PD-L1 TARGETED FUSION PROTEINS AND METHODS OF USE THEREOF

Abstract

The present invention provides PD-L1 targeted fusion proteins and methods of their use. The targeted fusion proteins comprise a trispecific killer engager molecule comprising a camelid or human CD16 natural killer (NK) cell engager domain, a wild-type or mutant IL-15 cytokine NK activation domain, and a PD-L1 targeting peptide light chain and heavy chain. Methods of use include methods of treating cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 245,135, filed September 16, 2021. The disclosure of the prior application is deemed to be part of the disclosure of this application in its entirety and is incorporated herein by reference.

[0002] Incorporating sequence tables The material in the attached sequence listing is hereby incorporated by reference into this application. The attached sequence listing xml file (named G1421US00_GTBIO2190-1WO.xml) was created on September 9, 2022 and is 58kb in size.

[0003] FIELD OF THEINVENTION The present invention relates generally to fusion proteins, and more specifically to PD-L1-targeted trispecific killer engager molecules and their use to treat cancer. [Background technology]

[0004] Background information Immunotherapy is a rapidly evolving personalized treatment to activate or suppress the immune system to amplify or reduce immune responses and treat various forms of cancer. Immunotherapies for cancer, such as chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, PD-1 and PD-L1 inhibitors, aim to help the patient's immune system fight cancer. T cell activation depends on both the specific combination of T cell receptor (TCR) and peptide-binding major histocompatibility complex (MHC), and the interaction of T cell co-stimulatory molecules with ligands on antigen-presenting cells (APCs).

[0005] Immune checkpoints, such as PD-1, PD-L1, PD-L2, and CTLA4, are molecules that maintain many receptor-ligand interactions to evade the immune system and promote proliferation. Several monoclonal antibodies (mAbs) that block these proteins have been developed to downregulate the suppressive immune response and promote the cytotoxicity of T cells to eliminate tumor cells. Among immune checkpoint blockers, inhibitors targeting PD-1 or CTLA4 have been successfully used to treat metastatic melanoma patients, improving response and prolonging survival. This success has led to the development of such agents to treat a wide range of malignancies, including renal cell carcinoma (RCC), NSCLC, and acute myeloid leukemia (AML), which further improved response rates and extended patient survival times compared to conventional treatments (Yang et al., Int J Biol Sci 2020;16(11):1767-1773 (Non-Patent Document 1)).

[0006] The programmed cell death-1 receptor (PD-1) and its ligands (PD-L1 / PD-L2) belong to a family of immune checkpoint proteins that function as co-inhibitory factors that can halt or limit the development of T cell responses. PD-1 is expressed on the surface of activated T cells, whereas PD-L1 and PD-L2 are expressed on the surface of dendritic cells or macrophages. The PD-1 / PD-L1 interaction ensures that the immune system is activated only at the right time to minimize the possibility of chronic autoimmune inflammation. Under normal conditions, the immune system executes a series of steps that lead to anti-cancer immune responses and cancer cell death, based on the activation of T cells by dendritic cells presenting tumor antigens, and the release by T cells of cytotoxins that induce apoptosis in their target cancer cells.

[0007] The PD-1 / PD-L1 pathway represents an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous immune antitumor activity. PD-L1 is overexpressed on tumor cells or on non-transformed cells in the tumor microenvironment. PD-L1 expressed on tumor cells binds to the PD-1 receptor on activated T cells, which results in the inhibition of cytotoxic T cells. These inactivated T cells remain inhibited in the tumor microenvironment.

[0008] Despite some success, there are limitations that reduce the overall efficiency of mAb therapy. With the development of CD16-directed bispecific and trispecific single-chain fragment variable (BiKE and TriKE) recombinant molecules, most of these undesirable limitations are avoided because they lack the Fc portion of the whole antibody and have target specificity for CD16, while inducing high effector function (Gleason et al., Mol Cancer Ther; 11(12); 2674-84, 2012). As a result, recombinant reagents are attractive for clinical use to enhance natural killer (NK) cell immunotherapy.

[0009] The ability of NK cells to recognize and kill targets is regulated by a sophisticated repertoire of inhibitory and activating cell surface receptors. NK cell cytotoxicity can occur by natural cytotoxicity mediated through natural cytotoxicity receptors (NCRs) or by antibodies such as rituximab that induce antibody-dependent cell-mediated cytotoxicity (ADCC) through CD16, an activating low-affinity Fc-γ receptor for immunoglobulin G (IgG), highly expressed by the CD56dim subset of NK cells. CD16 / CD19 BiKE and CD16 / CD19 / CD22 TriKE can induce NK cell activation through direct CD16 signaling, inducing directional secretion of lytic granules and target cell death. Furthermore, these reagents induce NK cell activation leading to cytokine and chemokine production. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Yang et al.,Int J Biol Sci 2020;16(11):1767-1773 [Non-Patent Document 2] Gleason et al.,Mol Cancer Ther;11(12);2674-84,2012 Summary of the Invention

[0011] The present invention is based on the development of a PD-L1-targeted fusion protein, specifically, the PD-L1-targeted trispecific killer engager molecule (TriKE).

