EA043910B1 - IMMUNOCYTOKINE FOR CANCER TARGETED DELIVERY OF IL-12 AND ITS APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present application relates to compositions containing a cytokine, an antigen binding domain and an improved linker.

State of the art

IL-12 is a heterodimeric cytokine containing two subunits, p35 and p40, linked by disulfide bonds. IL-12 stimulates the production of IFNy from T cells and natural killer cells, and also induces the differentiation of Th1 helper cells. IL-12 is a key mediator of innate and cellular immunity with potential antitumor and antimetastatic activity.

However, like many other cytokines, administration of IL-12 is associated with high toxicity (Car et al., 1999), even at doses as low as 1 μg/kg body weight per day, preventing its development as an anticancer drug.

The essence of the invention

The present invention provides, among other things, improved compositions and methods that can be used to effectively treat various diseases and disorders associated with the expression of EDB fibronectin.

In particular, the present invention relates to IL-12 associated with EDB binding domains having advantageous therapeutic properties compared to known recombinant IL-12 constructs. The compositions described herein are unexpectedly superior to previously known IL-12 constructs designed to target EDB and solve the long-standing problem of safely and effectively administering IL-12 for targeted treatment of a disease or disorder, such as cancer. The compositions and methods described herein provide improved therapeutic potential of IL-12 by improving one or more of its properties: its biodistribution profile, its tolerability, its therapeutic window, and its effectiveness in reaching the disease site. The designs described here also unexpectedly demonstrate superior manufacturability.

There remains a need in the field to improve tissue penetration of immunocytokines for treatment. There is also a need in the art for improved production of immunocytokines for treatment, as they are very complex proteins that are difficult to produce.

Therefore, it is an object of the present invention to provide improved versions of immunocytokines, for example, protein therapeutics containing IL-12 and a fibronectin EDB binding domain. Another object of the present invention is to provide immunocytokines that exhibit more efficient production. It is yet another object of the present invention to provide immunocytokines with improved in vivo efficacy, eg, target binding or tissue penetration.

The present invention provides compositions containing an antigen binding domain and a cytokine having such improved properties. The invention and the general advantages of its features, including corresponding linkers, will be discussed in detail below.

According to one aspect of the invention, there is provided a composition comprising:

a) an IL-12 protein comprising a first IL-12 subunit and a second IL-12 protein subunit;

b) a peptide or protein containing an EDB-binding domain; And

c) a linker between the IL-12 protein and a peptide or protein containing an EDB binding domain.

Preferably, the two IL-12 subunits are connected to each other by a specific linker according to the following scheme (N^C orientation): p40-linker 1-p35.

Preferably, the IL-12 is human IL-12. In some embodiments, the single chain diabody binds to the spliced isoform of fibronectin. Preferably, said extradomain B (EDB) of fibronectin is extradomain B of human fibronectin (UniProt: P02751).

In some embodiments, the linker between the IL-12 protein and the EDB-binding domain-containing peptide or protein comprises the sequence GSADGGSSAGGSDAG (SEQ ID NO: 4).

In some embodiments, the EDB-binding domain-containing peptide or protein includes scFv. In some embodiments, the peptide or protein containing the EDB binding domain is a diabody. In some embodiments, the peptide or protein containing the EDB binding domain is a single chain diabody.

In some embodiments, the first subunit of the IL-12 protein is p40, and the second subunit is p35.

In some embodiments, the first subunit of the IL-12 protein is p40, comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 1, or a fragment thereof, wherein the IL-12 protein can activate the IL-12 receptor.

In some embodiments, the second subunit of the IL-12 protein is p35, comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 3, or a fragment thereof, where the IL-12 protein can activate receptor

IL-12.

In some embodiments, the EDB-binding domain-containing peptide or protein is monospecific or bispecific.

In some embodiments, the peptide or protein containing the EDB binding domain binds to extra domain B (EDB) of fibronectin.

In some embodiments, the EDB-binding domain-containing peptide or protein contains an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% identity with one or more amino acid sequences presented in SEQ ID NO: 28-33.

In some embodiments, the EDB-binding domain-containing peptide or protein comprises any of the amino acid sequences of SEQ ID NOs: 28-33.

In some embodiments, the EDB-binding domain-containing peptide or protein contains an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 % 97%, 98%, 99% or 100% identity with one or more amino acid sequences presented in SEQ ID NO: 7 and 5.

In some embodiments, the EDB binding domain-containing peptide or protein comprises any of the amino acid sequences set forth in SEQ ID NOs: 7 and 5.

In some embodiments, the EDB-binding domain-containing peptide or protein comprises at least one of:

a) sequence pairs as described above, provided that at least one of the domains has >80% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, and/or

b) pairs of sequences as described above, provided that at least one of the domains has up to 10 amino acid substitutions relative to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, while maintaining its ability to bind to extra domain B (EDB) fibronectin.

In some embodiments, the peptide or protein contains at least one amino acid substitution, wherein the at least one amino acid substitution is a conservative amino acid substitution.

In some embodiments, the EDB-binding domain-containing peptide or protein: has a target binding affinity of >50% for fibronectin extra domain B (EDB) compared to one of the anti-EDB binding domain-containing peptides or proteins as described above, and/or competes for binding to the extra domain B (EDB) of fibronectin with one of the peptides or proteins containing an EDB binding domain, as described above.

In some embodiments, the EDB binding domain-containing peptide or protein comprises two L19 VH domains and two L19 VL domains.

In some embodiments, the two L19 VH domains have the same amino acid sequence:

the two L19 VH domains have different amino acid sequences;

two L19 VL domains have the same amino acid sequence or two L19 VL domains have different amino acid sequences.

In some embodiments, the EDB-binding domain-containing peptide or protein comprises one L19 VH domain and one L19 VL domain.

In some embodiments, the composition comprises a p40 domain linked to the p35 domain by a first linker (also referred to as linker 1);

the first VH domain of L19 linked to the p35 domain by the SAD linker;

a first L19 VL domain linked to the first L19 VH domain by a third linker (also referred to as linker 3);

a second L19 VH domain linked to the first L19 VL domain by a fourth linker (also called linker 4);

a second L19 VL domain linked to a second L19 VH domain by a fifth linker (also called linker 5).

In some embodiments, the third linker and the fifth linker contain the same amino acid sequence and/or can be replaced with each other.

In some embodiments, the p40 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 1, or its

- 2 043910 fragment.

In some embodiments, the p35 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 3, or a fragment thereof.

In some embodiments, the first linker (linker 1) is a GS linker.

In some embodiments, the first linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 2.

In some embodiments, the first L19 VH domain, the second L19 VH domain, or both comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence presented in SEQ ID NO: 28-30.

In some embodiments, the first L19 VL domain, the second L19 VL domain, or both comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence presented in SEQ ID NO: 31-33.

In some embodiments, the first L19 VH domain, the second L19 VH domain, or both comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 7.

In some embodiments, the first L19 VL domain, the second L19 VL domain, or both comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the SAD linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 4.

In some embodiments, the third linker (linker 3) is a GS linker.

In some embodiments, the third linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 6.

In some embodiments, the fifth linker (linker 5) is a GS linker.

In some embodiments, the fifth linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 6.

In some embodiments, the third linker (linker 3) and the fifth linker (linker 5) contain the same amino acid sequence and/or can be replaced with each other.

In some embodiments, the fourth linker (linker 4) is a GS linker.

In some embodiments, the fourth linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 8.

In some embodiments, the composition contains an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identity with the amino acid sequence presented in SEQ ID NO: 16.

In some embodiments, the composition consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence presented in SEQ ID NO: 16.

According to yet another aspect of the invention, there is provided the use of a composition according to any of the above claims (for the production of a medicament) in the treatment of a human or animal diagnosed with, suffering from, or at risk of developing a neoplastic disease, or for the prevention of such a pathological condition.

In some embodiments, the neoplastic disease is selected from the group consisting of malignant melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer with high microsatellite instability (MSI-H), or DNA error repair deficiency (dMMR), hepatocellular cancer, gastric cancer, cutaneous squamous cell carcinoma, cervical cancer and diffuse large B-cell lymphoma (DLBCL).

According to yet another aspect of the invention, there is provided the use of a composition in accordance with the above disclosure (for the manufacture of a medicament) for inhibiting angiogenesis in a human or animal.

According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising at least a composition as described above and, optionally, one or more pharmaceutically acceptable excipients.

According to yet another aspect of the invention, there is provided a combination comprising (i) a composition as described above or a pharmaceutical composition as described above and (ii) one or more therapeutically active compounds.

According to yet another aspect of the invention, there is provided a method of treating or preventing a disorder or condition associated with expression or overexpression of EDB fibronectin, comprising administering to a subject in need thereof an effective amount of a composition as described above, a pharmaceutical composition as described above, or a combination in accordance with the above description.

According to yet another aspect of the invention, there is provided a therapeutic kit comprising:

a) a composition as described above, a pharmaceutical composition as described above, or a combination as described above,

b) a device for administering the composition, pharmaceutical composition or combination, and

c) instructions for use.

Brief description of the figures

In fig. 1A-1B: results of protein expression experiments. See Materials and Methods below.

In fig. 1A: The 15-mer linker having the amino acid sequence SEQ ID NO: 4 (herein referred to as SAD) shows the best product yields for all variants (also referred to herein as clones). The yield is almost 100% higher compared to the 2nd best option, DDS.

The two sequences on line AKKAS represent SEQ ID NO: 9 and 18, the two sequences on line AP7 represent SEQ ID NO: 15 and 19, the two sequences on line DDS represent SEQ ID NO: 10 and 20, the two sequences on line AP6 represent SEQ ID NO: 14 and 21, two sequences in line G4S represent SEQ ID NO: 11 and 22, two sequences in line SES represent SEQ ID NO: 12 and 23, two sequences in line Alpha3 represent SEQ ID NO: 13 and 24, and two the sequences in line SAD represent SEQ ID NOs: 4 and 25.

The N-terminal and C-terminal residues (or 5' or 3' nucleotides) in FIG. 1, which are shown in gray. They are not relevant to the disclosures of this application for which a search is required. They simply show the structure into which the corresponding linkers can be inserted.

In fig. 1B, the results of SDS-PAGE analysis show a molecular mass of approximately 120 kDa for all variants.

In fig. 2 results of ELISA experiments. All variants bind to the 7B89 domain of human fibronectin at both 10 μg/ml and 1 μg/ml concentrations.

In fig. 3 results of experiments with Biacore. All variants exhibit similar binding behavior to the 7B89 domain of human fibronectin.

In fig. 4 size exclusion chromatography (SEC) results. All variants showed a comparable aggregation profile with a major peak at 13 ml, which corresponds to monomeric immunocytokine, and a smaller peak at 10 ml, which corresponds to aggregates.

In fig. 5 results of experiments with immunofluorescent staining. All variants specifically stained the vasculature of frozen F9 syngeneic teratocarcinoma samples compared to the negative control.

In fig. 6 results from in vivo tumor targeting evaluation. All variants and positive controls were labeled with radioactive iodine ¹²⁵ I and injected (at a rate of 4-9 μg protein/animal) into immunocompetent mice bearing subcutaneously implanted F9 murine teratocarcinoma. The results of radioactivity determination 24 hours after injection showed accumulation in the tumor for all variants. However, the SAD variant showed better tumor accumulation compared to the other seven clones (±2.9% ID/g (= percentage of injected dose per g tissue) compared to the second best variant (G4S) ₃ which showed ±2.4% ID/g).

