCR was performed to assemble the third and the fourth PCR products. Primers used were: “BamHI-L19VH fo” (CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 20) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19). The final PCR product was digested with BamHI and Notl restriction enzymes and ligated in the expression vector containing the first L19(VH-VL) digested with the same enzymes. The amino acid sequence of the L19(scDb)-SD-IL15 conjugate is shown in SEQ ID NO: 64.

4.1 Production of conjugates

The conjugates were expressed and purified as described in Example 1.5. The yield obtained for each of the conjugates is shown in Table 2 Table 2

The results of the production using TGE shows that the IL15-SD-F8(scDb) conjugate, unlike the other three conjugates, could not be produced at a level compatible with industrial development. The results also show that the yield of the F8(scFv)-SD-l L15 conjugate was surprisingly only approximately a third of the yield of the F8(scDb)-SD-IL15 conjugate and thus significantly lower.

Example 5 - In vitro characterization of conjugates comprising a scDb specific for the ED-A of fibronectin, a sushi domain and IL15 5.1 Protein characterization

Analysis of conjugates by SDS-PAGE, size-exclusion chromatography and ESI-MS was performed as described in Example 2.1.

The results of the AKTA FPLC analysis shows that F8(scDb)-IL15-SD, unlike the other two conjugates tested (F8(scDb)-SD-IL15 and SD-IL15-F8(scDb)), cannot be eluted with a purity profile compatible with industrial development as this particular conjugate forms too many aggregates

(Figure 11).

The results of the EMI-MS analysis showed that F8(scDb)-SD-IL15, unlike the three other conjugates tested, had a different glycosylation pattern (Figure 12A) which may be more suitable for industrial development. 5.2 Affinity measurement

Affinity measurements were performed as described in Example 2.2, except that samples were injected as serial-dilutions in a concentration range from 125nM to 1000nM.

5.3 Biological activity

The biological activity of IL15 in conjugates was determined as set out in Example 2.3, except that cells were incubated with the conjugates at 37°C for 48 hours.

The results of the biological activity analysis show that F8(scDb)-SD-IL15, unlike the other two conjugates tested (IL15-SD-F8(scDb) and SD-IL15-F8(scDb)), had excellent T-cell proliferation stimulation activity (Figure 13)

5.4 Results and Conclusions

Surprisingly, the F8(scDb)-SD-IL15 construct showed better biochemical properties compared to other sushi domain-containing formats in which the orientation of the cytokine / cytokine receptor complex (IL15 / sushi domain) was different, in terms of production yield, gel filtration profile, glycosylation pattern and biological activity.

The F8(scDb)-SD-IL15 conjugate was produced with excellent quality as evidenced by a single peak in gel filtration (Figure 11 A). The two bands seen in SDS-PAGE are due to the heavily glycosylation of IL15 (Figure 10A). The presence of N-linked glycans was confirmed by MS analysis (Figure 12A). BIAcore analysis confirmed the ability of F8(scDb)-SD-IL15 to recognize and bind ED-A (Figure 14). The activity assay based on CTLL2 cells showed that the F8(scDb)- SD-IL15 conjugate was able to induce T cell proliferation (Figure 13A).

Table 3: Summary of the in vitro characterization results of the different immunoconjugate formats comprising a scDb specific for the ED-A of fibronectin, a sushi domain, and IL15 tested

Example 6 - In vivo characterization of conjugates comprising a scDb specific for the ED-A of fibronectin, a sushi domain and IL15

6.1 Immunofluorescence studies

Antigen expression was confirmed on ice-cold acetone fixed 8-pm cryostat sections of CT26 tumors stained with a biotinylated preparation of F8(scDb)-SD-IL15 (final concentration 5pg/ml_) and detected with Streptavidin AlexaFluor488. For vascular staining rat anti-mouse CD31 and donkey anti-rat AlexaFluor594 antibodies were used. Slides were mounted with Fluorescence Mounting Medium and analyzed with TIRF microscope.

For ex vivo immunofluorescence analysis, mice were injected with 50pg biotinylated F8(scDb)-SD- IL15 and sacrificed 24 hours after injection. Tumors were excised and embedded in cryo- embedding medium and cryostat section (8pm) were stained using the following antibodies: rat anti-mouse CD31 and donkey anti-rat AlexaFluor594 and Streptavidin AlexaFluor488. Slides were mounted with Fluorescence Mounting Medium and analyzed with TIRF microscope.

6.2 Biodist bution studies

The ability of F8(scDb)-SD-IL15 to target ED-A in vivo was assessed by quantitative biodisthbution analysis and compared to F8(scFv)-SD-IL15, to assess the effect of the single-chain diabody format on ED-A targeting, with KSF(scDb)-SD-IL15 employed as a negative control (the KSF antibody is specific to an irrelevant antigen). Quantitative biodisthbution analysis was performed as set out in Example 3.1.

6.3 Results and Conclusion

Microscopic fluorescence analysis of CT26 tumor sections confirmed ED-A expression in vivo, by staining of the tumor sections with the F8(scDb)-SD-IL15 conjugate (Figures 15 and 16).

