WO2021239999A1 - Il-12 pd-l1 ligand fusion protein - Google Patents

Abstract

The invention relates to an IL-12 PD-L1 ligand recombinant protein, or a nucleic acid encoding said protein, for use as a locally administered medicament to treat neoplastic disease, particularly glioma.

Description

IL-12 PD-L1 Ligand Fusion Protein

The present invention relates to a fusion protein comprising an IL-12 polypeptide combined with PD-L1 ligand domain which selectively binds to the T cell inhibitory molecule PD-L1. The invention further relates to the use of the fusion protein as a treatment for cancer.

Background of the Invention

Patients diagnosed with Glioblastoma multiforme (GBM have a median survival of less than 2 years despite aggressive surgery, radiation and chemotherapy. Immunotherapy using checkpoint inhibitors (e.g. anti-PD1 , anti-PDL1 , anti-CTLA4) has thus far failed to improve the clinical outcome for glioblastoma patients, due to a highly immunosuppressive tumour- microenvironment (TME). Preclinical models of GBM are used as examples of immunosuppressive ‘cold’ solid tissue cancers which resist conventional immunotherapy.

Interleukin (IL)-12 is the prototype of a group of heterodimeric cytokines with inflammatory properties. IL-12 binds to the IL-12 receptor (IL-12R), a heterodimeric receptor formed by IL- 12R-b1 and Iί-12R-b2. The receptor complex is primarily expressed by T cells, but other lymphocyte subpopulations have also been found to be responsive to IL-12. IL-12 polarizes naive helper T-cells to adopt a TH1 phenotype and stimulates cells that can counter tumour growth such as cytotoxic T and natural killer-cells.

IL-12 has been shown to have potent anti-cancer effects in different pre-clinical models, as it can activate and expand T cells at tumour sites. However, systemic administration of IL-12 causes severe adverse effects, which has prevented its use in clinical applications. Local, contained administration of IL-12 may be one way to deliver potent anti-tumour signals to various solid tissue tumour types to overcome the problem of systemic toxicity.

PD-1 is expressed on the surface of activated T cells, B cells and macrophages. PD-1 (CD279; Uniprot QI 5116) has two ligands, PD-L1 (B7-H1 , CD274) and PD-L2 (B7-DC, CD273), which are members of the B7 family. Antagonising ligation of PD-1 with PD-L1 can greatly improve activation of immune cells, particularly T cells, and is a frontline immunotherapy for many types of cancer. However, side effects from PD-L1 targeted immunotherapy can arise driven by inflammatory responses to non-tumour molecules in sensitive tissues. Indeed, the serious nature of these side effects can sometimes cause patients to discontinue therapy. Various anti- PD-L1 antibodies (e.g. MDX-1105/BMS-936559) and anti-PD-1 antibodies are currently approved or undergoing clinical trials (e.g. MDX-1106/BMS-936558/ONO-4538 or MK- 3475/SCH 900475 or AMP-224).

Immunosuppressive PD-L1 is expressed by a majority of cancers. Although PD-1 and PD-L1 blockade has shown advantageous results in the treatment of melanoma, lung cancer and triple-negative breast cancer, it has failed to induce an immunogenic response and enhance survival rates in cancers characterised by a paucity of immune infiltration, in particular glioblastoma. The powerful antitumour action of IL-12 works in the context of the TME by increasing local T and natural killer cell responses, as well as making tumour cells more amenable to immunotherapy by increasing PD-L1 expression. Indeed, IL-12 has also been shown to act with dramatic synergy with systemically applied immunotherapy targeting the so called check-points (CPI, check-point inhibition).

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to treat immune “cold” tumours (i.e. tumours showing little or no response to checkpoint inhibitory therapeutic agents, particularly brain cancer). This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the Invention

A first aspect of the invention relates to a IL-12-PD-L1 ligand conjugate comprising, or essentially consisting, of two functional domains, an IL-12 polypeptide domain, and a nonagonist PD-L1 ligand polypeptide domain, in the form of a single polypeptide, or fusion protein. The PD-L1 ligand domain is capable of specifically and selectively binding to PD-L1 , thereby localizing the conjugate’s IL-12 activity at sites of PD-L1 expression in a tumour, limiting the potentially harmful activation of T cells in other locations, and inhibiting the interaction of PD- 1 with PD-L1 at the same time.

The two components of the conjugate combine the biological activity of synthetic, or recombinant human IL-12 and the biological activity of anti-PD-L1 antibodies (as exemplified by the antibodies atezolizumab, or avelumab). The inventors have found that combining the two activities into a single molecule can treat cancer, and reduce correlates of harmful systemic inflammation compared to recombinant IL-12. Particular embodiments relate to a IL-12-PD-L1 ligand conjugate with the protein sequence of SEQ ID NO 014, SEQ ID NO 015, SEQ ID NO 036 or SEQ ID NO 037.

Another aspect of the invention is a nucleic acid encoding the IL-12-PD-L1 ligand conjugate of the invention.

A further aspect of the invention is a recombinant cell comprising a nucleic acid encoding the IL-12-PD-L1 ligand conjugate of the invention.

Another aspect of the invention relates to the use of the IL-12-PD-L1 conjugate according to the invention as a medicament, particularly in treating cancer. Detailed Description of the Invention

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15 amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.

Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3^rd ed. p. 21 ). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

The terms gene expression, or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.

Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM). A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

The term antibody fragment refers to a polypeptide comprising the antigen binding fragment of an antibody, or single chains thereof and related or derived constructs, for example, a fragment of a natural antibody sequence comprising the binding domains defined above, or an artificial fusion protein comprising antibody binding domain sequences. The term encompasses a so-called nanobody or a single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain, and diabodies, or univalent or noncovalent dimers of single-chain Fragment variable (scFv) polypeptides.

A single-chain fragment variable, single chain Fv or ScFv in the context of the present specification refers to a recombinant fusion construct which consists of one, two, or several VH regions, each associated, or paired with a VL region, connected with either covalent bonds, or by a peptide linker. The term encompasses a univalent ScFv consisting of one VH and one VL region connected by a peptide linker. Other forms of scFv encompassed by the claims are bivalent scFv (such as diabodies, or tandem ScFv), or multivalent forms, consisting of two or more VH regions, and two or more VL regions, joined via covalent bonds or peptide linkers to provide two or more antigen binding sites, each antigen binding site comprising one VH region associated with one VL region. A bivalent tandem ScFv consists of two VH and two VL regions joined by peptide linkers in a single polypeptide chain. A diabody in the context of the invention refers to a bivalent ScFv which utilizes one or more short polypeptides (under 10 polypeptides, usually 5 polypeptides) to link a VH domain with a VL domain, in order to force their association and provide a more stable molecule. All ScFv lack the constant fragment (Fc) present in complete antibody molecules.

The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity / a Kd < 10E-8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich), an engineered antibody mimetic protein exhibiting highly specific and high-affinity target protein binding (see US2012142611 , US2016250341 , US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins. The term antibody-like molecule further encompasses a specifically non-agonist, PD-L1 binding polypeptide derived from a protein A domain, fibronectin domain FN3, consensus fibronectin domains, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1 -2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11 (7):803-813),

Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin,

Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2.

In the context of the present specification, the term fragment crystallizable (Fc) region is used in its meaning known in the art of cell biology and immunology; it refers to a fraction of an antibody comprising, if applied to IgG, two identical heavy chain fragments comprised of a CH2 and a CH3 domain, covalently linked by disulphide bonds.

The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of < 10^"7mol/l_ when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.

The term PD-L1 refers to the human programmed cell death ligand 1 , also known as CD271 (Uniprot Q0GN75).

The term non-agonist PD-L1 ligand, or inhibitor of PD-1 interactions with PD-L1 in the context of the present specification refers to a ligand which specifically binds to PD-L1 , and abrogates, or neutralizes the inhibition of the T cell receptor (TCR) mediated signalling and proliferation which occurs downstream of ligation of PD-L1 with PD-1 on the surface of T cells. The ability of the non-agonist PD-L1 ligand to inhibit PD-1/PD-L1 interactions according to the invention can be quantified, for example, with an assay such as the PD-1/PD-L1 luciferase assay used in the examples. This PD1/PDL1 blockade bioassay from Promega determines the ability of the IL12- anti-PDL1 fusion protein to interfere with PD1/PDL1 binding. In this assay, a nonagonist PD-L1 ligand according to the invention can bind to PD-L1 and drive more than a thousand-fold relative increase in luminescence at a concentration of 2 ug^mM.

In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitory antibody is meant to encompass an agent, particularly an antibody (or antibody-like molecule) capable of disrupting an inhibitory signalling cascade that limits immune cell activation, known in the art as an immune checkpoint mechanism. In certain embodiments, the checkpoint inhibitory agent or checkpoint inhibitory antibody is an antibody to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (PDL1 , Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), VISTA, TIGIT, TIM-3, CD158, TGF-beta.