[0012] In one embodiment, the present invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0013] In another embodiment, the present invention provides a protein encoded by the nucleic acid sequence shown in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0014] In one aspect, the amino acid sequence is selected from SEQ ID NO:6 or SEQ ID NO:7.

[0015] In an additional embodiment, the invention provides a fusion protein comprising the amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation.

[0016] In one embodiment, the protein comprises SEQ ID NOs: 6 and 7, with the C-terminus of SEQ ID NO: 6 directly linked to the N-terminus of SEQ ID NO: 7. In another embodiment, the protein comprises SEQ ID NOs: 7 and 6, with the C-terminus of SEQ ID NO: 7 directly linked to the N-terminus of SEQ ID NO: 6.

[0017] In a further embodiment, the present invention provides a fusion protein comprising a sequence as set forth in SEQ ID NO:1 or 15, and a sequence having 90% or greater identity to SEQ ID NO:1 or 15.

[0018] In one embodiment, the invention provides a fusion protein comprising, in operably linked form, SEQ ID NO: 2 or 23; SEQ ID NO: 4, 21 or 22; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

[0019] In one embodiment, SEQ ID NO:2 or 23 and SEQ ID NO:4, 21 or 22 are linked by SEQ ID NO:3 or SEQ ID NO:16. In another embodiment, SEQ ID NO:4, 17 or 18 and SEQ ID NO:6 or 7 are linked by SEQ ID NO:5 or SEQ ID NO:17. In other embodiments, SEQ ID NO:6 and SEQ ID NO:7 are operably linked in either orientation. In some embodiments, the fusion protein further comprises a half-life extension (HLE) molecule. In one embodiment, the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs:25-29. In some embodiments, SEQ ID NO:4 has an N72 substitution. In various embodiments, the N72 mutation is N72A or N72D. In one embodiment, the protein is set forth in SEQ ID NO:21 or 22.

[0020] In one embodiment, the invention provides a fusion protein comprising SEQ ID NO:23, SEQ ID NO:21 or 22, and SEQ ID NO:6 and 7 in either orientation. In one aspect, SEQ ID NO:23 is operably linked to SEQ ID NO:21 or 22 by a linker of SEQ ID NO:3 or 16. In another aspect, SEQ ID NO:21 or 22 is operably linked to sequences 6 and 7 in either orientation by a linker of SEQ ID NO:5 or 17. In some aspects, the fusion protein further comprises a half-life extension (HLE) molecule. In one aspect, the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs:25-29.

[0021] In additional embodiments, the invention provides an isolated nucleic acid sequence encoding any of the fusion proteins described herein.

[0022] In one embodiment, the sequence is SEQ ID NO:8 or SEQ ID NO:18.

[0023] In another embodiment, the invention provides a method of treating cancer in a subject, comprising administering to the subject any of the fusion proteins described herein, thereby treating the cancer.

[0024] In one aspect, the cancer is selected from non-small lung cancer, squamous cell carcinoma of the skin, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal carcinoma, prostate cancer, cervical cancer, ovarian cancer, melanoma, brain cancer, leukemia, lymphoma, myeloma, head and neck cancer, or breast cancer. In some aspects, an immune checkpoint inhibitor is further administered to the subject. In various aspects, the immune checkpoint inhibitors include programmed cell death 1 protein (PD-1) inhibitors, PD-1 ligand 1 (PD-L1) inhibitors, PDD-L2 inhibitors, cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitors, adenosine A2A receptor (A2AR) inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, B and T Lymphocyte Attenuator (BTLA) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, killer cell immunoglobulin-like receptor (KIR) inhibitors, lymphocyte activation gene-3 (LAG3) inhibitors, nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2) inhibitors, sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7) inhibitors, SIGLEC9 inhibitors, T cell immunoglobulin domain and mucin domain 3 (TIM-3) inhibitors, and V-domain Ig suppressor of activation of T cell (V ... The inhibitor is selected from the group consisting of (VISTA) inhibitors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention The present invention is based on the development of a PD-L1-targeted fusion protein, specifically, the PD-L1-targeted trispecific killer engager molecule (TriKE).

[0026] Before describing the compositions and methods of the present invention, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but it will be understood that modifications and variations are within the spirit and scope of this disclosure. Preferred methods and materials are now described.

[0030] In one embodiment, the present invention provides an isolated nucleic acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0031] As used herein, the term "nucleic acid" or "oligonucleotide" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded and linear or covalently circularly closed molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example, via polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, (iv) synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. The nucleic acids can be used for introduction into cells, i.e. for transfection into cells, in particular in the form of RNA, which can be prepared by in vitro transcription from a DNA template. Furthermore, the RNA can be modified before application by stabilizing sequences, capping and polyadenylation.