Moreover, microscopic fluorescence analysis of tumor sections, obtained from animals injected with F8(scDb)-SD-IL15 24 hours after administration of the conjugate, confirmed that the conjugate localized to its cognate ED-A antigen within the tumor mass in vivo (Figure 17). Biodistribution analysis showed good uptake of the F8(scDb)-SD-IL15 conjugate in the tumor (around 16% percent of the injected dose per gram (%l D/g)) compared to the negative control KSF(scDb)-SD-IL15 conjugate (Figure 18). Importantly, the F8(scDb)-SD-IL15 conjugate showed superior tumor uptake compared with the F8(scFv)-SD-IL15 conjugate (around 14% percent of the injected dose per gram (%ID/g)), as well as an improved tumor to organ ratio, as evidenced in particular by the reduced unspecific accumulation of the F8(scDb)-SD-IL15 conjugate in the healthy spleen compared with the F8(scFv)-SD-IL15 conjugate. Specifically, the F8(scFv)-SD-IL15 conjugate showed approximately the same level of accumulation in the healthy spleen as in the tumor, while the accumulation of the F8(scDb)-SD-IL15 conjugate in the tumor was significantly higher than in healthy spleen (compare Figures 18 A and B).

Example 7 - Therapeutic efficacy of the F8(scDb)-IL15 and F8(scDb)-SD-IL15 conjugates in a lung metastatic tumor model

7.1 Therapy experiments

Female BALB/c mice were injected i.v. with 10 ⁵ C51 cells. 24 hours after, mice were divided into 3 groups (n=6) and were injected 3 times every 48 hours with: PBS (100 mI), F8(scDb)-IL15 (5pg/g) or F8(scDb)-SD-IL15 (0.6pg/g). Body weight and breathing behaviour were monitored daily. At day 20, mice were sacrificed, lungs were removed, fixed in saline 4% formaldehyde and examined with a Zeiss stereomicroscope. Results were expressed as numbers of metastatic foci per lung.

7.2 Results and Conclusion

Mice treated with F8(scDb)-IL15 conjugate showed significantly fewer metastatic foci in the lungs, compared with mice treated with saline solution (PBS). The difference was even more striking in the group treated with F8(scDb)-SD-IL15 where there was almost a total absence of metastatic foci in the lungs of treated mice. These results demonstrate that both conjugates comprising an F8(scDb) and IL15 conjugated to the C-terminus of the scDb had anti-tumour activity but that inclusion of a sushi domain (SD) between the scDb and IL15 resulted in a significant increase in the anti-tumour activity of the conjugate (Figure 19A).

Toxicity due to the metastasis is visible as a reduction in body weight. Only mice in the control group, which were treated with PBS, started to lose body weight and show breathing disfunction from day 18. No toxicity was observed after injection of the conjugates, demonstrating that the conjugates were well-tolerated (see arrows in Figure 19B). Example 8 -Cloning and production of conjugates comprising a scDb specific for domain D of

Tenascin C and IL15

8.1 Cloning procedure for R6N(scDb)-IL 15

The genes encoding R6N(VH-VL) and human IL15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using Nhel/EcoRV/Notl restriction enzymes.

A first PCR was performed to amplify the hi L15 adding a Notl restriction site and a part of GDGSSGGSGGAS linker. Primers used were GDGS-IL15 fwd

(TCTTCAGGCGGCTCTGGCGGAGCTTCCAACTGGGTGAATGTAATAAG) (SEQ ID NO: 18) and Notl-STOP-I L15 ba (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A second PCR was performed to amplify the second R6N(VH-VL) adding a part of the

DIGGGSGGGGSGGGG linker, an EcoRV restriction site and a part of GDGSSGGSGGAS linker. Primers used were“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO: 82) and 26-RC VL GDS link Ba

(CTCCGCCAGAGCCGCCTGAAGAGCCGTCACCGCCTAGGACGGTCAGCTTGGTCCC) (SEQ ID NO: 83).

A third PCR was performed to assemble the first and the second PCR products. Primers used were an“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO: 82) and Notl-STOP-IL15 ba (AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

The final PCR product was digested with EcoRV and Notl restriction enzymes and ligated in the empty expression vector digested with the same enzymes.

In a fourth PCR reaction, primers were designed to amplify the first R6N(VH-VL) adding a Leader Sequence, a Nhel restriction site, a part of the DIGGGSGGGGSGGGG linker. Primers used were “Nhel-leader” >

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and Link Lambda EcoRV ba

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEQ ID NO: 84). The final PCR product was digested with Nhel and EcoRV restriction enzymes and ligated into an expression vector that contained the second R6N-IL15, digested with the same enzymes. The amino acid sequence of the R6N(scDb)-IL15 conjugate is shown in SEQ ID NO: 76.

8.2 Cloning procedure for IL 15-R6N(scDb)

The genes encoding R6N(VH-VL) and the human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using Nhel/EcoRV/Notl restriction enzymes.