In fig. 7 and 8 show formats of exemplary immunocytokines using IL-12 and anti-fibronectin antibody.

In fig. 9 SEC analysis results of various fusion proteins (A) huIL-12L19L19 SAD, batch A; (B) huIL-12L19L19 SAD, batch B; (C) huIL-12L19L19 old, batch A; (D) huIL-12L19L19 old, batch B.

In fig. 10 results of the experiment with Biacore. The SAD variant was shown to have increased apparent affinity compared to the old linker variant for the 7B89 domain of fibronectin (3.8 nM vs. 6.7 nM). This surprising result is unexpected because changing the linker can affect the stability of the protein but usually does not affect the affinity for its target.

- 4 043910

In fig. 11 results of an experiment to evaluate tumor targeting in vivo. The SAD and old variants were labeled with ^125I radioiodine and injected (at 10-11 μg protein/animal) into immunocompetent mice bearing subcutaneously implanted F9 murine teratocarcinoma. The SAD variant showed improved tumor targeting ability compared to the older linker variant.

Detailed Description of the Invention Definitions

The term antibody refers to a molecule of the immunoglobulin family containing a tetrameric structural unit. Each tetramer consists of two identical pairs of polypeptide chains, each having one light chain (with a molecular weight of approximately 25 kDa) and one heavy chain (with a molecular weight of approximately 50-70 kDa), linked by a disulfide bond. Known immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as many immunoglobulin variable region genes. Light chains are classified as k or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies can be of any isotype/class (eg, IgG, IgM, IgA, IgD, and IgE) or any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used in structural and functional terms. The N-terminus of each chain defines a variable (V) region or domain of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms light chain variable region ( _VL ) and heavy chain variable region (VH) refer to the indicated regions of the light and heavy chains, respectively. As a result of pairing of VH and V _L, one antigen-binding site is formed. In addition to V regions, both heavy chains and light chains contain a constant (C) region or domain. The C region of the secreted form of immunoglobulin consists of three C domains: CH1, CH2, CH3, optionally CH4 (Cμ) and a hinge region. The C-region of the membrane form of immunoglobulin also has membrane and intracellular domains. Each light chain has a V _L at the N-terminus followed by a constant domain (C) at the other end. The light chain (CL) and heavy chain constant domains (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental penetration, Fc receptor binding, complement fixation and the like. By convention, the numbering of constant region domains increases as they become more distant from the antigen-binding region or amino terminus of the antibody. The N-terminus contains a variable region, and the C-terminus contains a constant region; the CH3 and CL domains actually contain the carboxy-terminal heavy and light chain domains, respectively. VL pairs with VH, and CL pairs with the first heavy chain constant domain. As used herein, the term antibody covers traditional antibody structures and antibody variants. Thus, the scope of this concept includes full-length antibodies, chimeric antibodies, humanized antibodies, human antibodies and antibody fragments.

Antibodies are found as chains of intact immunoglobulins or as a series of well-characterized antibody fragments obtained by digestion with various peptidases. As used herein, the term antibody fragment refers to one or more regions of an antibody that retain the ability to specifically interact (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. For example, pepsin cleaves the antibody downstream of the disulfide bonds in the hinge region to form F(ab)' ₂ , a Fab' dimer, which itself is a light chain linked to VH-CH1 by a disulfide bond. F(ab)' ₂ can be reduced under mild conditions to break the disulfide bond at the hinge region, thereby converting the F(ab)' ₂ dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a hinge region portion (Paul, Fundamental Immunology 3d ed. (1993)). Although various antibody fragments are defined in terms of cleavage of the intact antibody, one of ordinary skill in the art will appreciate that such fragments can be synthesized de novo chemically or using recombinant DNA methodology. As used herein, the term antibody fragment refers to one or more antibody regions obtained by modifying whole antibodies by de novo synthesis using recombinant DNA methodologies that retain binding specificity and functional activity. Examples of antibody fragments include Fv fragments, single chain antibodies (ScFv), Fab, Fab', Fd (Vh and CH1 domains), dAb (Vh and dedicated CDR); diabodies and single-chain diabodies; and multimeric variants of these fragments (eg, F(ab') ₂ ,) with the same binding specificity. Antibody fragments can also be included in grafted cytokine proteins to achieve the specificity and binding activity provided herein.

As used herein, the Fab fragment includes a heavy chain variable domain, a CH1 constant region domain, a light chain variable domain, and a light chain constant region CL domain. The interaction of the domains is stabilized by a disulfide bond between the CH1 and CL domains. In some embodiments, the heavy chain domains of the Fab are in the order N-terminal to C-terminal, VH-CH, and the light chain domains of the Fab are in the order N-terminal to C-terminal, VL-CL. In some embodiments, the Fab heavy chain domains are in the order N-terminal to C-terminal, CH-VH, and the Fab light chain domains are in the order CL-VL. Although historically Fabs have been produced by papain digestion of intact immunoglobulin, as used herein, Fabs are typically produced recombinantly by any method. Each Fab fragment is monovalent with respect to antigen binding, i.e. it has one antigen-binding site.

Complementarity determining regions or complementarity determining regions (CDRs) are referred to interchangeably as the _VL and VH hypervariable regions. CDRs represent a target protein binding site of an antibody chain that has specificity for that target protein. Each human VL or _VH has three CDRs (CDR1-3, numbered sequentially from the N-terminus) comprising approximately 15-20% of the variable domains. CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for binding specificity. The remaining regions of VL or VH, the so-called framework regions (FR), show less variability in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W. H. Freeman & Co., New York, 2000).

The positions of the CDRs and framework regions can be determined using various numbering systems well known in the art, for example, Kabat, Chothia, the international immunogenetics database ImMunoGeneTics (IMGT) and AbM (see, for example, Johnson et al., Nucleic Acids Res. , 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al. , J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Determination of antigen binding sites is also described in the following publications: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996) and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

According to the Kabat numbering system, the CDRs in V _H consist of amino acid residues 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and CDRs in V _L have amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to the Chothia numbering system, the CDRs in V _H consist of amino acids 26–32 (HCDR1), 52–56 (HCDR2), and 95–102 (HCDR3); and CDRs in V _L have amino acid residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). Combining Kabat and Chothia's CDR definitions, CDRs consist of amino acid residues: 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues: 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3) in human VL.

As used herein, the term antibody light chain variable region or antibody heavy chain variable region refers to a polypeptide containing VL or _VH , respectively. Endogenous VL is encoded by the gene segments V (variable) and J (junction), and endogenous VH by V, D (diversity) and J. Each of the V _L or V _H includes CDRs as well as framework regions (FR). The term variable region or V region interchangeably refers to the heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The V region may be naturally occurring, recombinant, or synthetic. In this application, light chains of antibodies and/or heavy chains of antibodies may in some cases be collectively referred to as antibody chains. As presented and described hereinafter, the antibody light chain variable region or the antibody heavy chain variable region and/or the antibody variable region and/or chain optionally comprises a cytokine polypeptide sequence grafted into the CDR.

The C-terminal portion of an immunoglobulin heavy chain as described herein, containing, for example, the CH2 and CH3 domains, constitutes the Fc region. The term Fc region as used herein refers to the constant region of an antibody without the first immunoglobulin constant region domain (CH1). Fc refers to the last two constant region domains of the immunoglobulins IgA, IgD and IgG and to the last three constant region domains of the immunoglobulins IgE and IgM, and also to the N-terminus of the flexible hinge region of these domains. For IgA and IgM, the Fc may include the J chain. For IgG, the Fc includes the immunoglobulin domains Cy2 and Cy3 and the hinge region between Cy1 and Cy. It is understood in the art that the boundaries of the Fc region may vary, however, the Fc region of the human IgG heavy chain is generally defined as containing residues C226 or P230 up to its carboxyl terminus using the EU numbering system as described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The term Fc region can refer to this region alone or to this region in the context of an antibody or antibody fragment. The Fc region includes naturally occurring allelic variants of the Fc region, for example in the CH2 and CH3 region, including, for example, modifications that modulate effector function. Fc regions also include variants that do not result in changes in biological function. For example, deletion of one or more amino acids from the N-terminus or C-terminus of the Fc region of an immunoglobulin does not result in a significant loss of biological function. For example, in some embodiments, the C-terminal

- 6 043910 lysine is modified, replaced or deleted. In specific embodiments, one or more C-terminal residues in the Fc region are altered or deleted. In some embodiments, one or more C-terminal residues in the Fc (eg, terminal lysine) are deleted. In some further embodiments, one or more C-terminal residues in the Fc are replaced with an alternative amino acid (eg, a terminal lysine is substituted). Such options are selected in accordance with general rules known in the art to have minimal impact on activity (see, for example, Bowie et al., Science 247: 306-1310, 1990). The Fc region is a region of immunoglobulin (Ig) recognized by cellular receptors such as FcR and to which complement activation protein C1q binds. The lower hinge region, which is encoded in the 5' region of the CH2 exon, provides flexibility for the antibody to bind to FcR receptors.

A chimeric antibody is an antibody molecule in which: (a) the constant region or part thereof is altered, replaced or exchanged such that the antigen binding region (variable region) binds to a constant region of a different or altered class, effector function and/or species, or an entirely different one a molecule that imparts new properties to chimeric antibodies, for example, with an enzyme, toxin, hormone, growth factor and drug; or (b) the variable region or part thereof is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity.

A humanized antibody is an antibody that retains the reactivity (eg, binding specificity, activity) of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining portions of the antibody with human counterparts. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).

Human antibodies include antibodies having variable regions in which both framework regions and CDR regions are derived from human sequences. In addition, if the antibody contains a constant region, then the constant region is also derived from such human sequences, for example, human germline sequences, or mutated variants of human germline sequences or an antibody containing consensus framework sequences derived from human framework sequences, such as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000). Human antibodies may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutation in vivo, or conservative substitution to ensure stability or production).

The term isolated, when applied to a nucleic acid or protein, means that the nucleic acid or protein is substantially free of other cellular components with which it is naturally associated. Preferably they are in a homogeneous state. They can be in dry form or in aqueous solution. Purity and uniformity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-pressure liquid chromatography. The protein, which is the predominant species present in the preparation, is largely purified. In particular, the isolated gene is separated from open reading frames that flank the gene and encode a protein different from the gene of interest. The term purified means that the nucleic acid or protein produces essentially one band in gel electrophoresis. In particular, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95%, and most preferably at least 99%.

The term nucleic acid or polynucleotide refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the clearly stated sequence. In particular, substitutions of degenerate codons can be obtained by creating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 ( 1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)).

The terms polypeptide, peptide and protein are used interchangeably as used herein to refer to a polymer composed of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial, chemically produced mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term EDB-binding domain-containing peptide or protein refers to a peptide or protein that, as a whole or through a region/fragment thereof, binds to EDB, i.e. fibronectin containing extradomain B.

Typically, a peptide may be, for example, a monomeric molecule having a length of >3 amino acid residues and <50 amino acid residues (hence an oligo- or polypeptide), while a protein may, for example, be a monomeric or bi- or polymeric molecule with one or several proteins, each chain of which is > 50 amino acid residues long.