In certain embodiments, the immune checkpoint inhibitor agent is selected from the clinically available antibody drugs ipilimumab (Bristol-Myers Squibb; CAS No. 477202-00-9), nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), avelumab (Merck KGaA; CAS No. 1537032-82-8), durvalumab (Astra Zenaca, CAS No. 1428935-60-7), and cemiplimab (Sanofi Aventis; CAS No. 1801342-60-8).

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described herein.

A first aspect of the invention relates to an IL-12-PD-L1 ligand conjugate. This is a recombinant chimeric polypeptide made up of two domains which each mediate the following biological functions. The first domain of the IL-12-PD-L1 ligand conjugate according to the invention is an artificial IL-12 polypeptide which is a single polypeptide chain. IL-12 is sometimes referred to as IL-12 p70, in reference to the combined molecular weight of the p35 and p40 subunits. The second domain of the IL-12-PD-L1 ligand conjugate according to the invention is a non- agonist PD-L1 ligand polypeptide, which inhibits interactions between PD-L1 and its ligand PD-1.

The term conjugate in this context refers to an engineered fusion construct combining the biological functions of two discrete immunomodulatory molecules within a single polypeptide, namely a functional IL-12 domain, capable of signalling through it’s the IL-12 receptor, and a portion of a non-agonist PD-L1 binding ligand which specifically interacts with PD-L1.

The term domain is used here to denote separate functional regions of the IL-12-PD-L1 ligand conjugate protein. In other words, each modular component, both the IL-12 component, and the PD-L1 ligand component, are domains in the sense that each has independent folding and structure which are important for its biological function.

The data in the examples show that the non-agonist IL-12-PD-L1 ligand conjugate provides a benefit over IL-12 administration alone in a model of brain cancer, by targeting the IL-12 portion of the conjugate to PD-L1 + tumour cells, providing targeted activity of the cytokine IL-12 where T cell activation is required, as well as preventing the IL-12-containing conjugate from circulating systemically and causing toxic side effects.

The IL-12 polypeptide domain and the PD-L1 ligand domain of the IL-12-PD-L1 ligand conjugate are provided in the form of a single polypeptide, where the two domains are linked by a peptide linker, or bridging region.

Particular embodiments relate to a IL-12-PD-L1 ligand conjugate, in which the IL-12 polypeptide and the entire structure of the non-agonist PD-L1 ligand domain which make up the IL-12-PD-L1 ligand conjugate are part of one single polypeptide chain. In other words, the recombinant protein is expressed from an artificial genetic construct fusing the genes for IL-12 and a non-agonist PD-L1 ligand joined by means of spacer regions, or peptide linkers.

Particular embodiments relate to IL-12-PD-L1 ligand comprising a divalent PD-L1 ligand domain, in which the IL-12 polypeptide domain and a first and second non-agonist PD-L1 binding domain are joined via a peptide linker in a single polypeptide.

Some embodiments of the invention relate to an IL-12-PD-L1 ligand conjugate provided in the form of a single protein chain, wherein the IL-12 polypeptide, domain, or subunit, is positioned N-terminal relative to the non-agonist PD-L1 ligand polypeptide, domain, or subunit.

The IL-12 domain of the IL-12-PD-L1 ligand conjugate according to the invention, is a synthetic fusion of a p40 polypeptide domain, and a p35 polypeptide domain, joined together by a flexible peptide linker as part of the IL-12-PD-L1 ligand conjugate single polypeptide chain.

In certain embodiments of the IL-12-PD-L1 ligand conjugate the p40 polypeptide of the IL-12 domain is positioned N-terminal relative to the p35 polypeptide. In a further embodiment of the recombinant IL-12 PD-L1 ligand polypeptide, the sequence of the IL-12 domain comprises

- a polypeptide encoding the human IL-12 p35 subunit (for example, the natural sequence Uniprot Q92V6) altered to lack the naturally occurring leader sequence, particularly a polypeptide for which the sequence has > 85%, optionally, more particularly >90%, more particularly >95% identity to the sequence of SEQ ID NO 001 , or

- a polypeptide encoding the human IL-12 p40 subunit (for example, the natural sequence Uniprot P29460) altered to lack the naturally occurring leader sequence, particularly a polypeptide for which the sequence has > 85%, optionally, more particularly >90%, more particularly >95% identity to the sequence of SEQ ID No 002.

In addition, the domain comprising an IL-12 polypeptide is further characterized by having at least 70% of the biological activity of human or mouse IL-12.

The IL-12 polypeptide characterized by at least 70% of the biological activity of IL-12 in the context of the present invention relates to one of the main functions of the IL-12 protein, the ability to stimulate cytokine production by T cells. The biological activity of an IL-12 polypeptide can be measured in as assay such as that used in the examples. In short, 10⁵ splenocytes are cultured in the presence of indicated concentrations of the IL12-PDL1 ligand construct, IL-12, or an IL12-containing molecule with known functionality, and T cell activation is examined by measuring IFN gamma production by an ELISA after 48 h of culture.

The data in the examples shows that an IL-12-PD-L1 ligand conjugate having an IL-12 polypeptide domain made up of an IL-12 p40 and p35 domain joined by a flexible peptide linker, can increase overall survival rates in a mouse model of glioma, as well as reduce systemic toxicity reflecting in systemic production of IFN gamma by T cells when delivered by intracranial administration, and is retained within a solid colorectal cancer tumour when injected intratumorally.

A further aspect of the IL-12-PD-L1 ligand conjugate according to the invention, is the inhibition of the biological activity of PD-1 , by the binding of non-agonist PD-L1 ligand domain to PD-L1 , resulting in enhanced T cell activation.

Engagement of PD-1 by the ligands PD-L1 or PD-L2, on an adjacent cell inhibits TCR signalling and TCR-mediated proliferation, transcriptional activation and cytokine production. The non-agonist PD-L1 ligand domain, or inhibitor of PD-1 interactions with PD-L1 according to the invention is able to abrogate, or neutralize the inhibition of the T cell receptor (TCR) mediated signalling and proliferation which occurs downstream of ligation of PD-L1 with PD-1 on the surface of T cells. The ability of the non-agonist PD-L1 ligand to inhibit PD-1/PD-L1 interactions according to the invention can be measured by assays which detect binding, or the indirect immunological outcomes of said binding such as proliferation or cytokine release, for example, using an assay such as the PD-1/PD-L1 Blockade Bioassay used in the examples. This PD1/PDL1 blockade bioassay from Promega determines the ability of the IL12- anti-PDL1 fusion protein to interfere with PD1/PDL1 binding. The non-agonist PD-L1 ligand according to the invention can drive more than a thousand-fold relative increase in luminescence at a concentration of 2 ug^mM.

In particular embodiments of the IL-12-PD-L1 ligand conjugate according to the invention, the PD-L1 ligand domain comprises, or essentially is, an antibody-like molecule which binds to PD-L1 to inhibit of PD-1 as specified above, particularly an antigen-binding antibody fragment lacking the Fc region, and protein G binding capacity, such as a diabody or scFv.

Certain embodiments of this aspect of the invention relate to a PD-L1 ligand domain of the IL- 12-PD-L1 ligand conjugate which comprises a scFv made up of a VH domain, particularly a VH domain comprising or essentially consisting of the amino acid sequence SEQ ID NO 003, in addition to a VL domain, particularly a VL domain comprising or essentially consisting of the amino acid sequence SEQ ID NO 005.

According to certain embodiments of this aspect of the invention, the PD-L1 ligand domain of the IL-12-PD-L1 ligand conjugate comprises a scFv made up of a VH domain linked by a peptide linker to a VL domain, particularly a VH domain comprising or essentially consisting of the amino acid sequence SEQ ID NO 004, and a VL domain comprising or essentially consisting of the amino acid sequence SEQ ID NO 006.

According to certain embodiments of this aspect of the invention, the PD-L1 ligand domain of the IL-12-PD-L1 ligand conjugate comprises a scFv wherein the VH and VL domain are derived from a commercial non-agonist PD-L1 ligand antibody, for example a VH and a VL domain derived from atezolizumab, avelumab, or durvalumab. data in the examples shows that intracranial administration of an IL-12-PD-L1 ligand conjugate comprising a scFv with two binding sites specific for PD-L1 can impede the severity of cancer outcomes in a mouse model of glioma, as well as reducing systemic toxicity as measured by systemic production of INF gamma by T cells.

Some embodiments of the invention relate to a IL-12-PD-L1 ligand conjugate which comprises, or essentially consists of a PD-L1 ligand domain as specified above, joined to a IL-12 domain as specified above, via a inter domain peptide linker, particularly a peptide linker >30, or ³15 amino acids in length, more particularly a peptide linker 5 or 6 amino acids in length, most particularly a peptide linker of SEQ ID NO 007 or SEQ ID NO 033.