A first PCR was performed to amplify the hi L15 by adding a Nhel restriction site, the Leader sequence and a part of GDGSSGGSGGAS linker.

Primers used were“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEC ID NO: 15) and“25-RC I L15-GAS Ba”

(CTCCGCCAGAGCCGCCT GAAGAGCCGTCACCAGAAGT GTT GAT GAACATTT G) (SEC ID NO: 66).

A second PCR was performed to amplify the first R6N(VH-VL) by adding a part of the

GDGSSGGSGGAS linker, an EcoRV restriction site and a part of DIGGGSGGGGSGGGG linker. Primers used were“24-RC GAS-F8 Vh Fo”

(GGCTCTTCAGGCGGCTCTGGCGGAGCTTCCGAGGTGCAGCTGTTGGAGTCTG) (SEC ID NO: 65) and“Link Lambda EcoRV ba”

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEC ID NO: 84).

A third PCR was performed to assemble the first and the second PCR products. Primers used were“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEC ID NO: 15) and“Link Lambda EcoRV ba”

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEC ID NO: 84).

The final PCR product was digested with Nhel and EcoRV restriction enzymes and ligated into the expression vector that already contained the second R6N, digested with the same enzymes. The amino acid sequence of the IL15-R6N(scDb) conjugate is shown in SEQ ID NO: 77.

8.3 Production of conjugates

The conjugates were expressed and purified as described in Example 1.5. The yield obtained for each of the conjugates is shown in Table 4. Table 4

Example 9 - In vitro characterization of conjugates comprising a scDb specific for domain D of

Tenascin C and IL15

9.1 Protein characterization

SDS-PAGE analysis of the R6N(scDb)-IL15 and IL15-R6N(scDb) conjugates was performed with 4-12% Bis-Tris gel under reducing and non-reducing conditions.

The R6N(scDb)-IL15 and IL15-R6N(scDb) conjugates were analyzed by size-exclusion

chromatography using a Superdex 200 increase 10/300 GL column on an AKTA FPLC.

9.2 Biological activity

The biological activity of IL15 in the R6N(scDb)-IL15 and IL15-R6N(scDb) conjugates was determined by the ability of the conjugates to stimulate the proliferation of CTLL2 cells. Cells were seeded in 96-well plates in the culture medium supplemented with varying concentrations of the R6N(scDb)-IL15 or IL15-R6N(scDb) conjugates. After incubation at 37°C for 72 hours, cell proliferation was determined with Cell Titer Aqueous One Solution.

9.3 Results and Conclusions

The R6N(scDb)-IL15 (also named R6N-R6N-IL15) and IL15-R6N(scDb) (also named IL15-R6N- R6N) conjugates were expressed and purified in CHO cells exploiting the binding property of the VH domain of R6N antibody to Protein A resin. The R6N(scDb)-IL15 conjugate was produced with excellent quality as evidenced by a single peak in gel filtration (Figure 20A) whereas the IL15- R6N(scDb) conjugate was produced with an inferior quality, as seen in Figure 21A, which shows the presence of aggregates. The two bands seen in the SDS-PAGE analysis were shown to be due to the heavy glycosylation of IL15 (Figures 20B and 21 B). The activity assay based on CTLL2 cells showed that the R6N(scDb)-IL15 and IL15-R6N(scDb) conjugates were able to induce T cell proliferation, as shown in Figures 20C and 21 C respectively, with the R6N(scDb)-IL15 conjugate being more active.

These results demonstrate that the R6N(scDb)-IL15 conjugate was superior in terms of production yield, production quality and activity over the IL15-R6N(scDb) conjugate, confirming that the superiority of the (scDb)-IL15 conjugate format (where the IL15 moiety is conjugated to the C- terminus of the scDb) over the IL15-(scDb) conjugate format (where the IL15 moiety is conjugated to the N-terminus of the scDb) already shown in Examples 1 to 3 is independent of the target bound by the single-chain diabody (scDb). The R6N(scDb)-IL15 conjugate, as well as other (scDb)-IL15 conjugates wherein the scDb binds to an antigen capable of targeting the conjugate to the tumor or tumor microenvironment are therefore also expected to show good tumour uptake and tumour to organ ratio, as well as high anti-tumour efficacy as already demonstrated for the

F8(scDb)-IL15 and L19(scDb)-IL15 conjugates in Example 3 (tumour uptake/tumour to organ ratio) and the F8(scDb)-IL15 conjugate in Example 7 (anti-tumour efficacy).

Example 10 - Cloning and production of conjugates comprising a scDb specific for domain D of tenascin C, a sushi domain and IL15

10.1 Cloning procedure for R6N(scDb)-SD-IL 15

The genes encoding R6N(VH-VL), the Sushi Domain (SD) and the human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using

Nhel/EcoRV/Notl restriction enzymes.

A first PCR was performed to amplify the SD and hi L15 by adding a Notl restriction site and a part of (GGGGS)3 linker. Primers used were“28-RC New GGGG -SD link Fo”

(GTGGTGGGGGAGGTAGCGGTGGAGGGGGCTCCATCACGTGCCCTCCCCCCATG) (SEQ ID NO: 61) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19).