The term amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics, which function similarly to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as amino acids that are then modified, such as hydroxyproline, carboxyglutamine and O-phosphoserine. The term amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. have a carbon atom in the alpha position, which is bonded to a hydrogen atom, a carboxyl group, an amino group and an R group, for example homoserine, norleucine, methionine sulfoxide and methionine methylsulfonium. Such analogues have modified R groups (eg, norleucine) or a modified peptide backbone, but retain the same chemical structure as the naturally occurring amino acid. The term amino acid mimetics refers to chemical compounds that have a structure different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

The term conservatively modified variants refers to both amino acid sequences and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or in the case where the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, any specific protein is encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at each position at which alanine is encoded by a codon, the codon can be replaced by any corresponding known codons without changing the encoded polypeptide. Such nucleic acid variants are silent variations, which are a type of conservatively modified variant. As used herein, it is intended that each nucleic acid sequence that encodes a polypeptide also includes all possible silent variations of the nucleic acid. One skilled in the art will appreciate that every codon in a nucleic acid (except AUG, which is typically the only methionine codon, and TGG, which is typically the only tryptophan codon) can be modified to produce a functionally identical molecule. Therefore, for each sequence described, each silent nucleic acid variation that encodes a polypeptide is meant.

With respect to amino acid sequences, one skilled in the art will appreciate that single substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete one amino acid or a small percentage of amino acids in the encoded sequence result in a conservatively modified variant in which the change results in the replacement of an amino acid with a chemically similar amino acid. A list of conservative substitutions that provide functionally similar amino acids is well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecific homologs and alleles. Each of the following 8 groups contains amino acids that are conservative replacements for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T) and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)).

In this context, a conservative amino acid substitution, as used here, has less impact on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often divided into six main groups based on their structure and the general chemical characteristics of their R groups.

In some embodiments, a conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been identified in the art. These families include amino acids containing basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine , cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

Other conservative amino acid substitutions may also occur within families of amino acid side chains, such as replacing asparagine with aspartic acid to modify the charge of the peptide. Conservative changes may further include substitution of chemically homologous unnatural amino acids (ie, synthetic unnatural hydrophobic amino acid instead of leucine, synthetic unnatural aromatic amino acid instead of tryptophan).

Percent sequence identity is determined by comparing two optimally aligned sequences in a comparison window, where a portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide), which does not contain additions or deletions, for optimal alignment of two sequences. The percentage is calculated by determining the number of positions at which an identical nucleotide or amino acid residue is found in both sequences, resulting in the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

The terms identical or percentage identity with respect to two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same sequence. Two sequences are substantially identical if the two sequences have a certain percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99 % sequence identity in the specified region or, if not specified, in the entire sequence of the reference sequence), when compared and aligned for best fit in the comparison window or in a specified region measured using one of the following sequence comparison algorithms or manual and visual alignment analysis. The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. Optionally, the identity is in a region that is at least about 15, 25, or 50 nucleotides in length, or more preferably in a region that is 100 to 500 or 1000 nucleotides or more in length or the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity may exist over a region of at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids, optionally , at least approximately 150, 200 or 250 amino acids in length or the entire length of the reference sequence. For shorter amino acid sequences, such as amino acid sequences of 20 amino acids or less, significant identity occurs when one or two amino acid residues are conservatively substituted in accordance with the conservative substitutions defined herein above.

The terms subject, patient and individual interchangeably refer to a mammal, such as a human or a non-human mammalian primate. The mammal may also be a mammalian laboratory animal, such as a mouse, rat, rabbit, hamster. In some embodiments, the mammal may be a mammal, a farm animal (eg, a horse, a sheep, a bull, a pig, a camel) or a mammal, a domestic animal (eg, a dog, a cat).

As used herein, the terms treat, treat, or treat any disease or disorder refer in some embodiments to alleviating the disease or disorder (ie, slowing or stopping or reducing the progression of the disease or at least one of its clinical symptoms). In yet another embodiment, treating, treating, or treating refers to reducing or improving at least one physical parameter, including parameters that may not be noticeable to the patient himself. In yet another embodiment, treating, treating, or treating refers to modulating a disease or disorder physically (eg, stabilizing a noticeable symptom), physiologically (eg, stabilizing a physical parameter), or both. In yet another embodiment, treating, curing or treating or preventing refers to preventing or delaying the onset or development or progression of a disease or disorder.

The term co-administration refers to the simultaneous presence of two (or more) active agents in the body of a subject. Active agents that are coadministered may be delivered simultaneously or sequentially.

Various aspects of the invention are described in detail in the following sections. The division into sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. The invention is not limited to the specific constituents of the compositions described or the process steps of the methods described, since such compositions and methods may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting.

As used in the present specification and the accompanying claims, the singular articles a, an and the include references to the singular and/or plural unless the context clearly dictates otherwise. In this application, the use of or means and/or unless otherwise indicated. In addition, it should be understood that when parameter ranges are specified that are limited to numeric values, the ranges are deemed to include those restrictive values. All prior art disclosures cited herein are incorporated by reference in their entirety.

The term GS linker as used herein refers to a peptide linker that consists predominantly or exclusively of glycine and serine residues (also called a GlySer linker). In various embodiments, the GS linker is a linker with the sequence shown herein in any of SEQ ID NO: 2, 6, or 8.

In this context, a conservative amino acid substitution, as used here, has less impact on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often divided into six main groups based on their structure and the general chemical characteristics of their R groups.

As used herein, the term target binding affinity refers to the affinity of a binding molecule of the invention with its target and is expressed numerically using KD values. In general, a higher KD value corresponds to weaker binding. In some embodiments, KD is measured using a radiolabeled antigen (MA) binding assay or surface plasmon resonance (SPR) assay using, for example, the BIAcore™-2000 or BIAcore™-3000 systems. In some embodiments, the rate of association or kon and the rate of dissociation or koff are also determined using the surface plasmon resonance (SPR) method. In additional embodiments, KD, association rate, and dissociation rate are measured using Octet® systems.

As used here, the expression competes for binding is used in relation to one of the antibodies defined by the sequences specified above, meaning that the actual antibody has the activity that it binds to the same target or the same target epitope or domain or subdomain. that the specified antibody, defined by the sequence, is a variant of the latter. The efficiency (eg, kinetics or thermodynamics) of binding may be the same as, higher or lower than the efficiency of the latter. For example, the binding equilibrium constant for substrate binding may be different for two antibodies.

As used here, the expression retention of the ability to bind to a given target means, for example, that the corresponding variant has a binding affinity for the target of >50% compared to that of the unmodified peptide.

EDB fibronectin

Fibronectin (UniProt: P02751) is a high molecular weight (~440 kDa) extracellular matrix glycoprotein that binds to membrane receptor proteins called integrins. Like integrins, fibronectin binds to extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans (eg, syndecans).

Fibronectin is involved in the development of cancer. In lung carcinoma, fibronectin expression is increased, especially in non-small cell lung carcinoma. Adhesion of lung carcinoma cells to fibronectin increases tumorigenicity and imparts resistance to chemotherapeutic agents that induce apoptosis. Fibronectin may promote lung tumor growth/survival and therapy resistance and has been discussed as a novel target for the development of new anticancer drugs.

Fibronectin exists as a protein dimer consisting of two virtually identical monomers linked by a pair of disulfide bonds. Fibronectin protein is produced from a single gene, but alternative splicing of its mRNA precursor from one copy of the fibronectin gene occurs at three sites encoding the EDA, EDB, and IIICS domains, resulting in multiple isoforms.

Fibronectin isoforms containing EDA or EDB domains are known as oncofetal forms due to their role in embryonic development and their limited presence in normal tissues

- 10 043910 adult. These isoforms are also recognized as important markers of angiogenesis, a critical physiological process during development and essential for tumor cells during cancer progression. Fibronectin EDB is expressed in tumor tissues, especially breast carcinomas, brain tumors, lymphoma cells and prostate carcinoma. Due to its tissue-specific expression profile, fibronectin EDB is an attractive tumor antigen for use in targeted therapy.

IL-12

Interleukin-12 is a heterodimeric cytokine with numerous biological effects on the immune system. It consists of two subunits, p35 and p40, which are required for the secretion of the active form of IL-12, p70. Interleukin-12 is produced by dendritic cells (DCs), leading to increased maturation and presentation of antigens, allowing the initiation of T cell responses to tumor-specific antigens. It also regulates IL-12 secretion by DCs, creating a positive feedback mechanism to enhance the response. Once the response is initiated, IL-12 plays a fundamental role in regulating the immune system's Th1 cytokine profile, inducing CD4+ T cells to secrete interferon-gamma (IFN-γ) and leading to a cytotoxic CD8+ T cell response.

IL-12 is also a potent proinflammatory cytokine that leads to the secretion of other cytokines, including tumor necrosis factor-alpha (TNF-α), which, in combination with IFN-γ, is a prerequisite for the production of cytotoxic CD4+ T lymphocytes (CTLs). In addition, IL-12 can stimulate the activation of innate immune cells such as macrophages and eosinophils through the induction of IFN-γ and other cytokines. This activation then leads to the secretion of IL-12 by these cells and further amplification of both innate and adaptive immune responses. However, high levels of IL-12, and hence IFN-γ, have also been associated with the induction of antagonistic molecules such as IL-10 and the depletion of IL-12 downstream signaling molecules such as STAT4.

Previous attempts to use IL-12 as a therapeutic agent have been unsuccessful, as IL-12 has demonstrated modest antitumor effects at best, which are often accompanied by unacceptably high toxic side effects, including fever, fatigue, hematologic changes, hyperglycemia, and/or organ dysfunction.

The term p35 as used herein means a polypeptide that contains an amino acid sequence having at least 80% identity with the amino acid sequence given below:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTST

VEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTM NAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHA FRIRAVTIDRVMSYLNAS (SEQ ID NO: 3).

The term p40 as used herein means a polypeptide that contains an amino acid sequence having at least 80% identity with the amino acid sequence given below:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTL TIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAK NYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVEC QEDSACPAAEESLPIEVMVDAVHKL KYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVE VSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQD RYYSSSWSEWASVPCS (SEQ IDNO:1).

As used herein, the term IL-12 means a polypeptide that (i) includes both:

(a) p35 or a fragment thereof, wherein p35 contains an amino acid sequence having at least 80% identity with the amino acid sequence given below:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVE FKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLC ILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:3) and (b) p40 or a fragment thereof, wherein p40 contains an amino acid sequence having at least 80% identity with the amino acid sequence given below:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTL TIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAK NYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVEC QEDSACPAAEESLPIEVMVDAVHKL KYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVE VSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQD RYYSSSWSEWASVPCS (SEQ ID NO:1) and (ii) can activate the IL-12 receptor.

- 11 043910

Linkers

In some embodiments, one or more peptide linkers are independently selected from a (Glynn-Ser)m sequence, a (Glynn-Ala)m sequence, or any combination of a (Glyn _n -Ser)m/(Glyn _n -Ala)m sequence, wherein each is independently an integer from 1 to 5, and each m is independently an integer from 0 to 10. In some embodiments, the peptide linker is (Gly ₄ -Ser) _m , where m is an integer from 0 to 10. In some embodiments In embodiments, the peptide linker is (Gly ₄ -Ala) _m , where m is an integer from 0 to 10. Examples of linkers include, but are not limited to, some embodiments where one or more linkers include G ₄ S repeats, such as a Gly- linker Ser GGGGS (SEQ ID NO: 34), or (GGGGS) _m , where m represents a positive integer equal to or greater than 1. For example, m=1, m=2, m=3, m=4, m=5 and m=6, m=7, m=8, m=9 and m=10. In some embodiments, the linker includes multiple GGGGS repeats (SEQ ID NO: 34), including, but not limited to, (GGGGS) ₃ or (GGGGS)4. In some embodiments, Ser may be replaced by Ala, for example, G/A linkers such as (GGGGA) (SEQ ID NO: 35) or (GGGGA)m, where m is a positive integer equal to or greater than 1. B In some embodiments, the linker includes multiple GGGGA repeats (SEQ ID NO: 35). In yet other embodiments, the linker includes combinations of and a plurality of GGGGS (SEQ ID NO: 34) and GGGGA (SEQ ID NO: 35).