Some embodiments of the IL-12-PD-L1 ligand conjugate relate to a IL-12 polypeptide domain and a PD-L1 ligand domain joined by a conjugate domain peptide linker 6 amino acids in length wherein the amino acids are a G S, A or a D, more particularly a conjugate domain peptide linker with the amino acid sequence SEQ ID NO 007.

Some embodiments of the IL-12-PD-L1 ligand conjugate relate to a IL-12 polypeptide domain and a PD-L1 ligand domain joined by a conjugate domain peptide linker 5 amino acids in length wherein the amino acids are a G S, A or a D, more particularly a conjugate domain peptide linker with the amino acid sequence SEQ ID NO 033.

In certain embodiments of this aspect of the IL-12-PD-L1 ligand conjugate according to the invention, the two p40 polypeptide and the p35 polypeptide domains are connected by an IL- 12 domain peptide linker, particularly an IL-12 domain peptide linker >15 amino acids in length, particularly an IL-12 domain peptide linker 15 to 30 amino acids in length wherein the amino acids are a G S, A or a D, most particularly an IL-12 domain peptide linker with amino acid sequence SEQ ID NO 008.

The important characteristics of the conjugate peptide linkers as specified above are low immunogenicity, and a peptide length that allows the p35 and the p40 domains to interact to form a functional IL-12 subunit that can stimulate its receptor.

Examples of a non-agonist PD-L1 ligand, or anti-PD-L1 ligand include, but are not limited to, an antibody, camelid, diabody, or a mono, bi- or tri-valent scFv with specificity for PD-L1 .

Particular embodiments of the PD-L1 ligand conjugate provided by the invention relate to a PD-L1 ligand comprising two VH and two VL domains, arranged in a single polypeptide to form two functional PD-L1 binding sites. The intramolecular orientation of the VH domain and the VL domain in the scFv format is not key to the function of the bispecific scFv. However, the VH and VL domains as specified may be arranged in such a way that amino acid linkers which either restrict movement by being between 2 and 15 amino acids in length (-), particularly between 2 and 5, more particularly 5 or 6 amino acids in length, or permit flexibility by being >15 amino acids in length (--), particularly 15 to 30 amino acids in length, are alternated to enforce the interaction of each VH domain with its desired partner VL domain. Examples of such functional scFv arrangements may be selected from, but are not limited to:

VH-VL-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL, VH-VL-VL-VH, VL-VH-VL-VH, VL~ VH-VH-VL, VL-VL-VH-VH or VH-VH-VL-VL.

These aspects of the invention provide the possibility of more than one scFv domain as part of the IL-12-PD-L1 ligand, where at least one of the resulting functional antigen binding sites is a ligand for PD-L1 , i.e. the second functional antigen binding site could have another target desirable for cancer treatment, for example, a tumour antigen or hormone receptor. In certain particular embodiments of the IL-12-PD-L1 ligand conjugate provided by the invention, the non-agonist PD-L1 ligand is a bivalent, or multivalent, scFv which has the following important structural features a first scFv domain comprising a first VH domain and first VL domain, and a second scFv domain comprising a second VH domain and second VL domain, wherein at least one scFv domain in a non-agonist ligand for PD-L1 .

The bivalent or multivalent IL-12-PD-L1 ligand conjugate according to the embodiments of the invention comprising scFv as specified above, may be characterised by VH and VL domains joined sequentially by alternating placement of a first, flexible scFv peptide linker, wherein the flexible scFv peptide linker is >15 amino acids in length, particularly a flexible scFv peptide linker consisting of the amino acids G S, A or a D, more particularly a flexible scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 3, 4 or 5 adjacent motifs, most particularly a scFv domain peptide linker with the amino acid sequence SEQ ID NO 011 , and a second, rigid peptide linker, wherein the rigid scFv peptide linker is 2 to 15 amino acids in length, particularly a rigid scFv peptide linker consisting of the amino acids G S, A or a D, more particularly a rigid scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 1 or 2 adjacent motifs, most particularly a rigid scFv peptide linker with the amino acid sequence SEQ ID NO 012.

Optional embodiments of a bi- or multi-valent IL-12-PD-L1 ligand as specified above relate to a first and second scFv provided as two or more polypeptides, which interact via di-sulphide bonds between light and heavy antibody variable chain components. Alternative embodiments relate to first scFv and the second scFv which are provided as part of one single polypeptide chain with at least two functional antigen binding sites, particularly one single peptide chain with the amino acid sequence SEQ ID NO 009, or SEQ ID NO 010.

An alternative embodiment of the IL-12-PD-L1 ligand conjugate according to invention, comprises instead a PD-L1 ligand which is a univalent scFv specific for PD-L1 made up of a single VH domain and single VL domain only, and where the single VH domain is joined to single VL domain by a flexible scFv linker ³15 amino acids in length as specified above.

The embodiments concerning peptide linkers as specified above, encompass structures in which amino acids with similar characteristics are exchanged, for example, the amino acids V, L, I, P, S, C, or M replace G, S, or S, or where D is replaced by E. In particular desirable embodiments of the domain peptide linkers specified above, the sequences are primarily made up of stretches of small, polar amino acids such as glycine (G) and serine (S). In particular embodiments of the invention, any of the specified peptide linkers are made up of amino acids in the motif (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 1 , 3, 4 or 5 adjacent motifs, residues.

Certain embodiments of the IL-12-PD-L1 ligand conjugate provided by the invention as single polypeptide, the N-terminal amino acid sequence of said polypeptide is, or comprises a secretory leader signal, particularly a secretory leader signal derived from the human IgG variable heavy chain, most particularly a secretory leader signal consisting or comprising the sequence the amino acid motif SEQ ID NO 013.

Some embodiments of the invention relate to a IL-12-PD-L1 ligand conjugate which comprises, or essentially consists of a PD-L1 ligand sequence selected from SEQ ID NO 009, or SEQ ID NO 010, joined to a IL-12 domain as specified above, via a peptide linker of SEQ ID NO 007.

Some embodiments of the invention relate to a IL-12-PD-L1 ligand conjugate which comprises, or essentially consists of a PD-L1 ligand sequence selected from SEQ ID NO 009, or SEQ ID NO 010, joined to a IL-12 domain as specified above, via a peptide linker of SEQ ID NO 033.

Particular embodiments of the invention relate to a IL-12-PD-L1 ligand conjugate which comprises, or essentially consists of the polypeptide of SEQ ID NO 014, or SEQ ID NO 015,

Other particular embodiments of the invention relate to a IL-12-PD-L1 ligand conjugate which comprises, or essentially consists of the polypeptide of SEQ ID NO 036, or SEQ ID NO 037,

The data in the examples shows that a bivalent IL-12-PD-L1 ligand conjugate with two scFv PD-L1 binding domains has the same IL-12 signalling function as IL-12 conjugated to Fc, which has itself been shown to have similar functionality to the naturally occurring IL-12 cytokine. This molecule has heightened ability to induce immunity against glioma when injected locally, reducing disease severity, as well as lower systemic toxicity compared to a recombinant IL- 12-Fc construct.

Another aspect of the invention, is a nucleic acid molecule encoding the IL-12-PD-L1 ligand conjugate as specified in any one of the previous embodiments described above, particularly wherein the nucleic acid molecule comprises any one of SEQ ID NO 016 to 030, or SEQ ID NO 038 more particularly wherein the nucleic acid molecule comprises the sequence SEQ ID NO 029, SEQ ID NO 030, or SEQ ID NO 038.

A further related aspect of the invention, is a recombinant cell comprising the nucleic acid molecules, or the amino acid sequences according to the any one of the previous aspects of the invention.

Another aspect of the invention is an IL-12-PD-L1 ligand conjugate according to any one of the previously specified embodiments, for use as a medicament, or in the manufacture of a medicament, particularly a medicament for treatment of malignant neoplastic disease, more particularly for treatment of neoplastic disease selected from glioma, glioblastoma multiforme, meningioma, secondary brain cancer, brain metastases, melanoma, pancreatic cancer, lung cancer, prostate cancer, colorectal cancer or bladder cancer.

Another particular aspect of the invention, is an IL-12-PD-L1 ligand conjugate polypeptide, or an IL-12-PD-L1 ligand conjugate in the form of a nucleic acid molecule, for use as a medicament in the treatment of malignant neoplastic disease, particularly neoplastic disease selected from glioma, glioblastoma multiforme, meningioma, secondary brain cancer, brain metastases, melanoma, pancreatic cancer, lung cancer, prostate cancer, colorectal cancer or bladder cancer.

Further embodiments of this aspect of the invention relate to an IL-12-PD-L1 ligand conjugate in the form of a nucleic acid or polypeptide used to treat a cancer that is refractory to checkpoint inhibitory agent, particularly a PD-1 -specific or PD-L1 -specific checkpoint inhibitor. A cancer may be classed as refractory to checkpoint inhibitory treatment on the basis of biomarker testing, or the results of previous treatment regimes.