A second PCR was performed to amplify the second R6N(VH-VL) by adding a part of the

DIGGGSGGGGSGGGG linker, an EcoRV restriction site and a part of (GGGGS)3 linker.

Primers used were“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO: 82) and“29-RC VL New GGGGS link Ba”

(CCACCGCTACCTCCCCCACCACTGCCACCGCCCCCGCCTAGGACGGTCAGCTTGGT) (SEQ ID NO: 85).

A third PCR was performed to assemble the first and the second PCR products. Primers used were“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO:82) and“Notl-STOP-IL15 ba”

(AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT) (SEQ ID NO: 19). The final PCR product was digested with EcoRV and Notl restriction enzymes and ligated into the empty expression vector digested with the same enzymes.

In a fourth PCR reaction primers were designed to amplify the first R6N(VH-VL) by adding a Leader Sequence, a Nhel restriction site, a part of the DIGGGSGGGGSGGGG linker. Primers used were“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Link Lambda EcoRV ba”

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEQ ID NO: 84).

The final PCR product was digested with Nhel and EcoRV restriction enzymes and ligated into expression vector that contained the second R6N-SD-IL15, digested with the same enzymes. The amino acid sequence of the R6N(scDb)-SD-IL15 conjugate is shown in SEQ ID NO: 78.

10.2 Cloning procedure for R6N(scDb)-IL 15-SD

The genes encoding R6N(VH-VL), the Sushi Domain (SD) and the human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using

Nhel/EcoRV/Notl restriction enzymes.

A first PCR was performed to amplify the hi L15 and SD by adding a Notl restriction site and a part of (GGGGS)3 linker. Primers used were:“30-RC VL-E2 GGGGS link Fo”

(GTGGTGGGGGAGGTAGCGGTGGAGGGGGCTCCAACTGGGTGAATGTAATAAG) (SEQ ID NO: 86) and“Notl-STOP-SD ba”

(TTTCCTTTTGCGGCCGCTCATT AAGCT ATCT AATGCATTT GAGACT G) (SEQ ID NO: 87).

A second PCR was performed to amplify the second R6N(VH-VL) by adding a part of the

DIGGGSGGGGSGGGG linker, an EcoRV restriction site and a part of (GGGGS)3 linker.

Primers used were:“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO: 82) and“29-RC VL New GGGGS link Ba”

(CCACCGCTACCTCCCCCACCACTGCCACCGCCCCCGCCTAGGACGGTCAGCTTGGT) (SEQ ID NO: 85).

A third PCR was performed to assemble the first and the second PCR products. Primers used were:“EcoRV-link-fwd”

(CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG) (SEQ ID NO: 82) and“Notl-STOP-SD ba” (TTTCCTTTTGCGGCCGCTCATT AAGCT ATCT AAT GCATTT GAGACT G) (SEQ ID NO: 87). The final PCR product was digested with EcoRV and Notl restriction enzymes and ligated into the expression vector that already contained the Leader Sequence and the first R6N, digested with the same enzymes. The amino acid sequence of the R6N(scDb)-IL15-SD conjugate is shown in SEQ ID NO: 79.

10.3 Cloning procedure for IL 15-SD-R6N (scDb)

The genes encoding R6N(VH-VL), the Sushi Domain (SD) and the human I L15 were PCR amplified, PCR assembled and cloned into the mammalian expression vector using

Nhel/EcoRV/Notl restriction enzymes.

A first PCR was performed to amplify the hlL15 and the SD by adding a Nhel restriction site, the Leader sequence and a part of (GGGGS)3 linker. Primers used were“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGG C) (SEQ ID NO: 15) and“16-RC Ba”

(CGCCGCCAGAGCCACCTCCGCCT GAACCGCCTCCACCTCT AAT GCATTT GAGACTGG) (SEQ ID NO: 54).

A second PCR was performed to amplify the first R6N(VH-VL) by adding a part of the

GGGGSGGGGSGGGGS linker, an EcoRV restriction site and a part of DIGGGSGGGGSGGGG linker. Primers used were“17-RC Fo”

(CGGAGGTGGCTCTGGCGGCGGTGGGTCAGAGGTGCAGCTGTTGGAGTC) (SEQ ID NO: 55) and“Link Lambda EcoRV ba”

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEQ ID NO: 84).

A third PCR was performed to assemble the first and the second PCR products. Primers used were:“Nhel-leader >”

(CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC) (SEQ ID NO: 15) and“Link Lambda EcoRV ba”

(CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC) (SEQ ID NO: 84).

The final PCR product was digested with Nhel and EcoRV restriction enzymes and ligated into the expression vector that already contained the second R6N, digested with the same enzymes. The amino acid sequence of the IL15-SD-R6N (scDb) conjugate is shown in SEQ ID NO: 80. 10.4 Production of conjugates

The conjugates were expressed and purified as described in Example 1.5. The yield obtained for each of the conjugates is shown in Table 5.