In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 2). In some embodiments, the linker comprises the amino acid sequence GSADGGSSAGGSDAG (SEQ ID NO: 4). In some embodiments, the linker comprises the amino acid sequence GSSGG (SEQ ID NO: 6). In some embodiments, the linker comprises the amino acid sequence SSSSGSSSSGSSSSG (SEQ ID NO: 8). In some embodiments, the linker comprises the amino acid sequence GGGAKGGGGKAGGGS (SEQ ID NO: 9). In some embodiments, the linker contains the amino acid sequence GGGGDGGGGDGGGGS (SEQ ID NO: 10). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGEGGGGS (SEQ ID NO: 12). In some embodiments, the linker comprises the amino acid sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 13). In some embodiments, the linker comprises the amino acid sequence APPAPAPAPAP (SEQ ID NO: 14). In some embodiments, the linker comprises the amino acid sequence APPAPARAPARAP (SEQ ID NO: 15).

IL-12 bound to anti-EDB antibody

The present invention provides, among other things, methods and compositions for treating diseases or disorders associated with the expression of EDB fibronectin, including cancer. Described herein represents novel compositions and methods that utilize fibronectin as a target to achieve cancer-targeted delivery of IL-12. This approach promises to fully exploit the therapeutic potential of IL-12 while reducing systemic toxicity and extending the therapeutic window of IL-12.

Although other constructs containing IL-12 and the EDB targeting domain of fibronectin have been previously described, the currently disclosed compositions are surprisingly superior to them.

International application WO2006/119897, the contents of which are incorporated herein by reference, discloses three different molecular formats of IL-12 in combination with an antibody targeting fibronectin EDB termed L19.

One format was sc(IL-12)-scFv(L19), shown in FIG. 7A. This format, consisting of an IL-12 heterodimer in which two subunits are connected via a peptide linker (hence single-chain IL-12 or sc(IL-12)), and where said IL-12 is then linked, via a second peptide linker, to the L19 antibody , which is also in single-chain Fv format (hence scFv(L19)). This format showed moderate tumor targeting ability, which is consistent with prior art results.

Another format was the sc(IL-12)-SIP(L19) homodimer shown in FIG. 7B. The SIP (small immune protein) format was developed and presented by the applicants in WO 2003/076469 and is also called mini-antibody. SIP is a homodimer consisting of two subunits containing an scFv linked to a CH4 domain. The two CH4 domains are connected to each other by a disulfide bridge. Although the prior art indicates that the tumor targeting properties of L19 can be improved using the SIP format, increased tumor uptake of this conjugate has not been observed.

Another format was a heterodimer of IL-12 p40 and p35 subunits linked together by a disulfide bridge, and where each subunit was fused to scFv(L19), forming the scFv(L19)-p35/p40-scFv(L19) heterodimer shown in fig. 7C. With this heterodimeric format, a marked improvement in tumor uptake of the composition was achieved.

International application WO2013/014149, the contents of which are incorporated herein by reference - 12043910, discloses two new alternative molecular formats of IL-12 coupled to an anti-EDA fibronectin antibody called F8.

Here, another format of IL-12 immunoconjugates includes a single chain diabody. Essentially, it consists of two scFv antibodies with a short linker of 5 amino acids (thus forming a diabody), connected to each other by a longer peptide linker of 15 amino acids.

The molecular format comprising IL-12 fused to the monospecific single chain diabody F8 (see Fig. 8B) was shown to be an improved option in terms of tumor targeting due to: (i) the scFv(F8)-p35/p40 heterodimer -scFv(F8) (Fig. 8A), which in its L19 variant was the best format disclosed in WO 2006/119897, or (ii) two IL-12 molecules coupled to a diabody (Fig. 8C).

L19

Antibody L19 as used herein means any antibody that binds to the EDB of fibronectin or any region thereof and contains an amino acid sequence having at least 75% identity with one or more of the following amino acid sequences:

VHL19 (SEQ IDNO: 7)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSS

GTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVT VSS

VLL19 (SEQID NO: 5)

EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRA

TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

CDR1 VH (SEQ ID NO: 28)

SFSMS

CDR2 VH (SEQ ID NO: 29)

SISGSSGTTYYADSVKG

CDR3 VH (SEQ ID NO: 30)

PFPYFDY

CDR1 VL (SEQ ID NO: 31)

RASQSVSSSFLA

CDR2 VL (SEQ ID NO: 32)

YASSRAT

CDR3 VL (SEQ ID NO: 33)

QQTGRIPT

Diathelo LI9 (SEQ ID NO: 36)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSS GTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVT VSSGSSGGEIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYAS SRATGIPRDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQTGRIPPTFGQGTKVEIK

Pharmaceutical compositions

A further aspect of the present invention relates to a pharmaceutical composition containing at least one conjugate of the invention and, optionally, a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the present invention typically contain a therapeutically effective amount of a conjugate of the invention and optionally excipients such as pharmaceutically acceptable excipient(s). Said pharmaceutical compositions are prepared by methods well known in the field of pharmacy. The carrier or excipient can be a liquid substance that can serve as a carrier or vehicle for the active ingredient. Suitable carriers or excipients are well known in the art and include, for example, stabilizers, antioxidants, pH adjusting agents, controlled release excipients.

The pharmaceutical preparation of the invention can be adapted, for example, for parenteral use and can be administered to a patient in the form of solutions or the like. Formulations containing the composition of the present invention can be administered to a patient. Administration is preferably in a therapeutically effective amount that is sufficient to provide benefit to the patient. Such a beneficial effect may represent at least an improvement in at least one symptom. The actual amount to be administered and the rate and timing of administration will depend on the nature and severity of the disease being treated. Prescribing a treatment regimen, eg deciding on doses, etc., is the responsibility of general practitioners and other medical specialists. Treatment can be repeated daily, twice a week, once a week or once a month at the discretion of the doctor.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powder or liquid. The tablet may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petroleum, animal or vegetable fats, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.

For intravenous or intralesional injection, the active ingredient must be in the form of an aqueous solution suitable for parenteral administration, which is free of pyrogens and has a suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included.

In addition, it should be understood that the embodiments disclosed herein should not be construed as separate embodiments that are not related to each other. The features discussed in connection with a particular embodiment are intended to be disclosed also in connection with other embodiments provided herein. If in one case a particular feature is not disclosed in one embodiment but is disclosed in another, one skilled in the art will appreciate that this does not necessarily mean that said feature is not intended to be disclosed in combination with said other embodiment. One skilled in the art will appreciate that the intent of this application is to disclose this feature also for another embodiment, but this is done simply for purposes of clarity and to keep the application within an easily controllable scope.

In addition, the contents of the links mentioned herein are incorporated by reference. In particular, this applies to prior art documents that disclose standard or routine methods. In this case, incorporation by reference is primarily intended to provide sufficient disclosure and avoid lengthy repetition.

Typically, the composition of the invention is capable of binding to specific target structures in a cell, tissue, organ or patient, where these target structures are determined by the specificity of the peptide or protein containing the EDB binding domain.

Once at its target, IL-12 stimulates the production of IFNy from T cells and natural killer cells, and also induces the differentiation of Th1 helper cells. IL-12 is a key mediator of innate and cellular immunity. If the peptide or protein containing the EDB-binding domain in the construct is specific for a target structure, for example, a receptor or an extracellular matrix protein that characterizes a neoplasm, such as a tumor, a hematological disease or cells in the process of degenerating into cancer cells, then binding of the composition induces IL-12-mediated high antitumor and antimetastatic activity.

Applicants have been surprised to discover that when a linker containing an amino acid motif including GSADGGSSAGGSDAG is used to link IL-12 to a peptide or protein containing an EDB binding domain (i.e., the diabody disclosed in WO 2013/014149), it can Better tumor targeting efficiency as well as superior product yield can be achieved. At the same time, the binding behavior of this variant is superior to the binding behavior of the variant with the shorter linker GSADGG, tentatively called old here and disclosed in WO 2013/014149.

Applicants first evaluated and characterized eight clones of human IL-12 coupled to the L19 antibody in a single-chain diabody format (huIL-12L19L19) with different polypeptide linkers between the cytokine and the L19 single-chain diabody.

Five clones, designated: (i) AKKAS, (ii) DDS, (iii) (G4S) ₃ , (iv) SAD and (v) SES, contain linkers for conjugation of immunocytokines to recombinant antibodies, and were selected due to their different electric charge (neutral, positively charged, negatively charged).

Three additional clones were developed, designated: (vi) Alpha3, (vii) AP6, and (viii) AP7. For these three clones, the principles presented in Chen et al. were considered and implemented in this case. (2013). This review suggests that rigid linkers may have better stability and may maintain the correct distance between the cytokine and the antibody, thereby increasing therapeutic efficacy.

None of the linkers (i)-(viii) have been previously tested on this specific immunocytokine.

Surprisingly, the SAD linker was found to significantly increase (i) tumor targeting efficiency and (ii) yield of IL-12 product bound to an EDB binding domain-containing peptide or protein without (iii) disrupting EDB binding behavior compared to other clones . This is quite surprising since, despite considering and using the principles outlined in Chen's publication, only the SAD linker provided the composition with a number of superior properties

- 14 043910 compared to both the old clone (described in more detail below) and the other new variants described here.

Finally, a ninth clone, designated here as old, containing the linker disclosed in WO 2013/014149, was used for comparison with the SAD linker. Surprisingly, it was found that the SAD linker, despite having a similar first part of the sequence as the old linker, has superior binding affinity to EDB.

In addition, size exclusion chromatography results showed that the SAD linker has a higher relative monomer content compared to the old linker, meaning that the assembly of the complete conjugate is more efficient. The high relative monomer content provided by the SAD linker is expected to increase the overall product yield.

As used herein, the term single chain diabody refers to a construct of two single chain Fv(scFv) antibodies with a short linker, preferably 3-10 amino acids long, more preferably 5 amino acids long (also known as a diabody), connected to each other by a longer linker, preferably 5-20 amino acids long, more preferably 15 amino acids long, according to the following scheme (N^Q orientation: VHL9-linker 3-VLL19-linker 4-VHL19linker3-VLL19.

In some embodiments, the first subunit of the heterodimeric IL-12 protein is p40 and the second subunit is p35.

As discussed above, fibronectin isoforms containing EDA or EDB domains are known as oncofetal forms due to their importance in development and their reexpression in tumors, in contrast to their limited presence in normal tissues of adult subjects.

These isoforms are also recognized as important markers of angiogenesis, a critical physiological process during development and essential for tumor cells during cancer progression.

Therefore, extradomain B (EDB) of fibronectin is an attractive target for antitumor therapy, including the use of immunocytokines, as discussed here.

In some embodiments, the single chain diabody may comprise an antigen binding domain having complementarity determining regions (CDRs) or VH and/or VL domains of an antibody capable of specifically binding to an antigen of interest, e.g., one or more CDR domains or VH and/or VL domains of an antibody , capable of specifically binding to the extradomain B antigen of fibronectin.

The antigen binding domain can be formed through the arrangement of complementarity determining regions (CDRs). The structure for carrying a CDR or set of CDRs is typically an antibody heavy or light chain sequence, or a substantial portion thereof, in which the CDR or set of CDRs is located at a position corresponding to the CDR or set of CDRs of the naturally occurring antibody VH and VL variable domains encoded rearranged immunoglobulin genes. The structures and positions of immunoglobulin variable domains can be determined using the numbering system according to Kabat et al. (1987) (Sequences of Proteins of Immunological Interest. ^4th Edition. US Department of Health and Human Services) and its updated versions, now available on the Internet (at immuno.bme.nwu.edu or search Kabat using any search engine) .