Particular embodiments of this aspect of the invention relate to an IL-12-PD-L1 ligand conjugate as specified above, used to treat a glioblastoma whose tumour cells have been determined to express the wild type IDH1 gene (RefSeqlD:3417), rather than a mutated form. Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to a-ketoglutarate. Mutations in the IDH1/2 gene define distinct brain cancer subsets, including low-grade gliomas and secondary glioblastomas, chondrosarcomas, intrahepatic cholangiocarcinomas, and hematologic malignancies (Mondesir et al. J. Blood Med. 2016) (7): 171 -80). Conversely, high grade, malignant glioma or glioblastomas do not express IDH1 mutations, and are often characterised by SOX2 expression (Sampron et al. 2016 (6) Article 222).

The data in the examples demonstrates that the IL-12-PD-L1 ligand conjugate according to the invention is effective at reducing mortality rates in a mouse model of glioblastoma, a cancer which is normally refractive to immune checkpoint therapy treatment in mice and man. Furthermore, the data in the examples shows that human glioblastoma patients which express an unmated IDH1 gene are enriched for PD-L1 expression on the non-immune fraction of brain cells.

Particular embodiments relate to an IL-12-PD-L1 ligand conjugate as specified above, provided either as a polypeptide or nucleic acid for the treatment of cancer by direct administration of the IL-12-PD-L1 ligand conjugate into a solid tumour, or into the close vicinity of a tumour, or into the lymph node associated with a tumour.

Particular preferred embodiments relate to an IL-12-PD-L1 ligand conjugate administered by intracranial administration to treat brain cancer. This may be achieved either by a single, intermittent or a continuous infusion, for example by convection enhanced delivery, or by intrathecal administration, or in situ production from a nucleic acid, or by release from implantation of a slow release formulation, or by intranasal application, or by a formulation or cell-targeted transport which enhances transport across blood brain barrier into the CNS.

The data in the examples show that intracranial administration of a single-chain polypeptide IL-12-PD-L1 ligand conjugate provided by the invention was improve the survival rate in a high morbidity mouse model of glioma, while at the same time showing limited systemic toxicity.

Some embodiments relate to intratumoural injection of an IL-12-PD-L1 ligand conjugate according to the invention to treat colorectal cancer.

In the examples, intratumoural injection of a model IL-12-PD-L1 ligand conjugate according to the invention into a MC38 colon-derived solid tumour is demonstrated to have a desirable safety profile, containing the inflammatory effect of IL-12 within the tumour without leakage to the periphery.

Another aspect of the invention relates to a method of classifying a brain cancer patient as likely to respond favourably to treatment with an immune checkpoint inhibitor agent. As laid out herein, the invention provides data linking expression of PD-L1 in the tumour microenvironment, by tumour cells, to the expression of wild type IDH1 and SOX2. Consequently, the method according to this aspect of the invention has particularly in predicting the patient’s likelihood to respond to an immune checkpoint inhibitor agent selected from an antibody to PD-1 and PD-L1. The method according to this aspect of the invention comprises the steps of a. determining the expression level of a biomarker selected from IDH1 and SOX2 in a tumour sample obtained from the brain cancer patient; b. optionally, determining the expression level of the biomarker to a threshold, c. assigning to the patient a high probability of being responsive to said immune checkpoint inhibitor agent if the expression level of said biomarker is above the threshold.

The expression of a marker at a protein level may be assayed via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA or multiplex analyses.

The expression of a marker at an mRNA level may be assayed via techniques such genetic sequence, mRNA array, or quantitative, real-time polymerase chain reaction methods.

Methods for determining adequate threshold values are known in the art, such as comparison to previously analysed control samples with a known biomarker expression pattern and patient outcome, or mean, or median values of a cohort of patients suffering from the cancer in question. The method may entail comparison of the biomarker expression to the expression level of an easy to detect housekeeping gene (normalization). The biomarker may be detected as nucleic acid (RNA) or protein expression. Particular embodiments relate to the use of wild-type IDH1 , and/or SOX2 as biomarkers which indicate PD-L1 expression by the tumour. As laid out above, expression of these biomarkers in GBM is associated with high PD-L1 expression; at the same time, wt-IDH1 expressing GBM constitute an entity of particular malignancy, presenting an urgent need for rapid intervention.

In certain particular embodiments, the expression level of both wild-type IDH1 and of SOX2 are determined and compared to their respective threshold.

The method according to this aspect of the invention likewise presents an opportunity to selectively identify glioma patients most likely to benefit from treatment with anti-PD-1 or anti- PD-L1 agents, such as the commercial antibodies nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), Avelumab (Merck KGaA; CAS No. 1537032-82-8), Durvalumab (Astra Zenaca, CAS No. 1428935-60-7), and Cemiplimab.

Thus, the invention provides a pharmaceutical drug comprising an anti-PD-L1 -ligand, for use in treatment of a brain tumour, particularly in treatment of glioma, more particularly glioblastoma multiforme, wherein the pharmaceutical drug is administered to a patient identified as likely to respond favourably to treatment with the pharmaceutical drug by the method according to the aspect disclosed previously.

In a particular embodiment, the pharmaceutical drug for use in treatment of a brain tumour, particularly in treatment of glioma determined to actively express wild-type IDH1 , and/or the SOX2 according to this aspect of the invention, comprises an anti-PD-L1 oranti-PD-1 antibody, such as exemplified by the group comprised of ivolumab, pembrolizumab, pidilizumab, atezolizumab, Avelumab, Durvalumab and Cemiplimab. In a more particular embodiment, these antibody drugs are combined with local administration of IL-12 into the tumour, the vicinity of the tumour or a draining lymphocyte.

In another particular embodiment, the pharmaceutical drug for use in treatment of a brain tumour with the biomarker characteristics specified above, particularly in treatment of glioma according to this aspect is or comprises an IL-12-PD-L1 ligand conjugate according to any one of the aspects or embodiments of the present specification specified above, or a nucleic acid encoding the same.

Although the detection of IDH1/2 mutations and high SOX2 expression at diagnosis has been proposed as a biomarker suitable for prediction of poor patient prognosis, the data presented in the examples demonstrates further utility of wildtype IDH1 and SOX2 expression as biomarkers of a subset of tumours, that despite their aggressive nature, may surprisingly be amenable to immune checkpoint therapy, particularly in the form of an IL-12 and PD-L1 ligand fusion protein. The discovery that malignant IDH1^wt SOX2+ glioma cells express PD-L1 , suggests these cells are likely to be uniquely susceptible to novel treatment providing IL-12 to the tumour microenvironment, as the anti-PD-L1 ligand will target highly malignant SOX2+ PD- L1 + tumour cells to provide a potent local survival signal for tumour-specific T cells. Malignant glioma has previously been shown to be refractive to checkpoint inhibitor therapy, but the combination of the IL-12-PD-L1 construct as specified in the embodiments above, together with the single cell mass spectrometry analysis of PD-L1 on IDH1^wt SOC2+ glioma cells, provides a novel rational for treatment of this brain cancer patient subset, and a method for identifying patients which are likely to respond favourably to such therapy.

The invention further encompasses the following items:

1. An IL-12-PD-L1 ligand conjugate comprising, or essentially consisting of a single polypeptide chain, said single polypeptide chain comprising: an IL-12 domain comprising a p40 domain and a p35 domain, and a PD-L1 ligand domain.

2. The IL-12-PD-L1 ligand conjugate according to item 1 , wherein the IL-12 domain is N- terminal relative to the PD-L1 ligand domain.

3. The IL-12-PD-L1 ligand conjugate according to any one of the items 1 or 2, wherein the p40 domain is N-terminal relative to the p35 domain.

4. The IL-12 PD-L1 ligand conjugate according to any one of the items 1 to 3, wherein the IL-12 domain comprises

- a sequence at least (>) 85% identical, particularly >90%, more particularly >95% identical to the sequence of human p35 (SEQ ID NO 001 ), and

- a sequence > 85% identical, particularly >90%, more particularly >95% identical to the sequence of human p40 (SEQ ID NO 002), and wherein

- the IL-12 domain is characterized by at least 70%, particularly >85%, more particularly >90% of the biological activity of human IL-12.

5. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 4, wherein the PD- L1 ligand domain inhibits the binding of PD-L1 to PD-1.

6. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 5, wherein the PD- L1 ligand domain comprises or essentially is an antibody-like molecule, particularly an antigen-binding antibody fragment.

7. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 6, wherein the PD- L1 ligand domain comprises, or essentially consists of, a single chain variable fragment (scFv) comprising or consisting of

- a VH domain, particularly a VH domain comprising the sequence SEQ ID NO 003 or SEQ ID NO 004 and - a VL domain, particularly a VL domain comprising the sequence SEQ ID NO 005 or SEQ ID NO 006. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 7, wherein the IL-12 domain and the PD-L1 ligand domain are joined by a conjugate domain peptide linker 5 or 6 amino acids in length wherein the amino acids are a G S, A or a D, more particularly a conjugate domain peptide linker consisting of SEQ ID NO 007 or SEQ ID NO 033. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 8, wherein the p40 domain and the p35 domain are connected by an IL-12 domain peptide linker, particularly an IL-12 domain peptide linker >15 amino acids in length, particularly an IL-12 domain peptide linker 15 to 30 amino acids in length wherein the amino acids are a G S, A or a D, most particularly an IL-12 domain peptide linker consisting of SEQ ID NO 008. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 9, wherein the PD- L1 ligand is a bivalent, or multivalent, scFv comprising or consisting of

- a first scFv domain comprising a first VH domain and first VL domain, and

- a second scFv domain comprising a second VH domain and second VL domain,

- wherein at least one of the scFv inhibits the binding of PD-L1 to PD-1 and wherein optionally,

- the first scFv and the second scFv are part of one single polypeptide chain with at least two functional antigen binding sites, particularly one single peptide chain of SEQ ID NO 009 or SEQ ID NO 010 , The IL-12-PD-L1 ligand conjugate according to item 10, wherein the VH and VL domains are joined sequentially by alternating placement of

- a flexible scFv peptide linker, wherein the flexible scFv peptide linker is >15 amino acids in length, particularly a flexible scFv peptide linker consisting of the amino acids G S, A or a D, more particularly a flexible scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 3, 4 or 5 adjacent motifs, most particularly a scFv domain peptide linker of SEQ ID NO 011 , and

- a rigid peptide linker, wherein the rigid scFv peptide linker is <15 amino acids in length, particularly a rigid scFv peptide linker consisting of the amino acids G S, A or a D, more particularly a rigid scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 1 or 2 adjacent motifs, most particularly a rigid scFv peptide linker of SEQ ID NO 012. 12. The IL-12-PD-L1 ligand conjugate according to any of the items 1 to 9, wherein the PD- L1 ligand is a univalent scFv that inhibits the binding of PD-L1 to PD-1 , said scFv comprising

- a single VH domain and single VL domain only, and wherein

- the single VH domain is joined to single VL domain by a flexible scFv linker as specified in claim 14.

13. The IL-12-PD-L1 ligand conjugate according to any one of the items 1 to 12, wherein the N-terminal amino acid sequence of the single polypeptide chain is, or comprises a secretory leader signal, particularly wherein said secretory leader signal is derived from the human IgG variable heavy chain, most particularly wherein said secretory leader signal comprises SEQ ID NO 013.

14. The IL-12-PD-L1 ligand conjugate according to any one of the items 1 to 13, wherein the IL-12-PD-L1 ligand conjugate comprises, or essentially consists of, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 036, or SEQ ID NO 037.

15. A nucleic acid molecule encoding the IL-12-PD-L1 ligand conjugate as specified in any one of items 1 to 14, particularly wherein the nucleic acid molecule comprises any one of the sequences SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, or SEQ ID NO 038 more particularly wherein the nucleic acid molecule comprises, or essentially consists of, SEQ ID NO 29, SEQ ID NO 030, or SEQ ID NO 038.

16. A recombinant cell comprising the nucleic acid molecules according to item 15.

17. An IL-12-PD-L1 ligand conjugate according to any one of item 1 to 14, or a nucleic molecule acid according to item 15, for use as a medicament.

18. An IL-12-PD-L1 ligand conjugate according to any one of items 1 to 14, or a nucleic molecule acid according to item 15, for use in treatment of malignant neoplastic disease, particularly neoplastic disease selected from glioma, glioblastoma multiforme, meningioma, secondary brain cancer, brain metastases, melanoma, pancreatic cancer, lung cancer, prostate cancer, colorectal cancer, or bladder cancer.

19. The IL-12-PD-L1 ligand conjugate, or the nucleic molecule acid, for use in treatment of malignant neoplastic disease according to item 18, wherein the malignant neoplastic disease is refractive to treatment with a checkpoint inhibitory agent.

20. The IL-12-PD-L1 ligand conjugate, or the nucleic molecule acid, for use in treatment of malignant neoplastic disease according to item 18, wherein the neoplastic disease is glioblastoma characterized by expression of wild type IDH1. 21. The IL-12-PD-L1 ligand conjugate for use according to any one of items 18 to 20, wherein said IL-12-PD-L1 ligand conjugate is provided by direct administration into

- a solid tumour, or

- into the close vicinity of a tumour, or

- into the lymph node associated with a tumour.

22. The IL-12-PD-L1 ligand conjugate for use according to any of the items 18 to 21 , wherein the conjugate is administered by intracranial administration to treat brain cancer.

23. A method of classifying a brain cancer patient as likely to respond favourably to treatment with an immune checkpoint inhibitor agent, particularly wherein the immune checkpoint inhibitor agent is selected from an antibody to PD-1 or PD-L1 , wherein the method comprises the steps of a. determining the expression level of a biomarker selected from IDH1 and SOX2 in a tumour sample obtained from the brain cancer patient; b. optionally, determining the expression level of the biomarker to a threshold, c. assigning to the patient a high probability of being responsive to said immune checkpoint inhibitor agent if the expression level of said biomarker is above the threshold.

24. The method according to item 23, wherein the biomarker is wild-type IDH1 .

25. The method according to item 23 or 24, wherein the expression level of wild-type IDH1 and the expression level of SOX2 are determined and compared to their respective threshold.

26. A pharmaceutical drug comprising an anti-PD-L1 -ligand, particularly: a. an anti-PD-L1 antibody or b. an IL-12-PD-L1 ligand conjugate according to any one of items 1 to 14, or a nucleic acid encoding the same, for use in treatment of a brain tumour, particularly in treatment of glioma, more particularly glioblastoma multiforme, wherein the pharmaceutical drug is administered to a patient identified as likely to respond favourably to treatment with the pharmaceutical drug by the method according to any one of the items 23 to 25.

27. A pharmaceutical drug for treatment of a brain tumour according to item 26, wherein the brain tumour is characterized by expression of wild type IDH1. Medical treatment.

Similarly, within the scope of the present invention is a method of treating cancer in a patient in need thereof, comprising administering to the patient an IL-12-PD-L1 ligand conjugate as a polypeptide, or nucleic acid sequence vector according to the above description.

Pharmaceutical Compositions and Administration

Another aspect of the invention relates to a pharmaceutical composition comprising a IL-12- PD-L1 ligand conjugate of the present invention and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the IL-12-PD-L1 ligand conjugate of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handled product.

The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows (A) a representative UMAP map showing the FlowSOM-guided metaclustering of CD45+ subsets, and CD45- cells including tumour cells and other unmarked cell types. (B) a representative UMAP map as in A, overlaid with PD- L1 expression with high intensity in red, and lower intensity in black. PD-L1 expression was normalized between 0 and 1 to the 99-999th percentile. (C) Boxplots quantify the mean antigen intensity of PD-L1 across the indicated cell types in (A), each point indicates data from one patient, boxplots represent the interquartile range (IQR) 50% and whiskers 25%. Fig. 2 shows a representative UMAP maps from human IDH1 mutant and wildtype glioma samples, overlaid with PD-L1, or SOX2 expression, with high intensity in red, and lower intensity in black.

Fig. 3 shows a map of the (A) primary and (B) secondary structure of a representative engineered IL12-anti-PDL1 construct. (C) SDS-PAGE analysis of purified fusion proteins comprising mouse (SEQ ID NO 032, left) or human (SEQ ID NO 037, right) IL-12 linked to a bivalent ScFv anti-PDL1 construct under nonreducing (NR) and reducing (R) conditions. M, Ladder of molecular weight.

Fig. 4 shows that IL12-anti-PDL1 has no loss of IL-12 signalling capacity. 10⁵ splenocytes were cultured in the presence of concentrations of the two different IL12 fusion molecules indicated, and T cell activation was measured by IFN-g production by ELISA after 48h. (A) and (B) mouse IL-12 fused to anti-PDL1 diabody versions 1 or 2 (SEQ ID NO 032 or SEQ ID NO 035), respectively.

Fig. 5 shows that intratumoural injection of IL12-anti-PDL1 fusion protein (SEQ ID NO

032) increases survival in a murine model of glioblastoma. (A) Schematic overview of experimental procedure: Mice were inoculated with a luciferase- expressing GL261 glioma cell line and tumour size was quantified using an IVIS 100 imaging system. Animals with an ROI flux of less than 10⁵ p/s were excluded. 14 days after inoculation, mice were intracranially injected with PBS or IL12-anti-PDL1 (0.5 pg) and (B) survival was monitored overtime. N = 14 for PBS and 13 for IL12-anti-PDL1 from two independent experiments.