Table 5

The IL15-SD-R6N(scDb) conjugate was successfully cloned into the expression vector. However, it was not possible to produce sufficient material for biochemical characterization. This conjugate was therefore not developed further.

Example 11 - In vitro characterization of conjugates comprising a scDb specific for domain D of Tenascin C, sushi domain and IL15

11.1 Protein characterization

Analysis of conjugates by SDS-PAGE and size-exclusion chromatography was performed as described in Example 9.1.

11.2 Results and Conclusions

The R6N(scDb)-SD-IL15 (also named R6N-R6N-SD-IL15) conjugate showed better biochemical properties compared to other sushi domain-containing formats in which the orientation of the cytokine / cytokine receptor complex (IL15 / sushi domain) was different, both in terms of production yield and gel filtration profile. The results of the FPLC analysis showed that R6N(scDb)- IL15-SD (also named R6N-R6N-IL15-SD), unlike the R6N(scDb)-SD-IL15 conjugate, cannot be eluted with a purity profile compatible with industrial development as this conjugate forms too many aggregates (Figure 22). The two bands seen in SDS-PAGE analysis for both conjugates were due to heavily glycosylation of IL15 (Figure 23).

These results demonstrate the superiority of the scDb-SD-IL15 conjugate format over conjugates comprising a sushi domain and IL15 in other orientations (e.g. at the N-terminus of the scDb or at the C-terminus of the scDb but with IL15 rather than the sushi domain linked to the scDb) and therefore confirm the findings obtained using the F8(scDb)-SD-IL15 conjugate in Example 5. The superior biochemical properties of this conjugate format are therefore independent of the target bound by the scDb. The R6N(scDb)-SD-IL15 conjugate, as well as other (scDb)-SD-IL15 conjugates wherein the scDb binds to an antigen capable of targeting the conjugate to the tumor or tumor microenvironment are therefore expected to show good tumour uptake and tumour to organ ratio, as well as high anti-tumour efficacy as demonstrated for the F8(scDb)-SD-IL15 conjugate in Examples 6 and 7.

Sequence Listing

SEQ ID NO: 1 (F8 VH)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS

SEQ ID NO: 2 (F8 VL)

EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGS GSGTDFTLTISRLEPEDFA VYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 3 (CDR1 F8 VH)

LFT

SEQ ID NO: 4 (CDR2 F8 VH)

SGSGGS

SEQ ID NO: 5 (CDR3 F8 VH)

STHLYL

SEQ ID NO: 6 (CDR1 F8 VL)

MPF

SEQ ID NO: 7 (CDR2 F8 VL)

GASSRAT

SEQ ID NO: 8 (CDR3 F8 VL)

MRGRPP

SEQ ID NO: 9 (F8 single-chain diabody [F8 scDb])

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGS GGEIVLTQ

SPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTD

FTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLL ESGGGLVQ

PGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKN

TLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTL SLSPGERA

TLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFA

VYYCQQMRGRPPTFGQGTKVEIK SEQ ID NO: 10 (linker between the single-chain diabody, or diabody, and IL15; linker between the sushi domain and IL15)

GDGSSGGSGGAS

SEQ ID NO: 11 (linker between the two sets of VH and VL in a single-chain diabody)

SSSSGSSSSGSSSSG

SEQ ID NO: 12 (linker between the VH and VL domains of the F8 single-chain diabody and of R6N single-chain diabody)

GGSGG

SEQ ID NO: 13 (Interleukin-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 14 (F8(scDb)-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGS GGEIVLTQ

SPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTD

FTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLL ESGGGLVQ

PGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKN

TLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTL SLSPGERA

TLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFA

VYYCQQMRGRPPTFGQGTKVEIKGDGSSGGSGGASNWVNVISDLKKIEDLIQSMHID ATLYTESD

VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE CEELEEKNIKE

FLQSFVHIVQMFINTS

SEQ ID NO: 15 (“Nhel-leader >” primer)

CTAGCTAGCGTCGACCATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGC

SEQ ID NO: 16 (“L19-SSG Ba” primer)

CGGAAGAGCT ACT ACCCG AT G AGG AAG ATTT G ATTTCCACCTT GGTCC

SEQ ID NO: 17 (“Link-BamHI ba” primer)

GCCGCT GGACGAGGATCCGGAAGAGCT ACT ACCCGAT GAGGAAGA SEQ ID NO: 18 (“GDGS-IL15 fwd” primer)

TCTTCAGGCGGCTCTGGCGGAGCTTCCAACTGGGTGAATGTAATAAG

SEQ ID NO: 19 (“Notl-STOP-IL15 ba” primer)

AT AGTTT AGCGGCCGCATTCTT ATTCAAGAAGT GTT GAT GAACATTT

SEQ ID NO: 20 (“BamHI-L19VH fo” primer)

CTCTTCCGGATCCTCGTCCAGCGGCGAGGTGCAGCTGTTGGAGTC

SEQ ID NO: 21 (“GDGS-IL15-kappa ba” primer)

CCGCCAGAGCCGCCTGAAGAGCCGTCACCTTTGATTTCCACCTTGGTCC

SEQ ID NO: 22 (IL15-F8(scDb))