By CDR region or CDR is meant the hypervariable region of the heavy and light chains of an immunoglobulin, as defined in the monograph by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, ^4th Edition, US Department of Health and Human Services (Kabat et al., (1991a), Sequences of Proteins of Immunological Interest, ^5th Edition, US Department of Health and Human Services, Public Service, NIH, Washington and later editions). Typically an antibody contains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used herein to indicate, as the case may be, one or more of these regions, or even all of these regions, which contain the majority of the amino acid residues responsible for binding to an antigen or epitope, due to the affinity of the antibody which it recognizes.

Thus, the single chain diabody may comprise an antigen binding region having one, two, three, four, five or six CDRs or VH and/or VL domains of the L19 antibody.

In some embodiments, the single chain diabody may comprise an antigen binding domain having the antibody L19 complementarity determining regions (CDRs) set forth in SEQ ID NOs: 28-33. The antigen binding region may comprise the VH and/or VL domains of the L19 antibody shown in SEQ ID NO: 7 and 5, respectively.

The antigen binding region may comprise one, two, three, four, five or six L19 antibody CDRs. The amino acid sequences of CDR L19 represent

SEQ ID NO: 28 (CDR1 VH);

SEQ ID NO: 29 (CDR2 Vh);

SEQ ID NO: 30 (CDR3 VH);

SEQ ID NO: 31 (CDR1 VL);

SEQ ID NO: 32 (CDR2 VL) and/or

SEQ ID NO: 33 (CDR3 VL).

- 15 043910

SEQ ID NOs: 28-30 represent the amino acid sequences of the CDR VH regions (1-3, respectively) of the human monoclonal antibody L19. Sequences

SEQ ID NOs: 31-33 represent the amino acid sequences of the CDR VL regions (1-3, respectively) of the human monoclonal antibody L19. The amino acid sequence of the VH and VL domains of antibody L19 corresponds to SEQ ID NO: 7 and 5, respectively.

In some embodiments, the single chain diabody comprises at least one of:

a) a set containing 3 heavy chain CDRs, defined herein as SEQ ID NO: 28-30, and 3 light chain CDR, defined herein as SEQ ID NO: 31-33;

b) a set of 3 heavy chain CDRs in VH, defined herein as SEQ ID NO: 7, and a set of 3 light chain CDRs in VL, defined herein as SEQ ID NO: 5;

c) a heavy chain CDR/light chain CDR combination according to point a) or b), provided that at least one of the CDRs has up to 3 amino acid substitutions relative to the corresponding CDR specified in point a) or b), while simultaneously maintaining its ability to bind to extradomain B (EDB) of fibronectin;

d) a heavy chain CDR/light chain CDR combination according to claim a) or b), provided that at least one of the CDRs has a sequence identity of >66% relative to the corresponding CDR specified in claim a) or b) , while retaining its ability to bind to extradomain B (EDB) of fibronectin;

wherein the CDRs are inserted into a suitable protein scaffold to be able to bind to the extra domain B (EDB) of fibronectin.

In some embodiments, at least one of the CDRs has a sequence identity of >67, preferably >68, more preferably any of >69, >70, >71, >72, >73, >74, >75, > 76 , >77, >78, >79, >80, >81, >82, >83, >84, >85, >86, >87, >88, >89, >90, >91, >92, >93, >94, >95, >96, >97, >98, or most preferably >99% relative to the corresponding CDRs.

In yet another embodiment, at least one of the CDRs has been modified through affinity maturation or other modifications, resulting in a sequence modification compared to the sequences disclosed above.

In some embodiments, at least one of the CDRs has up to 2, and preferably 1, amino acid substitution relative to the corresponding CDR specified in paragraph a) or b).

In some embodiments, the single chain diabody comprises at least one of:

a) the VH and VL domains of antibody L19, shown in SEQ ID NO: 7 and 5;

b) the heavy chain/light chain variable domain sequence pairs of claim a), provided that at least one of the domains has >80% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, and/or

c) a pair of heavy chain/light chain variable domain sequences according to claim a), provided that at least one of the domains has up to 10 amino acid substitutions relative to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, while simultaneously maintaining its ability bind to extradomain B (EDB) of fibronectin.

In some embodiments, at least one of the domains has a sequence identity of >81, preferably >82, more preferably >83, >84, >85, >86, >87, >88, >89, >90, >91 , >92, >93, >94, >95, >96, >97, >98, or most preferably at a level of >99% relative to SEQ ID NO: 7 or SEQ ID NO: 5, respectively.

In some embodiments, at least one of the domains has up to 9, preferably up to 8, more preferably up to 7, 6, 5, 4, 3 or 2, and most preferably 1 amino acid substitution relative to SEQ ID NO: 7 or SEQ ID NO : 5 respectively.

According to some embodiments of the invention, at least one amino acid substitution in the single chain diate is a conservative amino acid substitution.

In another embodiment, the composition has the full-length structure [p40]-[linker1]-[p35]-[linkerSAD]-[VHL19]-[linker3]-[VLL19]-[linker4]-[VHL19]-[linker3][VLL19 ].

In yet another embodiment, the composition has the full-length sequence of SEQ ID NO: 16.

Disorders

According to yet another aspect of the invention, there is provided the use of a composition as described above (for the production of a medicament) in the treatment of a human or animal diagnosed with, suffering from, or at risk of developing a neoplastic disease, or for the prevention of such a pathological condition.

According to another aspect of the invention, there is provided the use of a composition according to any of the above claims (for the production of a medicament) for inhibiting angiogenesis in

- 16 043910 person or animal.

Thus, the conjugate described herein can be used in a method of treating a neoplastic disease or inhibiting angiogenesis by targeting IL-12 to neovascularization in vivo.

The term neoplastic disease covers malignant transformation of cells and cancer, including tumors and hematological diseases.

Also provided is a method of treating cancer or inhibiting angiogenesis by targeting an agent, particularly a therapeutic agent, such as IL-12, to neovascularization in a patient, comprising administering a therapeutically effective amount of a conjugate described herein to the patient. Pathological conditions amenable to treatment using the composition described herein include cancer, other tumors and neoplastic conditions. The composition can be used to inhibit angiogenesis and therefore to treat rheumatoid arthritis, diabetic retinopathy, age-related muscular dystrophy, angiomas, tumors and cancer. Treatment may include preventative treatment. The composition can also be used for administration in diagnostic methods, for example, in targeting and diagnosing angiogenesis, which may be associated with any of the above pathological conditions. Other diseases and pathological conditions can also be diagnosed and treated in accordance with the nature of the protein therapeutic or diagnostic agent contained in the composition and the specificity of the targeting site.

Types of cancer suitable for treatment as described herein include any type of solid or non-solid tumor or malignant lymphoma and, in particular, liver cancer, lymphoma, leukemia (eg, acute myeloid leukemia), sarcoma, skin cancer, bladder cancer, cancer breast, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, stomach cancer and brain cancer. Cancer may have a family history or be sporadic. Cancer can be metastatic or non-metastatic.

Preferably, the cancer is a cancer selected from the group consisting of kidney cancer, breast cancer, liver cancer, lung cancer, lymphoma, sarcoma (eg, gastrointestinal stromal tumor), skin cancer (eg, melanoma), colorectal cancer and neuroendocrine tumors.

In some embodiments, the neoplastic disease is characterized by expression or overexpression of EDB fibronectin.

The compositions of the invention can be administered to a patient in need of treatment by any suitable route, typically by injection into the bloodstream and/or directly into the site to be treated, for example, a tumor or tumor vasculature. The exact dose and frequency of administration will depend on a number of factors, the route of administration, and the size and location of the area to be treated (eg, tumor).

For response, a subject responds to treatment if the subject's cancer score (e.g., hematologic cancer, e.g., cancer cell growth, proliferation, and/or survival) slows or decreases by a detectable amount, e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, as determined by any suitable indicator, such as cell weight, number or volume. In one example, a subject responds to treatment if its life expectancy is increased by about 5%, 10%, 20%, 30%, 40%, 50% or more above its life expectancy if no treatment is given. In yet another example, a subject responds to treatment if the subject has increased progression-free survival, overall survival, or increased time to disease progression. Several methods can be used to determine whether a patient is responding to treatment, including, for example, the criteria found in the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®).

Combination therapy

The composition may be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the pathological condition being treated. Other treatments may include administering appropriate doses of pain medications such as nonsteroidal anti-inflammatory drugs (such as aspirin, ibuprofen, or ketoprofen), or opiates such as morphine, or antiemetics.

According to a further aspect of the invention, a combination of a composition or pharmaceutical composition as described above and one or more therapeutically active compounds is provided.

According to a further aspect of the invention, there is provided a method of treating or preventing a disorder or condition associated with expression or overexpression of EDB fibronectin, comprising administering to a subject in need thereof an effective amount of a composition, pharmaceutical composition or combination as described above.

- 17 043910

Sets

According to yet another aspect of the invention, there is provided a set of items comprising:

a) a composition, pharmaceutical composition or combination as described above,

b) a device for administering the composition, pharmaceutical composition or combination, and

c) instructions for use.

In some embodiments, such a kit includes a pre-filled syringe provided with a suitable patient information leaflet. In yet another embodiment, such a set of items contains infusion bottles with suitable instructions for the user.

The components of the kit are preferably sterile and are contained in hermetically sealed vials or other containers.

The kit may further contain instructions for using the components in the method described herein. The kit components may be contained or packaged in a container, such as a bag, box, can, tin or blister pack.

Examples

Although the invention has been illustrated and described in detail in the figures and in the foregoing description, such illustration and description are to be considered as illustrative or exemplary and not limiting; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments of the claimed invention can be understood and practiced by those skilled in the art upon examination of the figures, disclosure, and appended claims. In the claims, the word containing does not exclude other items or steps, and the indefinite article a or an does not exclude the plural. The fact that certain measures are set out in mutually distinct dependent clauses does not mean that a combination of these measures cannot be used to obtain an advantage. Any reference numbers in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed here are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown in 5'^3' orientation.

Example 1.

Applicants have surprisingly discovered that when certain linkers are used to link IL-12 to a single chain diabody (i.e., an improved format than disclosed in WO 2013/014149), better tumor targeting efficiency as well as higher product yield can be achieved .

Applicants evaluated and characterized eight clones of human IL-12 coupled to the L19 antibody in a single-chain diabody format (huIL-12L19L19), with a different polypeptide linker between the cytokine and the L19 single-chain diabody.

The five clones, which were designated: (i) AKKAS, (ii) DDS, (iii) << (G4S) ₃ , (iv) SAD and (v) SES, contain linkers for conjugation of immunocytokines with recombinant antibodies and were selected due to their different electrical charge (neutral, positively charged, negatively charged).

Three additional clones were developed, designated: (vi) Alpha3, (vii) AP6 and (viii) AP7. With regard to these three clones, in this case the ideas presented in the publication of Chen et al. were considered and put into practice. (2013). This review suggests that rigid linkers may have better stability and may maintain the correct distance between the cytokine and the antibody, thereby increasing therapeutic efficacy.

None of the linkers (i)-(viii) have been previously tested on this specific immunocytokine.

Surprisingly, the SAD linker was found to significantly improve tumor targeting efficiency and single chain diabody bound IL-12 product yield, while being equally capable of binding to EDB compared to other clones.