Fig. 6 shows that IL12-anti-PDL1 fusion protein improved retention in the tumour environment compared to recombinant IL12. (A) Mice were inoculated with a luciferase-stable GL261 glioma cell line and tumour size was quantified using IVIS 100 imaging system. 14 days after inoculation, mice were intracranially injected with recombinant mouse IL12p70 or IL12-anti- PDL1 (SEQ ID NO 032, 2 pg). Three days later, brain and serum were isolated and ELISA was used to measure IL12 and IFN-y. (B) Mice were inoculated with MC38 colon cancer cell line and tumour size was measured. 7-12 days after inoculation mice were intratumorally injected with PBS, IL12-anti-PDL1 (SEQ ID NO 035, 12 pg) and equimolar amounts of recombinant mouse IL12p70. Three days later, tumour and serum were isolated and ELISA was performed to measure IFN-g.

Fig. 7 shows that IL12-anti-PDL1 fusion protein blocks PD1-PDL1 interactions.

PD1/PDL1 blockade bioassay from Promega was used to determine the ability of the IL12- anti-PDL1 fusion protein (SEQ ID NO 032) to interfere with PD1/PDL1 binding. In short, CHO-K1 cells stably expressing human PD-L1 and a cell surface protein designed to activate cognate Jurkat T cells were plated overnight. The following day serial dilutions of the IL12-anti-PDL1 fusion protein and PD1 expressing Jurkat T effector cells were added. 6 hours later, increasing bioluminescence indicates active blocking of PD1/PDL1 interaction.

Fig. 8 shows in vitro binding of mouse IL-12-anti-PDL1 fusion protein construct (SEQ

ID NO 035) to PDL-1 measured by surface plasmon resonance analysis (singlecycle kinetics). IL-12 anti-PDL1 on mouse (A) and human (B) PDL1 -coated sensor chip.

Examples

Methods

Human Brain Tissue Samples

Human brain tissue samples were collected in the Department of Neurosurgery at the University Hospital Zurich after written informed patient consent following the local ethical requirements. 18 patients (including glioma IDH1 ^mut and IDH1^wt), male and female subjects between the ages of 10 - 80 years old were included in the present study.

Processing of Human Samples for Cytometry Analysis

Resected human brain tissue samples were thoroughly washed with phosphate-buffered saline (PBS), minced, and digested (1 mg/mL Collagenase IV (Sigma-Aldrich), 10 pg/mL DNase (Sigma-Aldrich), 10% Foetal Bovine Serum (FBS) (Thermo Fisher Scientific), RPM1 1640 (Seraglob)) at 37°C for45 minutes using the gentle MACS Octo Dissociator. Myelin and red blood cells were removed by Percoll gradient (Sigma-Aldrich). The cells were stained for viability with Cell-IDTM Cisplatin (Fluidigm) (3 minutes at 4°C), fixed with 1.6% Paraformaldehyde aqueous solution (PFA; Electron Microscopy Sciences), and cryopreserved in FACS buffer (EDTA 2mM (StemCell Technologies, Inc.), FBS 0.5%, PBS) complemented with 10% DMSO (Sigma-Aldrich). Cell fixation before cryopreservation allowed preservation of the myeloid compartment (including microglia and neutrophils) and cell frequencies. Cryopreserved samples were stored at -80°C (maximum 30 days) or in liquid nitrogen -160°C until analysis.

Mass-Tag Cellular Barcoding

To minimize inter-sample staining variation, two strategies of mass-tag barcoding were applied to fixed cells (Zunder et al., 2015). Intracellular barcoding consisted of a ten sample barcoding scheme composed with unique combinations of three out of five palladium metals (^^Pd,

(j_race Sciences International). Palladium isotopes were conjugated to Bromoacetamidobenzyl-EDTA (Dojindo Laboratories) and adjusted to 100 nM. After thawing, cells were washed once with Cell Staining Medium (CSM: PBS, 0.5% Bovine Serum Albumin (BSA) (Sigma-Aldrich) and 0.02%NaN3), once with PBS and once with 0.03% Saponin (Sigma-Aldrich) in PBS and diluted barcoding reagent in PBS with 0.03% saponin was thoroughly and quickly mixed with the sample and incubated at RT for 15 minutes. Cells were then washed three times in CSM, and ten samples were pooled for antibody surface staining.

Metal-Isotope-Tagged Antibodies

All anti-human antibodies, and tagged metal isotopes for mass cytometry analysis are listed in Table 1. Pre-conjugated antibodies to metal isotope were purchased from Fluidigm or commercial suppliers in purified form and conjugated in house using the Maxpar X8 chelating polymer kit (Fluidigm) according to the manufacturer’s instructions.

Cell Preparation and Mass Cytometry Acquisition

The sample was incubated at 4°C for 15 minutes in Human TruStain FcXTM (Fc Receptor Blocking Solution; Biolegend). Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. After cell surface and intracellular antibody staining the cells were incubated in 4% PFA (Electron Microscopy Sciences) overnight. Prior to acquisition the cells were pelleted without washing and resuspended in up to 1 ml of diluted 1 :3000 Cell-ID™ Intercalator-lr

(Fluidigm) + Maxpar® Fix and Perm Buffer (Fluidigm) for 1 .5-3 hours. After washing, 1.5-10^D cells/ml in H20 containing 10% EQTM Four Element Calibration Beads (Fluidigm) were analysed with a Helious CyTOF2 (Fluidigm).

Pre-processing of Cytometry Data

Raw mass cytometry data were normalized using the MATLAB version of the Normalizer tool.

191 193

Cells were assigned by manually gating on Event length and DNA ( Ir and Ir) channels,

195 followed by the dead cell discrimination analysing Pt expression using FlowJo Software (Tree Star). Data were concatenated and de-barcoded using Boolean gating in FlowJo software. The normalized data containing live cells from individual patients were analysed with R packages “flowCore” and “flowWorkspaceData” (R Foundation for Statistical Computing). Before automated high-dimensional data analysis, the mass cytometry data were transformed with a cofactor in the range of 5 and 60 using an inverse hyperbolic sine (arcsinh) function. For the mass cytometry data, the marker expression distributions were verified between two batches of the acquisition applying R package “flowStats” Automated Population Identification. To identify CD45+ and CD45- cell populations, FlowSOM clustering was used to generate a starting point of 100 nodes, on pre-processed and combined mass cytometry datasets, followed by expert-guided manual metaclustering. The respective k-value was manually chosen (in the range of between 20 and 30); identified clusters were annotated and merged based on a similarity of antigen expression in order to uphold the biological relevance of the dataset. Manually-annotated clusters were used to calculate the relative frequencies of immune populations. For PD-L1 intensity analysis, the median antigen expression among selected cell types of the second mass cytometry batch was calculated.

6 - 12 week-old C57BL/6 mice (female and male) were used for preclinical models. All animal experiments performed in this study were approved by the Swiss Veterinary Office. Animals were monitored once a week to assess weight loss and physical/neurological abnormalities. In vivo measurements to assess tumour growth were performed once a week.

Orthotopic Glioma Cell Injection

GL-261 cells stably transfected with pGI3-ctrl and pGK-Puro (Promega) were selected with puromycin (Sigma-Aldrich) to generate luciferase-stable GL-261 cells. A single clone was isolated by limiting dilution and passaged in vivo by intracranial tumour inoculation. Mice were anesthetized with 2-5% Isoflurane (Minrad) on a stereotactic frame (David Kopf Instruments). A blunt-ended syringe (Hamilton; 75N, 26s/272, 5 pi; Sigma-Aldrich) was injected 1.5 mm lateral and 1 mm frontal from the bregma. A 5ml syringe (Hamilton; Sigma-Aldrich) was injected with a depth of 4 mm below the skull and retracted 1 mm, forming a reservoir. Using a microinjection pump (UMP-3; World precision Instruments Inc.), 5 * 10⁴ GL-261 cells were injected in a volume of 2 pi at 1 mI/minute. After resting the needle for 2 minutes, it was retracted at a speed of 1 mm/minute. The injection hole was closed and sealed.

Colon cancer model

MC38 tumor cells (3* 10⁵ cells/mouse, 1 :1 PBS/Matrigel) were implanted subcutaneously (s.c.) in the right flank of female C57BI/6 mice. Tumour volume was calculated as follows: [length (mm) * width (mm) * width (mm)]/2. When tumour volumes reached approx. 100mm³, the mice were randomized into groups receiving intratumorally injected with recombinant mouse IL12-anti-PDL1 (12 pg) or IL12p70 (equimolar dose). Functional subunit testing in vitro assays

A PD1/PD-L1 blockade bioassay from Promega was used to measure the ability of IL12-anti- PD-L1 to neutralise PD-1 interactions with PD-L1 according to the manufacturer’s instructions. PD-L1 expressing aAPC cells were plated overnight and the following day serial dilutions of the IL12-anti-PDL1 fusion protein were added together with PD1 expressing effector cells. 6 hours later, bioluminescence was measured. Increasing bioluminescence indicates active blocking of PD1/PDL1 interaction. To measure IL-12 activity, 10⁵ murine splenocytes were cultured in the presence of the IL12-anti-PDL1 construct or IL12-Fc fusion molecule and T cell activation was examined by measuring IFN-g production by ELISA after 48h according to the manufacturer’s instruction (Biolegend).