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGDGSSGGSGGAS EVQLLESG

GGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVK GRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQ SPGTLSLS

PGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRL

EPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLESGGGLVQ PGGSLRL

SCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN TLYLQMN

SLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTLSLSPGER ATLSCRAS

QSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPED FAVYYCQQ

MRGRPPTFGQGTKVEIK

SEQ ID NO: 23 (L19(scDb)-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGG EIVLTQSP

GTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFT

LTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLES GGGLVQPG

GSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISR DNSKNTLY

LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPG ERATLSCR

ASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEP EDFAVYYCQ

QTGRIPPTFGQGTKVEIKGDGSSGGSGGASNWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCK

VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFV

HIVQMFINTS SEQ ID NO: 24 (L19 VH)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

SEQ ID NO: 25 (L19 VL)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIP DRFSGS

GSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

SEQ ID NO: 26 (Linker between VH and VL of L19 single-chain diabody)

GSSGG

SEQ ID NO: 27 (CDR1 L19 VH)

SFSMS

SEQ ID NO: 28 (CDR2 L19 VH)

SISGSSGTTYYADSVKG

SEQ ID NO: 29 (CDR3 L19 VH)

PFPYFDY

SEQ ID NO: 30 (CDR1 L19 VL)

RASQSVSSSFLA

SEQ ID NO: 31 (CDR2 L19 VL)

YASSRAT

SEQ ID NO: 32 (CDR3 L19 VL)

QQTGRIPPT

SEQ ID NO: 33 (L19 single-chain diabody)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGG EIVLTQSP

GTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFT

LTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLES GGGLVQPG

GSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISR DNSKNTLY

LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPG ERATLSCR ASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQ

QTGRIPPTFGQGTKVEIK

SEQ ID NO: 34 (“GDGS-IL15-kappa ba 2” primer)

GGACCAAGGTGGAAATCAAAGGTGACGGCTCTTCAGGCGGCTCTGGCGG

SEQ ID NO: 35 (F8(Db)-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGS GGEIVLTQ

SPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTD

FTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKGDGSSGGSGGASNWVNVISD LKKIEDLIQ

SMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSL SSNGNVTESGC

KECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 36 (linker between the F8 or R6N single-chain diabodies and the sushi domain of IL15R alpha or between the F8 single-chain diabody and IL15)

GGGGSGGGGSGGGGS

SEQ ID NO: 37 (linker between IL15 and a sushi domain of IL15R alpha)

GGGSGGGGSGGGSGGGGSLQ

SEQ ID NO: 38 (mutant Interleukin-15 -> N79Q mutation)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 39 (IL15 Receptor alpha)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS LKCIRD PALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLM PSKSPSTG TTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAV SLLACY LKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

SEQ ID NO: 40 (Sushi Domain of IL15 Receptor alpha)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS LKCIR

SEQ ID NO: 41 (F8(scDb)-SD-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGS GGEIVLTQ SPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTD

FTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLL ESGGGLVQ

PGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKN

TLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTL SLSPGERA

TLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFA

VYYCQQMRGRPPTFGQGTKVEIKGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSY SLYSRE

RYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGGGSGGGG SLQNWV

NVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNS

LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 42 (F8(scDb)-IL15-SD)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGS GGEIVLTQ

SPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGSGSGTD

FTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLL ESGGGLVQ

PGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKN

TLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGSGGEIVLTQSPGTL SLSPGERA

TLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFA

VYYCQQMRGRPPTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSM HIDATLY

TESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEE

KNIKEFLQSFVHIVQMFINTSGGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWV KSYSLYSR

ERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

SEQ ID NO: 43 (IL15-SD-F8(scDb))

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGSGGGGSGGG SGGGGSL

QITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHW TTPSLKCIR

GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPG KGLEW

VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYL FDYWGQGT

LVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPR LLIYGASS

RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSS SGSSSSGS

SSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISG SGGSTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVS SGGSGGEI

VLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSG

SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK SEQ ID NO: 44 (SD-IL15-F8(scDb))

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS LKCIRG

GGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLE

LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS

GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPG KGLEW

VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYL FDYWGQGT

LVTVSSGGSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPR LLIYGASS

RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKSSS SGSSSSGS

SSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISG SGGSTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVS SGGSGGEI

VLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSG

SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

SEQ ID NO: 45 (KSF(scDb)-SD-IL15 )

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPKVSLFDYWGQGTLVTVSSGG SGGSSEL

TQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGN

TASLTITGAQAEDEADYYCNSSPLNRLAVVFGGGTKLTVLGEFSSSSGSSSSGSSSS GEVQLLES

GGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV KGRFTIS

RDNSKNTLYLQMNSLRAEDTAVYYCAKSPKVSLFDYWGQGTLVTVSSGGSGGSSELT QDPAVSV

ALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNT ASLTITGA

QAEDEADYYCNSSPLNRLAVVFGGGTKLTVLGGGGGSGGGGSGGGGSITCPPPMSVE HADIWV

KSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGG SGGGSGG

GGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISL ESGDASIHDT

VENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 46 (“RC-9 Fo” primer)