Materials and methods

The options tested in the examples have the following general structure:

Domain (N- >C) P4 0 Linke P 1 rZ 5 Linke p2 VH L1 9 Linke rZ L1 9V L Linke p4 VH L19 Linker 3/5 VL L19 SEQ ID NO 1 2 3 4.9-15 7 6 5 8 7 6 5

The various variants (also referred to herein as clones) differ from each other in the sequence of linker 2, as detailed in Table 1. 2.

- 18 043910

table 2

Linker 2 SEQ ID NO Subsequence AKKAS 9 GGGAKGGGGKAGGGS DDS 10 GGGGDGGGGDGGGGS (G4S) ₃ eleven GGGGSGGGGSGGGGS S.A.D. 4 GSADGGSSAGGSDAG SES 12 GGGGSGGGGEGGGGS Alpha3 13 AEAAAI<EAAAAI<EAAAI<A AP6 14 APAPAPAPAPAP AP7 15 APAPAPAPAPAPAP

Cloning of eight fusion proteins with different linkers

The coding sequence of huIL-12L19L19 was obtained by assembling various fragments using PCR: the L19 antibody and the IL12 payload. The L19 gene was amplified by PCR from a previously generated L19-IL2 fusion protein template using appropriate primers. The second L19 gene was amplified by PCR using appropriate primers.

At the same time, the IL-12 p35 domain gene region was amplified by PCR from the previously prepared IL12-based immunocytokine using appropriate primers. The two intermediate fragments were assembled by PCR (to produce the P35-L19 fragment), double digested with BamHI/BspEI, and cloned into a double digest vector containing p35. Then the newly obtained p35-L19 vector was double-digested using BspEI/NotI-HF restriction enzymes and ligated with the gene of the second fragment of the L19 diabody. The p35L19L19 fragment was digested with BamHI/NotI-HF and cloned into a previously double-digested expression vector traditionally used for mammalian cells pcDNA3.1 (+) carrying the p40 subunit gene, resulting in full-length IL12-L19L19.

Various linkers between the IL12 fragments and the L19 single-chain diabody were inserted by PCR assembly of fragment A (p35 coding region with linker) and fragment B (coding linker and antibody region). Various A fragments and B fragments were amplified from IL12L19L19 as a template using appropriate primers.

The cloning strategy developed for clone AP7 resulted in a mutant clone (named AP6). All PCR products were double digested with BamHI-HF and BspEI restriction enzymes and ligated into the p35-L19L19 pcDNA3.1 plasmid. The resulting plasmids were amplified, double digested with NotI-HF and BamHI-HF, and the insert was subcloned into the pcDNA3.1 plasmid containing IL12. The resulting DNA plasmids were amplified and used for cell transfection.

Expression and purification of eight fusion proteins with different linkers

CHO-S cells were used in suspension culture to produce various human IL-12 fusions. The huIL-12L19L19 variants were expressed using transient gene expression. For 1 ml of product, 4x10 ⁶ CHO-S cells in suspension were centrifuged and resuspended in 1 ml of medium suitable for CHO-S. Next, 0.625 μg of plasmid DNA was added to the cells, followed by 2.5 μg of polyethylenimine (PEI; 1 mg/ml solution in water at pH 7.0) per million cells and mixed gently. Transfected cultures were incubated in a shaker-thermostat at 31°C for 6 days.

Finally, the fusion proteins produced by transient gene expression were purified from cell culture medium by protein A affinity chromatography and then dialyzed against PBS.

SDS-PAGE

The correct molecular weight of the fusion proteins was analyzed under reducing and non-reducing conditions using SDS-PAGE 10% and SDS-PAGE 12%.

ELISA

To test for proper binding of the various IL-12 fusion proteins, ELISA plates were coated overnight with 50 μg/ml fibronectin domain 7B89 (see WO 2001/062800 A1, the contents of which are incorporated herein by reference). Immunocytokines were tested at concentrations of 10 and 1 μg/ml. Horseradish protein A peroxidase was used as a secondary reagent. The assay product was developed using soluble substrate BM-Blue POD. The colorimetric reaction was stopped by adding 333 mM H ₂ SO ₄ and the absorbance was measured at wavelengths of 450 and 650 nm using a plate reader.

Size exclusion chromatography and Biacore

Size exclusion chromatography was performed on an AKTA FPLC system using a Superdex Increase 200 column. Affinity experiments were performed using surface plasmon resonance on a Biacore X100 instrument with purified huIL-12L19L19 clones on a CM5 biosensor chip coated with the 7B89 fibronectin domain. Samples were injected as serial dilutions ranging in concentration from 1 μM to 250 nM.

Immunofluorescence

To confirm the ability of various huIL-12 fusion proteins to bind to tumor cells, immunofluorescence analysis was performed on frozen sections of an F9 syngeneic teratocarcinoma sample (8 μm). Tumor sections were fixed in ice-cold acetone (5 min). After fixation, coverslips were washed and blocked with 20% fetal bovine serum in PBS for 45 min. huIL-12L19L19 clones at a concentration of 5 μg/ml were added to a 2% BSA/PBS solution for 1 hour at room temperature. The coverslips were then washed twice with PBS and a final dilution of 1:1000 mouse anti-human interleukin-12 secondary antibody was applied in 2% BSA/PBS at room temperature for 1 h. The coverslips were then washed twice with PBS and a final dilution of goat anti-mouse tertiary antibody was applied. dilution 1:500. DAPI was used to counterstain the nuclei.

Radiolabeling and in vivo tumor targeting assessment

To confirm the ability of various IL-12 fusion proteins to bind to tumors in vivo, their targeting ability was assessed using a biodistribution assay. 100 μg of each IL-12L19L19 clone was labeled with radioactive iodine ¹²⁵ I and chloramine hydrate T, and purified on a PD10 column. Radiolabeled proteins were injected into the lateral tail vein of immunocompetent mice bearing subcutaneously implanted F9 murine teratocarcinoma. The administered dose per mouse varied from 4 to 9 μg. Mice were euthanized 24 h after injection. The organs were weighed and radioactivity measured using a Packard Cobra gamma counter. The level of radioactivity in representative organs was expressed as a percentage of the injected dose per gram of tissue (%ID/g ± standard error).

Results Cloning, expression and SDS-PAGE

Eight huIL-12L19L19 fusion protein variants were successfully cloned, each containing a different polypeptide linker between the cytokine and the L19 single-chain diabody. SDSPAGE analysis showed molecular weights of approximately 120 kDa for all variants, confirming the expected size of the protein (approximately 109 kDa, unglycosylated). Expression yields (transient gene expression in CHO-S cells) varied for all variants from 3.5 to 5 mg/L. Surprisingly, the clone designated SAD showed a yield of 9 mg/L, which was significantly higher than the yield with the other 7 clones. The results are shown in Fig. 1.

ELISA

ELISA results from eight clones Alpha3, AP6, AP7, DDS, SES, AKKAS, (G4S) ₃ and SAD confirmed binding (both 10 μg/ml and 1 μg/ml) to the 7B89 domain of human fibronectin. The results are shown in Fig. 2.

BIAcore

A more precise determination of the affinity constant was performed using the Biacore assay on a biosensor chip coated with the 7B89 domain of human fibronectin (Fig. 3). Samples were injected as serial dilutions at concentrations of 1000, 750, 500 and 250 nM (Figure 3). Apparent KD was determined using Biacore X100 Evaluation software.

Size exclusion chromatography

Eight clones Alpha3, AP6, AP7, DDS, SES, AKKAS, (G4S) ₃ and SAD were characterized using size exclusion chromatography (SEC-200), where all clones showed a comparable profile with the presence of a major peak corresponding to a monomeric immunocytokine (Fig. 4 ).

Immunofluorescence

Immunofluorescence experiment was performed with clones Alpha3, AP6, AP7, DDS, SES, AKKAS, (G4S) ₃ and SAD on a frozen section of F9 syngeneic teratocarcinoma sample (8 μm). All clones showed specific binding to the vasculature compared to the negative control (Fig. 5).

In vivo tumor targeting assessment

In vivo targeting was assessed using a biodistribution assay. Eight clones Alpha3, AP6, AP7, DDS, SES, AKKAS, (G4S) ₃ and SAD, as well as a positive control (single chain diabody L19 coupled to mouse IL-12), were labeled with radioactive iodine ¹²⁵ I and injected (at a rate of 4- 9 μg protein/animal) to immunocompetent mice bearing subcutaneously implanted F9 murine teratocarcinoma. Radioactivity measurements 24 hours after injection indicated tumor accumulation for all clones, but clone SAD showed higher tumor accumulation compared to the other seven clones (Fig. 6).

Example 2.

In additional serine comparison experiments, it was unexpectedly found that the SAD linker was also superior to the older (and shorter) GSADGG linker (SEQ ID NO: 26) disclosed in WO 2013/014149 in terms of binding ability, monomeric profile and tumor targeting ability .

- 20 043910

Material and methods

The options tested in this example have the following general structure:

Home n р4 0 Linke P 1 rZ 5 Linke p2 VH L1 Linke rZ VL L1 Linke p4 VH L19 Linker 3/5 VL L19 (NC) 9 9 SEQ ID NO 1 2 3 4.26 7 6 5 8 7 6 5

The different variants (also referred to here as clones) differ from each other by the sequence of linker 2:___________________________________________________________

Linker 2 SEQ Ш NO Subsequence S.A.D. 4 GSADGGSSAGGSDAG "Old" 26 GSADGG

Cloning of fusion proteins

Fusion proteins containing huIL-12 fused via a 6- or 15-amino acid linker to the L19 antibody in single-chain diabody format (namely, the huIL-12L19L19 old and huIL12L19L19 SAD variants, respectively) were cloned as described above.

Expression of Fusion Proteins

Fusion proteins containing huIL-12 fused via a 6- or 15-amino acid linker to antibody L19 in a single-chain diabody format (namely, the huIL-12L19L19 old and huIL12L19L19 SAD variants, respectively) were generated by transient gene expression in a suspension of adapted CHO cell cultures . After transfection, cells were maintained in ProCHO-4 medium (supplemented with 4 mM ultraglutamine) for 6 days at 31°C with shaking, after which the culture supernatant was collected by centrifugation and further processed to purify the fusion protein.

Purification of Fusion Proteins Using Protein A Resin

Suspension cultures of transfected CHO cells were centrifuged for 30 min at 5000 rpm at 4°C. The supernatant was further clarified by filtration using 0.45 μm filters. Protein A resin was added to the filtered supernatant and the mixture was incubated with shaking for approximately 1 hour. The resin was then collected on a liquid chromatography column and washed with buffer A (100 mM NaCl, 0.5 mM EDTA, 0.1% Tween 20 in PBS , pH 7.4) followed by a second wash with buffer B (500 mM NaCl 0.5 mM EDTA in PBS, pH 7.4). Fusion proteins containing huIL-12 were eluted by gravity using 100 mM TEA. Aliquots were collected and fractions containing the fusion protein, confirmed by UV spectrometry, were pooled and dialyzed against PBS overnight.

Size exclusion chromatography of fusion proteins

Size exclusion chromatography of the fusion proteins was performed using a Superdex 200 Increase 10/300 GL column with PBS as the running buffer in an AKTA-FPLC system. 100 μL of protein solutions were injected into the loop and automatically injected onto the column. UV absorbance at 280 nm was assessed over time. SEC profiles of the fusion proteins were analyzed using the peak integration function of UNICORN software to quantify the percentage of monomeric fraction relative to the total % area or % peak area. To exclude peak artifacts caused by sample loading or salts present in the sample buffers, only the retention volume interval of 5–17.5 mL was considered for quantitation.