IL-12 toxicity assay

Mice were inoculated with luciferase-stable GL261 glioma cell line. 14 days after inoculation mice were intracranially injected with recombinant mouse IL12p70 or IL12-anti-PDL1 (2 pg). Three days later, brain and serum were isolated and ELISA was performed for IL12 and IFN- g according to the manufacturer’s instructions (Biolegend).

Example 1. PD-L1 expression by CD45- fraction in human glioma patients.

Tumour tissue from 18 patients undergoing neurosurgery for the treatment of gliomas was characterized according to IDH1 R132H mutation (IDHI mut and IDHIwt) and methylation status of the 06-methylguanine DNA methyltransferase (MGMT) promoter. To map the complexity of the cell compartment of the TME, a mass cytometry (CyTOF) panel measured 37 parameters at the single-cell level (Table 1 ). The combined antibodies captured the major leukocyte populations and their relative cellular frequencies along with frequency and diversity of CD45^" cells. To visualize immune populations isolated from the different brain tumour samples, a two-dimensional graph using the dimensionality reduction algorithm UMAP was used (Fig. 1A). Next, embedded cell clusters were categorized using self-organizing maps (FlowSOM) (Van Gassen et al., 2015 Cytometry A. 87(7):636-645; Hartmann et al., 2016 J. Exp. Med. 213(12):2621 -2633) to create a map of diverse immune and non-immune cells including, CNS-resident and invading TAMs/monocytes (CD64⁺, CD11c⁺, CD11b⁺), neutrophils (CD66b⁺, CD16⁺), two subsets of dendritic cells (CD141⁺ and CADM1⁺ for cDCI and CD1c⁺ for cDC2), T cells (CD3⁺), NK cells (CD56⁺CD16⁺), B cells (CD19⁺, HLA-DR⁺), plasma cells (CD19⁺,

_and CD45^" cells (including tumour and CNS-resident cells). The expression level of PD-L1 was assessed on each cell subset, showing that PD-L1 was expressed on CD45^" cells (not immune cells), a population which includes SOX2+ glioma tumour cells, in addition to tumour infiltrating macrophages (Fig. 1B) Furthermore, division of the samples into those classed as either IDH1^wt or IDH1 ^mut gliomas revealed that PD-L1 expression was strongest among I D H 1 ^wt tumour cells, identified by SOX2 expression, a pattern that was not observed in matched WT IDH1 ^mut patient samples.

Example 2. Design of IL-12-anti-PDL1 fusion protein.

IL-12 has long been recognised for its potent anti-tumour immunity across various preclinical models of cancer. However, one major concern regarding IL-12 as a therapeutic agent to treat cancer patients, is its ability to elicit a toxic cytokine storm, a major adverse reaction leading in some cases to mortality. Hence, the systemic accumulation of IL-12 and associated toxicities must be avoided in order to exploit its anti-tumour activity. PD-L1 was demonstrated to specifically localise on tumour cells in the glioma samples assessed above, and is known to be present in the tumour microenvironment (TME) of many solid tissue cancers. It is also widely held that this TME-enriched expression of PD-L1 underpins the observed tumour-immune escape of solid tissue cancers and metastasis. Thus, IL-12 tethered to a PD-L1 binding unit was expected to have superior targeting and retention into the TME, maximising the anti- tumoural effect of IL-12 effect whilst minimising it’s potential to elicit systemic toxicity.

To improve the therapeutic index of IL-12 by “binding” it to PDL1 positive tumour cells and other cells in the tumour microenvironment using anti-PDL1 as an anchor, an IL12-anti-PDL1 ligand conjugate was designed for use in preclinical mouse models by sequential fusion of the p35 and p40 subunit of mouse IL-12, joined by a 5 amino acid peptide linker (SEQ ID NO 033), or a 6 amino acid peptide linker (SEQ ID NO 007 encoded by sequence ID NO 022), with one of two versions of a tandem arrangement of two linked anti-human PDL1 antibody scFv fragments (SEQ ID NO 009, or 010, encoded by SEQ ID NO 024, and 025 respectively) in single chain format (Fig. 3, DNA sequence of SEQ ID NO 031 and SEQ ID 034, encoding SEQ ID NO 035 and SEQ ID NO 032 respectively). An IL12-anti-PDL1 ligand conjugate comprising the human IL-12 p35 (SEQ ID NO 001 encoded by SEQ ID NO 016) and p40 (SEQ ID NO 002 encoded by SEQ ID NO 017) subunits linked by the 5 amino acid peptide linker to the scFv above was also successfully expressed and purified (SEQ ID NO 037 encoded by SEQ ID NO 038) and showed a similar SDS page profile to the experimental mouse molecule (Fig. 3C). Combinatorial therapy with the two linked biological agents IL-12 and PD-L1 was aimed at retaining IL12 in the TME to limit harmful side effects, while also inhibiting PDL1 immune blockade to improve overall patient survival and tumour regression in comparison to therapy with single agents.

Example 3. Fusion with anti-PDL1 does not impair the bioactivitv of IL12 in vitro.

The functionality of the IL-12 subunit of the IL12-anti-PDL1 fusion protein was compared to IL- 12-Fc (IL-12 fused with the crystallizable fragment (Fc region) of a human immunoglobulin to facilitate purification (Vom Berg et al., 2013, J. Exp. Med. 210(13):2803-2811 ). IL-12Fc has previously been shown to be a) of equal activity when compared to commercially available recombinant IL-12 heterodimer and b) to have a dramatically extended half-life compared to commercially available recombinant IL-12 heterodimer. Splenocytes were co-cultured in the presence of either of the two IL12 molecules and T cell activation was examined by measuring IFN-g production by ELISA. The levels of IFN-g were comparable between the two constructs (Fig. 4A), showing that the addition of the mAb fragment against PDL1 does not diminish the bioactivity of IL-12. The second mouse construct bearing variable regions derived from a commercial PDL1 antibody performed similarly in the assay, demonstrating equivalent IL-12 function (Fig 4B).

Example 4. IL12-anti-PDL1 fusion protein has a significant benefit on the overall survival in a murine model of glioblastoma.

Mice were inoculated with GL261 glioma cell line and tumour size was assessed to ensure comparable tumour burden. Subsequently, mice were intracranially injected with PBS or IL12- anti-PDL1 and survival was monitored over time. The results showed that the fusion protein had a significant benefit on the overall survival in comparison to a PBS-treated group (Fig. 5).

Example 5. IL12-anti-PDL1 is retained in the tumour, reducing systemic toxicity.

One of the leading cytokines of the toxic cytokine storm is IFN gamma which can be used as a surrogate of systemic toxicity. To evaluate the ability of IL12-anti-PDL1 to retain in the TME, IL12-anti-PDL1 or recombinant mouse IL-12 were intracranially injected into tumour-bearing mice 14 days upon inoculation with GL261. Three days later, the levels of IL-12 and IFN-g in the serum and brain were measured, to obtain information about the system implication of the local injection. Significantly lower levels of IFN-g were detected in the serum of mice treated with IL12-anti-PDL1 compared to recombinant IL-12, demonstrating the local retention of IL- 12 by means of linkage to an anti-PDL1 domain reduced systemic activation of T cells (Fig. 6A). However, equivalent upregulation of IL12 was observed in the brain tissue of mice treated with IL12-anti-PDL1 compared to recombinant IL-12, confirming that IL12 persists in the tumour at the same level as recombinant heterodimeric IL-12 when coupled to anti-PDL1 (Fig. 6A). A similar pattern of intratumoural inflammation combined with limited systemic IFN-g release following intratumoural injection of IL-12-anti-PDL1 was observed in the MC38 model of peripheral solid tumour (Fig. 6B). Taken together, IL-12:aPD-L1 triggers a strong anti- tumoural response whilst its retention within the TME avoids systemic toxicity

Example 6. IL-12-anti-PDL1 efficiently blocks PD-1 interactions with PD-L1.

PD1/PDL1 blockade bioassay from Promega was used to determine the ability of an IL12- anti- PDL1 fusion protein to interfere with PD1/PDL1 binding. PDL1 aAPC cells were plated overnight, and the following day serial dilutions of the IL12-anti-PDL1 fusion protein were added together with PD1 expressing effector cells. 6 hours later, Bioluminescence was measured. Increasing bioluminescence indicates active blocking of PD1/PDL1 interaction (Fig.