CGGATCGGGAGGCGGGGGTAGTCTGCAAAACTGGGTGAATGTAATAAG

SEQ ID NO: 47 (“RC-10 Fo” primer)

GAGGTAGCGGCGGTGGGGGAAGTGGTGGCGGATCGGGAGGCGGGGGTAG

SEQ ID NO: 48 (“RC-1 1 Fo” primer)

CAGGCGGAGGTGGCTCTGGCGGCGGTGGGTCAATCACGTGCCCTCCCCCCATG

SEQ ID NO: 49 (“RC-12 Ba” primer)

CTTCCCCCACCGCCGCT ACCTCCCCCTCT AAT GCATTT GAGACTGG SEQ ID NO: 50 (“GGGGS3-VL-kappa ba” primer)

CAGAGCCACCTCCGCCTGAACCGCCTCCACCTTTGATTTCCACCTTGGTCC

SEQ ID NO: 51 (“13-RC Ba” primer)

CACCACTTCCCCCACCGCCGCT ACCTCCCCCAGAAGT GTT GAT GAACATTT G

SEQ ID NO: 52 (“14-RC Ba” primer)

TTGCAGACTACCCCCGCCTCCCGATCCGCCACCACTTCCCCCACCGCCGCTAC

SEQ ID NO: 53 (“15-RC Fo” primer)

CGGGAGGCGGGGGTAGTCTGCAAATCACGTGCCCTCCCCCCATGTC

SEQ ID NO: 54 (“16-RC Ba” primer)

CGCCGCCAGAGCCACCTCCGCCT GAACCGCCTCCACCTCT AAT GCATTT GAGACTGG

SEQ ID NO: 55 (“17-RC Fo” primer)

CGGAGGTGGCTCTGGCGGCGGTGGGTCAGAGGTGCAGCTGTTGGAGTC

SEQ ID NO: 56“Not Stop DPK Ba<” primer)

T AGTTT AGCGGCCGCATTCTT ATTCACT ATTT GATTTCCACCTTGGTCCCTTGGCC

SEQ ID NO: 57 (“18-RC Fo” primer)

CTGTTCCTCGTCGCTGTGGCTACAGGTGTGCACTCGATCACGTGCCCTCCCCCC

SEQ ID NO: 58 (“19-RC Ba” primer)

CCGCCAGAGCCACCTCCGCCT GAACCGCCTCCACCAGAAGT GTT GAT GAACATTT G

SEQ ID NO: 59 (F8(scFv)-SD-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSGGG GSGGGGS

GGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGAS SRATGIPD

RFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIKGGGGSGGGGS GGGGSIT

CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRGG

GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTA MKCFLLEL

QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS SEQ ID NO: 60 (“30-RC GGGG -SD F8 Ba” primer)

CCACCGCTACCTCCCCCACCACTGCCACCGCCCCCTTTGATTTCCACCTTGGTCC

SEQ ID NO: 61 (“28-RC New GGGG -SD link Fo” primer)

GTGGTGGGGGAGGTAGCGGTGGAGGGGGCTCCATCACGTGCCCTCCCCCCATG

SEQ ID NO: 62 (“RC-20 Fo” primer)

TTCAGGCGGAGGTGGCTCTGGCGGCGGTGGGTCAAACTGGGTGAATGTAATAAG

SEQ ID NO: 63 (“GGGGS3-SD fwd” primer)

TTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAATCACGTGCCCTCCCCCCAT

SEQ ID NO: 64 (L19(scDb)-SD-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYY ADSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGG EIVLTQSP

GTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFT

LTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKSSSSGSSSSGSSSSGEVQLLES GGGLVQPG

GSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISR DNSKNTLY

LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPG ERATLSCR

ASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEP EDFAVYYCQ

QTGRIPPTFGQGTKVEIKGGGGSGGGGSGGGGITCPPPMSVEHADIWVKSYSLYSRE RYICNSG

FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGDGSSGGSGGASNWVNVISDLKKI EDLIQSMHI

DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNG NVTESGCKEC

EELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 65 (“24-RC GAS-F8 Vh Fo” primer)

GGCTCTTCAGGCGGCTCTGGCGGAGCTTCCGAGGTGCAGCTGTTGGAGTCTG

SEQ ID NO: 66 (“25-RC I L15-GAS Ba” primer)

CTCCGCCAGAGCCGCCT GAAGAGCCGTCACCAGAAGT GTT GAT GAACATTT G

SEQ ID NO: 67 (CDR1 R6N VH)

GFTFSQYSMS

SEQ ID NO: 68 (CDR2 R6N VH)

AISGSGGSTYYADSVKG SEQ ID NO: 69 (CDR3 R6N VH)

GRRIFDY

SEQ ID NO: 70 (CDR1 R6N VL)

QGDSLRPTMAS

SEQ ID NO: 71 (CDR2 R6N VL)

GKNNRPS

SEQ ID NO: 72 (CDR3 R6N VL)