BIAcore

Affinity measurements for surface plasmon resonance experiments were performed on a BiacoreX100 instrument with purified old and SAD clones on a CM5 biosensor chip freshly coated with 7B89 fibronectin. Samples were injected as serial dilutions at 250, 125 nM and 62.5 mM (Figure 10). Apparent KD was determined using Biacore X100 Evaluation software.

Radiolabeling and in vivo tumor targeting assessment

Purified protein samples huIL-12L19L19 SAD (with linker GSADGGSSAGGSDAG, SEQ ID NO 4) and huIL-12L19L19 old (with linker GSADGG, SEQ ID NO 26) (100 μg) were labeled with radioactive iodine ¹²⁵ I and chloramine hydrate T, and purified on a column PD10. Proteins were labeled with radioactive iodine after protein A affinity chromatography. Proteins were injected into the lateral tail vein of immunocompetent (129/Sv) mice bearing subcutaneously implanted F9 murine teratocarcinoma. The administered dose per mouse varied from 10 to 11 μg. Mice were euthanized 24 h after injection. Organ samples were weighed and radioactivity measured using a Packard Cobra gamma counter. Protein absorption in various organs was calculated and expressed as the percentage of injected dose per gram of tissue (% ID/g ± standard error). Tumor protein uptake was correlated

- 21 043910 with tumor growth, as described in Tarli et al. (1999).

Results Expression and purification of fusion proteins and size exclusion chromatography

Two variants huIL-12L19L19 SAD and huIL-12L19L19 old were produced by transient gene expression in CHO cells. Experiments were performed in duplicate, where two serine product production experiments were performed on different days, resulting in batches A and B, respectively. After one-step purification by protein A affinity chromatography and dialysis against PBS, the homogeneity of the protein samples was assessed by size exclusion chromatography (Fig. 9). Both protein variants showed a certain degree of protein aggregation, reflected by the presence of high molecular weight variants eluting at early retention volume. The huIL-12L19L19 SAD variant showed the best profile in both cases, as confirmed by quantification of the monomeric fraction of the proteins using the peak integration function of the UNICORN software. Indeed, the huIL-12L19L19 SAD variant showed a lower tendency to aggregate compared to the huIL-12L19L19 old variant, considering monomer peak area as a percentage of the total area under the curve above the baseline (mean values: 54.57% vs. 46.69% respectively) or monomer peak area as a percentage of the sum of all integrated peaks (mean values: 58.83% vs. 52.74%, respectively) (Table 1).

Table 1

Quantification of the monomeric fraction of different fusion proteins assessed by peak integration function using UNICORN software. (*) Peak area as a percentage of the total area under the curve above the baseline. (°) Peak area as a percentage of the sum of all integrated peaks

Protein Linker length (amino acids) Steam Tia Monomeric peak retention volume (ml) Monomeric peak area/Total area (*) (%) Average area/Total area (*) (%) Monomeric peak area/peak area (°) (%) Average area/peak area (°) (%) huIL- 12L19L19 "SAD" 15 A 11.70 54.06 54.57 58.51 58.83 huIL- 12L19L19 "SAD" 15 IN 11.72 55.08 59.14 huIL- 12L19L19 "old" 6 A 11.87 46.73 46.69 48.99 52.74 huIL- 12L19L19 "old" 6 IN 11.86 46.65 56.48

Biacore

The apparent KD was estimated by Biacore X100 Evaluation software to be 6.7 nM for the old clone (huIL-12L19L19, old with linker GSADGG) and 3.8 nM for the SAD clone (huIL12L19L19, SAD with linker GSADGGSSAGGSDAG) (Fig. 10).

In vivo tumor targeting assessment

Radioactivity measured 24 hours after injection showed that the SAD clone had unexpectedly better tumor uptake compared to the old clone (Fig. 11).

Example 3.

The efficacy of the huIL-12L19L19 SAD variant is being evaluated in human patients with malignant melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer with high microsatellite instability (MSI-H), or DNA error repair deficiency (dMMR), hepatocellular cancer, gastric cancer, cutaneous squamous cell carcinoma, cervical cancer and diffuse large B-cell lymphoma (DLBCL). At least one cohort of patients demonstrates disease progression on an immune checkpoint blockade regimen administered as an immediate pretreatment.

- 22 043910

Patients receive the huAD-12L19L19 SAD variant intravenously once a week for 8 weeks. Patients receive doses of 4 mcg/kg; 8 mcg/kg; 12 mcg/kg; 16 mcg/kg or 20 mcg/kg.

Patients are followed for 6 months from the start of treatment or until consent to participate in the trial is withdrawn or until disease progression.

Pharmacokinetic analysis of IL12-L19L19L19-L12 was performed using a sandwich fusion and IL12 fragment capture assay. Antibodies against human fusion proteins (HAFA) are tested using surface plasmon resonance assay and sandwich capture assay. Antitumor activity, such as efficacy, is assessed at weeks 8, 16, and 24 using the RECIST Solid Tumor Response Criteria (version 1.1) or the LUGANO Malignant Lymphoma Response Criteria.

Additional embodiments

According to the first embodiment, the following is provided:

1. Conjugate containing:

a) a heterodimeric IL-12 protein having a first and second subunit,

b) single chain body and

c) a linker between the IL-12 protein and the single-chain diabody, where the linker contains an amino acid motif containing SAD.

2. The conjugate according to claim 1, wherein the SAD linker contains the amino acid motif GSADGGSSAGGSDAG (SEQ ID NO: 4).

3. The conjugate according to any one of claims 1 or 2, wherein the first subunit of the heterodimeric IL-12 protein is p40 and the second subunit is p35.

4. A conjugate according to any one of claims 1 to 3, wherein the single-chain diabody is monospecific or bispecific.

5. A conjugate according to any one of claims 1 to 4, wherein the single-chain diabody binds to extra domain B (EDB) of fibronectin.

6. The conjugate according to any one of claims 1 to 5, wherein the single-chain diabody contains two VH L19 domains and two VL L19 domains.

7. A conjugate according to any one of claims 1 to 6, which has the full-length structure [p40]-[linker1]-[p35][SAD linker]-[VHL19]-[linker3]-[VLL 19]-[linker4]-[ VHL 19]-[linker3]-[VLL 19].

8. The conjugate according to any one of claims 1 to 7, which has the full-length sequence presented in SEQ ID NO: 16.

9. Use of a conjugate according to any of the above points (for the production of a medicinal product) in the treatment of humans or animals:

diagnosed with, suffering from, or at risk of developing a neoplastic disease, or for the prevention of such a pathological condition.

10. Use of a conjugate according to any of the above paragraphs (for the production of a medicinal product) in the inhibition of angiogenesis in humans or animals.

11. Pharmaceutical composition containing at least a conjugate according to any one of paragraphs. 1-8 and optionally one or more pharmaceutically acceptable excipients.

12. A combination comprising (i) a conjugate according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 11 and (ii) one or more therapeutically active compounds.

13. A method of treating or preventing a disorder or pathological condition associated with the expression or overexpression of EDB fibronectin, comprising administering to a subject in need thereof an effective amount of a conjugate according to any one of claims 1 to 8, a pharmaceutical composition according to claim 11, or a combination according to claim 13. 12.

14. Therapeutic set of items, including:

a) a conjugate according to any one of claims 1 to 8, a pharmaceutical composition according to claim 11 or a combination according to claim 12,

b) a device for administering the conjugate, composition or combination, and

c) instructions for use.

According to the second embodiment, the following is provided:

1. Conjugate containing:

a) a heterodimeric IL-12 protein having a first and second subunit,

b) single chain diabody and

c) a linker between the IL-12 protein and the single-chain diabody, where the linker contains an amino acid motif containing GSADGGSSAGGSDAG (SEQ ID NO: 4).

2. The conjugate of claim 1, wherein the first subunit of the heterodimeric IL-12 protein is p40 and the second subunit is p35.

3. Conjugate according to any one of claims 1 or 2, wherein the single-chain diabody is monospecific or bispecific.

4. Conjugate according to any one of claims 1 to 3, wherein the single-chain diabody binds to extradomain B

- 23 043910 (EDB) fibronectin.

5. The conjugate according to any one of claims 1 to 4, wherein the single-chain diabody contains two VH L19 domains and two VL L19 domains.

6. A conjugate according to any one of claims 1 to 5, which has the full-length structure [p40]-[linker1]-[p35][linkerSAD]-[VHL19]-[linker3]-[VLL19]-[linker4]-[VHL19] -[linker3]-[VLL19].

7. A conjugate according to any one of claims 1 to 6, which has the full-length sequence presented in SEQ ID NO: 16.

8. Use of a conjugate according to any of the above claims (for the production of a medicinal product) in the treatment of a human or animal diagnosed as suffering from or at risk of developing a neoplastic disease, or for the prevention of such a pathological condition.

9. Use of a conjugate according to any of the above points (for the production of a medicinal product) in the inhibition of angiogenesis in humans or animals.

10. A pharmaceutical composition containing at least a conjugate according to any one of claims 1 to 7 and, optionally, one or more pharmaceutically acceptable excipients.

11. A combination comprising (i) a conjugate according to any one of paragraphs. 1 to 7 or a pharmaceutical composition according to claim 10 and (ii) one or more therapeutically active compounds.

12. A method of treating or preventing a disorder or pathological condition associated with the expression or overexpression of EDB fibronectin, comprising administering to a subject in need thereof an effective amount of a conjugate according to any one of claims 1 to 7, a pharmaceutical composition according to claim 10, or a combination thereof according to claim 12. .eleven.

13. Therapeutic set of items, including:

a) a conjugate according to any one of claims 1 to 7, a pharmaceutical composition according to claim 10 or a combination according to claim 11,

b) a device for administering the conjugate, composition or combination, and

c) instructions for use.

According to the third series of embodiments, the following is provided:

1. Conjugate containing:

a) a heterodimeric IL-12 protein having a first and second subunit,

b) a single chain diabody, and

c) a linker between the IL-12 protein and the single-chain diabody, where the linker contains an amino acid motif containing GSADGGSSAGGSDAG (SEQ ID NO: 4).

2. The conjugate of claim 1, wherein the first subunit of the heterodimeric IL-12 protein is p40 and the second subunit is p35.

3. Conjugate according to any one of claims 1 or 2, wherein the single-chain diabody is monospecific or bispecific.

4. A conjugate according to any one of claims 1 to 3, wherein the single-chain diabody binds to extra domain B (EDB) of fibronectin.

5. The conjugate according to any one of claims 1-4 above, wherein the single-chain diabody comprises an antigen binding domain having the L19 antibody complementarity determining regions (CDRs) set forth in SEQ ID NOs: 28-33.

6. The conjugate according to any one of claims 1 to 5, wherein the single-chain diabody contains the VH and VL domains of antibody L19 presented in SEQ ID NO: 7 and 5.

7. Composition according to any one of claims 1 to 6, wherein the single-chain diabody contains at least one of:

a) the heavy chain/light chain variable domain sequence pairs of claim 6, provided that at least one of the domains has >80% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, and/or

b) the heavy chain/light chain variable domain sequence pairs of claim 6, provided that at least one of the domains has up to 10 amino acid substitutions relative to SEQ ID NO: 7 or SEQ ID NO: 5, respectively, while maintaining its ability to bind with extradomain B (EDB) of fibronectin.