To confirm the binding of human-anti-PDL1 domain used in the mouse IL-12-PD-L1 conjugate ligand to bind to both mouse and human PD-L1 , the binding kinetics of the interaction was measured using Surface Plasmon Resonance. Mouse or human PD-L1 proteins were immobilized as ligand on a chip and single-cycle kinetics of a series of ascending analyte concentrations was measured in one cycle (Fig.8). This demonstrates that the addition of the sequential fusion of both p35 and p40 subunits of IL-12 does not diminish the binding of aPDL1 to mouse or human ligand PD-L1 .

SEQUENCES

In cases of doubt or discrepancy between any supplementary sequence material, the sequences provided in the specification below should be used.

SEQ ID NO 002: (human p40)

SEQ ID NO 003: VH domain 1

SEQ ID NO 004: VH domain 2

SEQ ID NO 005: VL domain 1

SEQ ID NO 006: VL domain 2

SEQ ID NO 007: conjugate domain linking element 1

SEQ ID NO 008: IL-12 domain peptide linker

SEQ ID NO 009: single chain scFv 1 , two functional PD-L1 binding sites

SEQ ID NO 0010: single chain scFv 2, two functional PD-L1 binding sites

SEQ ID NO 011 : first intra scFv Linker element

SEQ ID NO 012: second intra scFv Linker element

SEQ ID NO 013: secretory leader signal

SEQ ID NO 014: hlL-12-PD-L1 ligand conjugate v1

SEQ ID NO 015: hlL-12-PD-L1 ligand conjugate v2

SEQ ID NO 016: human p35

SEQ ID NO 017: human p40

SEQ ID NO 018: VH v1

SEQ ID NO 019: VH v2

SEQ ID NO 020: VL v1

SEQ ID NO 021 : VL v2

SEQ ID NO 22: conjugate domain linking element 1

SEQ ID NO 023: IL-12 domain peptide linker

SEQ ID NO 024: ScFv anti-PD-L1 ligand v1

SEQ ID NO 025: ScFv anti-PD-L1 ligand v1

SEQ ID NO 026: first intra scFv anti-PD-L1 Linker element

SEQ ID NO 027: second intra scFv anti-PD-L1 Linker element

SEQ ID NO 028:

SEQ ID NO 029: hlL-12-PD-L1 ligand conjugate v1

SEQ ID NO 030: hll_-12-PD-L1 ligand conjugate v2

SEQ ID NO 031 : mlL-12-PD-L1 ligand conjugate v1

SEQ ID NO 032: mlL-12-PD-L1 ligand conjugate v1

SEQ ID NO 033: conjugate domain linking element 2

GSADG

SEQ ID NO 034: mlL-12-PD-L1 ligand conjugate v2

SEQ ID NO 035: mlL-12-PD-L1 ligand conjugate v1

SEQ ID NO 036: hlL-12-PD-L1 ligand conjugate v3

SEQ ID NO 037: hlL-12-PD-L1 ligand conjugate v4 (v2 anti PD-L1 AB, short linker)

SEQ ID NO 038: : hlL-12-PD-L1 ligand conjugate v4

Table 1.

Table 1 continued

Claims

Claims

1. An IL-12-PD-L1 ligand conjugate comprising, or essentially consisting of a single polypeptide chain, said single polypeptide chain comprising: an IL-12 domain comprising a p40 domain and a p35 domain, and a PD-L1 ligand domain.

2. The IL-12-PD-L1 ligand conjugate according to claim 1 , wherein the IL-12 domain is N- terminal relative to the PD-L1 ligand domain.

3. The IL-12-PD-L1 ligand conjugate according to claims 1 or 2, wherein the p40 domain is N-terminal relative to the p35 domain.

4. The IL-12 PD-L1 ligand conjugate according to any one of the claims 1 to 3, wherein the IL-12 domain comprises

- a sequence at least (>) 85% identical, particularly >90%, more particularly >95% identical to the sequence of human p35 (SEQ ID NO 001 ), and

- a sequence > 85% identical, particularly >90%, more particularly >95% identical to the sequence of human p40 (SEQ ID NO 002), and wherein

- the IL-12 domain is characterized by at least 70%, particularly >85%, more particularly >90% of the biological activity of human IL-12.

5. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 4, wherein the PD-L1 ligand domain inhibits the binding of PD-L1 to PD-1.

6. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 5, wherein the PD-L1 ligand domain comprises or essentially is an antibody-like molecule, particularly an antigen-binding antibody fragment.

7. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 6, wherein the PD-L1 ligand domain comprises, or essentially consists of, a single chain variable fragment (scFv) comprising:

- a variable heavy chain (VH) domain, and a variable light chain (VL) domain, particularly wherein the VH domain comprises or consists of the sequence SEQ ID NO 003 and the VL domain comprises or consists of the sequence SEQ ID NO 005, or the VH domain comprises or consists of the sequence SEQ ID NO 004, and the VL domain comprises or consists of the sequence SEQ ID NO 006.

8. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 7, wherein the IL-12 domain and the PD-L1 ligand domain are joined by a conjugate domain peptide linker, particularly wherein the conjugate domain peptide linker is 5 or 6 amino acids in length and wherein the amino acids are a G, S, A or a D, more particularly a conjugate domain peptide linker consisting of SEQ ID NO 007, or SEQ ID NO 033.

9. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 8, wherein the p40 domain and the p35 domain are connected by an IL-12 domain peptide linker, particularly an IL-12 domain peptide linker >15 amino acids in length, more particularly an IL-12 domain peptide linker 15 to 30 amino acids in length wherein the amino acids are a G, S, A or a D, most particularly an IL-12 domain peptide linker consisting of SEQ ID NO 008.

10. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 9, wherein the PD-L1 ligand is a bivalent, or multivalent, scFv that inhibits the binding of PD-L1 to PD- 1 , said scFv comprising or consisting of

- a first scFv domain comprising a first VH domain and first VL domain, and

- a second scFv domain comprising a second VH domain and a second VL domain, and wherein optionally,

- the first scFv and the second scFv are part of one single polypeptide chain with at least two functional antigen binding sites, particularly one single peptide chain of SEQ ID NO 009 or SEQ ID NO 010.

11. The IL-12-PD-L1 ligand conjugate according to claim 10, wherein the VH and VL domains are joined sequentially by alternating placement of

- A first scFv peptide linker, wherein the first scFv peptide linker is >15 amino acids in length, particularly a first scFv peptide linker consisting of the amino acids G, S, A or a D, more particularly a first scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 3, 4 or 5 adjacent motifs, most particularly a first scFv domain peptide linker of SEQ ID NO 011 , and

- a second scFv peptide linker, wherein the second scFv peptide linker is <15 amino acids in length, particularly a second scFv peptide linker consisting of the amino acids G, S, A or a D, more particularly a second scFv peptide linker comprising a motif selected from (GGGGS)_n, (GGSGG)_n, or (SSSSG)_n, with n being 1 or 2 adjacent motifs, most particularly a second scFv peptide linker of SEQ ID NO 012.

12. The IL-12-PD-L1 ligand conjugate according to any of the claims 1 to 9 wherein the PD-L1 ligand is a univalent scFv comprising

- a single VH domain and single VL domain only, and wherein

- the single VH domain is joined to single VL domain by the first scFv peptide linker as specified in claim 11 .

13. The IL-12-PD-L1 ligand conjugate according to any one of the claims 1 to 12, wherein the IL-12-PD-L1 ligand conjugate comprises, or essentially consists of, SEQ ID NO 014, or SEQ ID NO 015, SEQ ID NO 036, or SEQ ID NO 037.

14. A nucleic acid molecule encoding the IL-12-PD-L1 ligand conjugate as specified in any one of claims 1 to 13, particularly wherein the nucleic acid molecule comprises a sequence selected from SEQ ID NO 016, SEQ ID NO 017, SEQ ID NO 018, SEQ ID NO 019, SEQ ID NO 020, SEQ ID NO 021 , SEQ ID NO 022, SEQ ID NO 023, SEQ ID NO 024, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 027, SEQ ID NO 028, SEQ ID NO 038, SEQ ID NO 029 and/or SEQ ID NO 030; more particularly wherein the nucleic acid molecule comprises, or essentially consists of, SEQ ID NO 038, SEQ ID NO 029 or SEQ ID NO 030.

15. A recombinant cell comprising the nucleic acid molecule according to claim 14.

16. An IL-12-PD-L1 ligand conjugate according to any one of claims 1 to 14, or a nucleic molecule acid according to claim 15, for use as a medicament.

17. An IL-12-PD-L1 ligand conjugate according to any one of claims 1 to 14, or a nucleic molecule acid according to claim 15, for use in treatment of malignant neoplastic disease, particularly neoplastic disease selected from glioma, glioblastoma multiforme, meningioma, secondary brain cancer, brain metastases, melanoma, pancreatic cancer, lung cancer, prostate cancer, colorectal cancer or bladder cancer.