QSSRMDVPTVV

SEQ ID NO: 73 (R6N VH)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSS

SEQ ID NO: 74 (R6N VL)

SSELTQDPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDR FSGSS

SGNTASLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLG

SEQ ID NO: 75 (R6N single-chain diabody [R6N scDb])

EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSG GSSELTQ

DPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG SSSGNTA

SLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLGDIGGGSGGGGSGGGGEVQ LLESGG

GLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG RFTISRD

NSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDPA VSVALGQ

TVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLT ITGAQAED

EADYYCQSSRMDVPTVVFGGGTKLTVLG

SEQ ID NO: 76 (R6N(scDb)-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSG GSSELTQ

DPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG SSSGNTA

SLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLGDIGGGSGGGGSGGGGEVQ LLESGG

GLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG RFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDPAVSV ALGQ TVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAED EADYYCQSSRMDVPTVVFGGGTKLTVLGGDGSSGGSGGASNWVNVISDLKKIEDLIQSMH IDATL YTESDVH PSCKVTAM KCFLLELQVISLESGDASI H DTVEN LI I LAN NSLSSNGN VTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 77 (IL15-R6N(scDb))

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGDGSSGGSGGAS EVQLLESG

GGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVK GRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDP AVSVALG

QTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASL TITGAQAE

DEADYYCQSSRMDVPTVVFGGGTKLTVLGDIGGGSGGGGSGGGGEVQLLESGGGLVQ PGGSL

RLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNS KNTLYLQ

MNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDPAVSVALGQTV RITCQGD

SLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDE ADYYCQSS

RM DVPTVVFGGGTKLTVLG

SEQ ID NO: 78 (R6N(scDb)-SD-IL15)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSG GSSELTQ

DPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG SSSGNTA

SLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLGDIGGGSGGGGSGGGGEVQ LLESGG

GLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG RFTISRD

NSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDPA VSVALGQ

TVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLT ITGAQAED

EADYYCQSSRMDVPTVVFGGGTKLTVLGGGGGSGGGGSGGGGSITCPPPMSVEHADI WVKSYS

LYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGGG SGGGGSL

QNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLII

LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID NO: 79 (R6N(scDb)-IL15-SD)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYY ADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSG GSSELTQ

DPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG SSSGNTA

SLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLGDIGGGSGGGGSGGGGEVQ LLESGG

GLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG RFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSSGGSGGSSELTQDPAVSV ALGQ

TVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLT ITGAQAED

EADYYCQSSRMDVPTVVFGGGTKLTVLGGGGGSGGGGSGGGGSNWVNVISDLKKIED LIQSMHI

DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNG NVTESGCKEC

EELEEKNIKEFLQSFVHIVQMFINTSGGGSGGGGSGGGSGGGGSLQITCPPPMSVEH ADIWVKSY

SLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

SEQ ID NO: 80 (IL15-SD-R6N(scDb))

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIIL

ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGSGGGGSGGG SGGGGSL

QITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHW TTPSLKCIR

GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPG KGLE

WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIF DYWGQGTL

VTVSSGGSGGSSELTQDPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVI YGKNNRP

SGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLGDI GGGSGGG

GSGGGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAI SGSGG

STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVT VSSGGSG

GSSELTQDPAVSVALGQTVRITCQGDSLRPTMASWYQQKPGQAPVLVIYGKNNRPSG IPDRFSG

SSSGNTASLTITGAQAEDEADYYCQSSRMDVPTVVFGGGTKLTVLG

SEQ ID NO: 81 (Linker between the two sets of VH and VL in a single-chain diabody)

DIGGGSGGGGSGGGG

SEQ ID NO: 82 (“EcoRV-link-fwd” primer)

CCTAGGCGATATCGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGG

SEQ ID NO: 83 (“26-RC VL GDS link Ba” primer)

CTCCGCCAGAGCCGCCTGAAGAGCCGTCACCGCCTAGGACGGTCAGCTTGGTCCC

SEQ ID NO: 84 (“Link Lambda EcoRV ba” primer)

CCTGAACCGCCTCCGATATCGCCTAGGACGGTCAGCTTGGTCC

SEQ ID NO: 85 (“29-RC VL New GGGGS link Ba” primer)

CCACCGCTACCTCCCCCACCACTGCCACCGCCCCCGCCTAGGACGGTCAGCTTGGT

SEQ ID NO: 86 (“30-RC VL-E2 GGGGS link Fo” primer)

GTGGTGGGGGAGGTAGCGGTGGAGGGGGCTCCAACTGGGTGAATGTAATAAG SEQ ID NO: 87 (“Notl-STOP-SD ba” primer)

TTTCCTTTTGCGGCCGCTCATT AAGCT ATCT AATGCATTT GAGACT G) SEQ ID NO: 88 (linker between single-chain diabody and sushi domain) GGGGSGGGGSGGGG

References

Alter ed a/., Targeted IL-15-based Protein Fusion Complexes as Cancer Immunotherapy

Approaches. J. Immunol .Sci. (2018); 2(1): 15-18.

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