8. The conjugate according to any one of claims 1 to 7, wherein the at least one amino acid substitution in the single chain diator is a conservative amino acid substitution.

9. The conjugate according to any one of claims 1 to 8, wherein the single-chain diabody has a target binding affinity of >50% with the extra domain B (EDB) of fibronectin compared to one of the antibodies according to claim 5 or 6, and/or competes for binding with extradomain B (EDB) of fibronectin with one of the antibodies according to claim 5 or 6.

10. The conjugate according to any one of claims 1 to 9, wherein the single-chain diabody contains two VH L19 domains and two VL L19 domains.

11. The conjugate according to any one of claims 1 to 11, which has the full-length structure [p40]-[linker1][p35]-[SAD linker]-[VHL 19]-[linker3]-[VLL 19]-[linker4]- [VHL 19]-[linker3]-[VLL 19].

- 24 043910

12. The conjugate according to any one of claims 1 to 11, which has the full-length sequence presented in SEQ ID NO: 16.

13. Use of a conjugate according to any of the above claims (for the production of a medicinal product) in the treatment of a human or animal diagnosed as suffering from or at risk of developing a neoplastic disease or for the prevention of such a pathological condition.

14. Use of a conjugate according to any of the above paragraphs (for the production of a medicinal product) for inhibiting angiogenesis in humans or animals.

15. A pharmaceutical composition containing at least a conjugate according to any one of claims 1 to 12 and, optionally, one or more pharmaceutically acceptable excipients.

16. A combination comprising (i) a conjugate according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 15 and (ii) one or more therapeutically active compounds.

17. A method of treating or preventing a disorder or pathological condition associated with the expression or overexpression of EDB fibronectin, comprising administering to a subject in need thereof an effective amount of a conjugate according to any one of claims 1 to 12, a pharmaceutical composition according to claim 15, or a combination thereof according to claim 17. .16.

18. Therapeutic set of items, including:

a) a conjugate according to any one of claims 1 to 12, a pharmaceutical composition according to claim 15 or a combination according to claim 16,

b) a device for administering the conjugate, composition or combination, and

c) instructions for use.

In yet another embodiment, the SAD linker contains the amino acid motif GSADGGSSAGGSDAG (SEQ ID NO: 4). As used herein, the term single chain diabody refers to a construct of two single chain Fv(scFv) antibodies with a short linker, preferably 5 amino acids long, conjugated to each other by a longer linker, preferably 15 amino acids long, according to the following scheme (N^C orientation ): VHL19-linker 3-VLL19-linker 4-VHL19-linker 3-VLL19.

In one embodiment, linker 3 is GSSGG (SEQ ID NO: 6) and linker 4 is SSSSGSSSSGSSSSG (SEQ ID NO: 8). According to one embodiment of the invention, the first subunit of the heterodimeric IL-12 protein is p40, and the second subunit is p35. Preferably, the two subunits are conjugated to each other via this linker according to the following scheme (N^C orientation): p40-linker1-p35.

Preferably, the IL-12 is human IL-12. In a preferred embodiment, linker 1 is GGGGSGGGGSGGGGS (SEQ ID NO: 2). According to one embodiment of the invention, the single chain diabody is monospecific or bispecific. In one embodiment, the single chain diabody binds to the spliced isoform of fibronectin. In one embodiment, the single chain diabody binds to extra domain B (EDB) of fibronectin.

References (disclosures that have been incorporated by reference herein in their entirety)

Car et al., Toxicologic Pathology (1999), 27(1), 58-63

Chen et al., Adv Drug Deliv Rev. (2013), 65(10), 1357-1369

WO 2013/014149

WO 2006/119897

Tarli et al., Blood (1999), 94(1), 192-198.

Sequences

The following sequences form part of the disclosure of this application. Also provided with this application is a list of electronic sequences compatible with WIPO ST 25. For the avoidance of doubt, if there are discrepancies between the sequences in the following table and the list of electronic sequences, then the sequences in this table should be considered correct.

- 25 043910

SEQ ID NO Identifier Subsequence 1 P40 MVDAV HKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNS RQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKK DRVFTDKTS AT VICRKNASIS VRAQDRYYS S SWSEWAS VPCS 2 Linker 1 GGGGSGGGGSGGGGS 3 P35 Rnlpvatpdpgmfpclhsqnlravsnmlqkarqtl EfypCtseidhedkdtStstveaclpleltknesclns RETSFITSFMALCL S SIYEDLKVMYQVEF KTMNAKLMDPKRQIFLD Qnmlavidelmqalnfnse tvpqsssleepdfyktkiklCillhafrivtidrvmsy Lnas 4 Linker 2 (“SAD”) GS ADGGS S AGGSD AG 5 VLL19 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL

- 26 043910

EPEDFAVYYCQQTGRIPPTFGQGTKVEIK 6 Linker 3/Linker 5 GSSGG 7 VHL19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVR QAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVT VSS 8 Linker 4 SSSSGSSSSGSSSSG 9 Linker 2 (“AKKAS”) GGGAKGGGGKAGGGS 10 Linker 2 (“DDS”) GGGGDGGGGDGGGGS AND Linker 2 (“G4S ₃ ”) GGGGSGGGGSGGGGS 12 Linker 2 (“SES”) GGGGSGGGGEGGGGS 13 Linker 2 (“Alpha 3”) AAAAAAAAAAAAAAA 14 Linker 2 (“AP6”) AR AR ARAR AR AR 15 Linker 2 (“AP7”) AR AR ARAR AR AR AR 16 Full Size SAD MVDAV HKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNS RQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKK DRVFTDKTS AT VICRKNASIS VRAQDRYYS S SWSEWAS VPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHH SQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKT STVEACLPLELT KNESCLNSRETSFITNGSCLASRKTSFM MALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLD QNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFRIRAVTIDRVMSYLNASGSADGGSSAGGSDA GEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWV RQAPGKGLEW VS SISGS SGTT Y YADS VKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVT

- 27 043910

VSSGSSGGEIVLTQSPGTLLSLSPGERATLSCRASQSVSSS FLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTD FTLTISRLEPEDF AVYYCQQTGRIPPTFGQGTKVEIKS S S SGSSSSGSSSSGEVQLLESGGGLVQPGGSLRLSCAASGF TF S SFSMSW VRQ APGKGLEWVS SISGS SGTT YY ADS VK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYF DYWGQGTLVTVSSGSSGGEIVLTQSPGTLSLSPGERATL SCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIP DRFSGSGGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEIK 17 Full size muIL-12-L19-L19 MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDIT WTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETL SHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGR FTC S WL VQRNMDLKFNIKS S S S SPD SRA VTCGMASL SA EKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELAL EAR QQNKYENYSTSFFIRDIIKPDPPKNLQMRPLKNSQVEVS WEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQ KGAFLVERTSTEVQCKGGNVCVQAQDRYYNSSCSKW ACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQ SRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQ TSTLK TCLPLELHKNESCLATRETSSTTRGSCLPPQKTSL MMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILD KGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKM KLCILLHAFSTRVVTINRVMGYLSSAGSADGEVQLLESG GGLVQPGGSLRLSC AASGFTF S SF SMSWVRQ APGKGLE W VSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDT AVYYC AKPFP YFDYWGQGTL VTVS SGS SGG EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDF A VYYCQQTGRIPPTFGQGTKVEIKS S S SGS S S SGS SSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMS WVRQ APGKGLEWVS SISGS SGTT YYADS VKGRFTISRD NSKNTLYLQMNSLRAEDT AVYYC AKPFP YFDYWGQGT LVTVSSGSSGGEIVLTQSPGTLLSLSPGERATLSCRASQSV S S SFL AW YQQKPGQ APRLLIYYAS SRATGIPDRF SGSGS

- 28 043910

GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK 18 Nucleotide sequence of the linker AKKAS ggagggggag ctaaaggtgg cggtggcaag gcaggggggag ggagt 19 Nucleotide sequence of the AP7 linker gcaccagcac cagcaccagc accagcacca gcaccagcac ca 20 Nucleotide sequence of the DDS linker ggaggtgggg gtgatggtgg gggaggtgac ggcggaggtg ggtet 21 Nucleotide sequence of AP6 linker gcaccagcac cagcaccagc accagcacca gcacca 22 Linker nucleotide sequence (G4S) ₃ ggtggaggcg ggteaggegg agggggttct ggcggtggcg gateg 23 Nucleotide sequence of the SES linker ggtggggggtg ggteeggagg eggaggegaa ggcggaggtg ggteg 24 Alpha3 linker nucleotide sequence gcagaagcag cagcaaaaga agcagcagca aaagaagcag cagcaaaagc a 25 Nucleotide sequence of the SAD linker gggtctgcag aeggeggate atcagctggg ggaagtgacg cagga 26 Linker 2 ("old") GSADGG 27 Full-size “old” version MVDAV HKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNS

Claims (6)

RQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKK DRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWAS VPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHH SQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKT STVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFM MALCLSSIYEDL KMYQVEFKTMNAKLLMDPKRQIFLD QNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFRIRAVTIDRVMSYLNASGSADGGEVQLLESG GGLVQPGGSLRLSC AASGFTF S SF SMSWVRQ APGKGLE WVS SISGS SGTT YYADS VKGRFTISRDNSKNTLYLQMN SLRAEDT AVY YC AKPFPYFDYWGQGTL VTVS SGS SGG EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDF AVYYCQQTGRIPPTFGQGTKVEIKS S S SGSS S SGS SSSGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMS WVRQ A PGKGLEWVS SISGS SGTT YYADS VKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGT LVTVSSGSSGGEIVLTQSPGTLLSLSPGERATLSCRASQSV S S SFL AW YQQKPGQ APRLLIYYAS SRATGIPDRF SGSGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVE IK 28 CDR1 VHL19 SFSMS 29 CDR2 VHL19 SISGSSGTTYYADSVKG thirty CDR3 VHL19 PFPYFDY 31 CDR1 VLL19 RASQSVSSSFLA 32 CDR2 VLL19 YASSRAT 33 CDR3 VLL19 QQTGRIPT 34 Linker GGGGS GGGGS 35 Linker GGGGA GGGGA 36 Diathelo L19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVR QAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVT VSSGSSGGEIVLTQSPGTLLSLSPGERATLSCRASQSVSSS FLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTD FTLTISRLE PEDFAVYYCQQTGRIPPTFGQGTKVEIK CLAIM 1. An immunocytokine for tumor-targeted delivery of IL-12, where the cancer expresses extra-domain B of fibronectin (EDB-FN) containing the full-length sequence of SEQ ID NO: 16. 2. The use of an immunocytokine according to claim 1 for the treatment of a human or animal diagnosed with, suffering from, or at risk of developing a neoplastic disease caused by malignant cells expressing EDB-FN, or for the prevention of such a pathological condition. 3. The use of claim 2, wherein the neoplastic disease is selected from the group consisting of malignant melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer with high microsatellite instability ( MSI-H) or DNA error repair deficiency (dMMR), hepatocellular cancer, gastric cancer, cutaneous squamous cell carcinoma, cervical cancer and diffuse large B-cell lymphoma (DLBCL). 4. Use of an immunocytokine according to claim 1 to inhibit angiogenesis in tumors expressing EDB-FN. 5. A pharmaceutical composition containing the immunocytokine according to claim 1 and one or more pharmaceutically acceptable excipients. 6. A combination for tumor-targeted delivery of IL-12, where the cancer expresses EDB-FN, comprising: (i) an immunocytokine according to claim 1 or a pharmaceutical composition according to claim 5 and (ii) one or more therapeutically active compounds for the treatment of malignant tumors. -