[0032] As used herein, "amplified DNA" or "PCR product" refers to an amplified fragment of DNA of a defined size. A variety of techniques for detecting PCR products are available and well known in the art. PCR product detection methods include gel electrophoresis using agarose or polyacrylamide gels and adding ethidium bromide staining (DNA intercalant), labeled probes (radioactive or non-radioactively labeled, Southern blotting), labeled deoxyribonucleotides (radioactive or non-radioactively labeled, for direct incorporation), or silver staining for direct visualization of the amplified PCR product; restriction endonuclease digestion relying on agarose or polyacrylamide gels or high performance liquid chromatography (HPLC); detection on specific labeled probes (radioactive or non-radioactively labeled); These include, but are not limited to, dot blots using hybridization of amplified DNA; high pressure liquid chromatography using ultraviolet detection; electro-chemiluminescence combined with voltage-initiated chemical reactions / photon detection; and direct sequencing using radioactively or fluorescently labeled deoxyribonucleotides for determination of the exact order of nucleotides comprising a DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE), and / or direct hybridization.

[0033] In general, nucleic acids can be extracted, isolated, amplified or analyzed by a variety of techniques, such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012), or those described in U.S. Patent No. 7,957,913, U.S. Patent No. 7,776,616, U.S. Patent No. 5,234,809, U.S. Patent Publication No. 2010 / 0285578, and U.S. Patent Publication No. 2002 / 0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing can be by any method known in the art. DNA sequencing techniques include classical dideoxy sequencing reactions (Sanger method) using labeled terminators or primers, and gel separation in slabs or capillaries, as well as next-generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, sequencing by synthesis using allele-specific hybridization to a library of labeled oligonucleotide probes followed by ligation to a library of labeled clones, real-time monitoring of the incorporation of labeled nucleotides during the polymerization step, polony sequencing, and SOLiD sequencing. The separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases, as well as by single or sequential differential hybridization with a library of probes.

[0034] The terms "sequence identity" or "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). In some embodiments, the length of the reference sequence (e.g., SEQ ID NO: 13 or 14) aligned for comparison purposes is at least 80% of the length of the comparison sequence, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are the same length.

[0035] The desired range of sequence identity is about 80% to 100% and integer values ​​therebetween. The percent identity between the disclosed sequences and the claimed sequences can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. Generally, a perfect match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NO: 13 or 14). Preferably, sequences that are not 100% identical to the sequences provided herein retain the function of the original sequence (e.g., the ability to bind PD-L1 or CD16).

[0036] Polypeptides and polynucleotides that are about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more identical to the polypeptides and polynucleotides described herein are embodied within the present disclosure. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 13 or 14.

[0037] Variants of the disclosed sequences also include peptides or full-length proteins that contain substitutions, deletions or insertions into the protein backbone, and still retain at least about 70% homology with the original protein over the corresponding portions. Larger deviations from homology are permitted, where similar amino acids, i.e., conservative amino acid substitutions, are not counted as sequence changes. Examples of conservative substitutions include amino acids with the same or similar properties. Exemplary amino acid conservative substitutions include changes from alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, glutamine, or glutamic acid, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, valine to isoleucine, and leucine.

[0038] In another embodiment, the present invention provides a protein encoded by the nucleic acid sequence shown in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

[0039] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by a covalent chemical bond. As used herein, a polypeptide can refer to a complete amino acid sequence that encodes an entire protein or a portion thereof. A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0040] In one aspect, the amino acid sequence is selected from SEQ ID NO:6 or SEQ ID NO:7.

[0041] The nucleic acid sequences provided herein can, for example, encode a light or heavy chain of an antibody, and confer upon the encoded polypeptide a binding domain or a targeting domain to a particular target. Such polypeptides can be referred to as targeting peptides.

[0042] The term "antibody" generally refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. "Native antibodies" and "intact immunoglobulins" and the like are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0043] In a typical antibody molecule, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. Each variable region contains three segments called complementarity determining regions (CDRs) or hypervariable regions, and the more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of the heavy and light chains each contain four FR regions, mainly in a β-sheet configuration, connected by three CDRs, which form loops that connect and sometimes form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding or targeting domain of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669

[1991] ). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0044] Antibodies can be experimentally cleaved with the proteolytic enzyme papain, which cleaves each heavy chain and produces three separate antibody fragments. Two units, consisting of a light chain and a fragment of the heavy chain approximately equal in mass to the light chain, are called Fab fragments (i.e., "antigen-binding" fragments). The third unit, consisting of two equal segments of the heavy chain, is called the Fc fragment. The Fc fragment typically does not participate in antigen-antibody binding, but is important in subsequent processes involved in removing antigens from the body. As used herein, "antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab' and F(ab')2, Fc fragments or Fc fusion products, single chain Fv (scFv), disulfide-linked Fv (sdfv), as well as fragments containing either the VL or VH domains, diabodies, tribodies, and the like (Zapata et al. Protein Eng. 8(10):1057-1062

[1995] ).

[0045] Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have the hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0046] The Fc region of an antibody is the tail region of the antibody that interacts with cell surface receptors and several proteins of the complement system. This property allows the antibody to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody, whereas the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are mainly complex-type, core-fucosylated, biantennary structures. In addition, a small proportion of these N-glycans also have bisected GlcNAc and α-2,6-linked sialic acid residues.

[0047] Fc-fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins and Fc-tagged proteins) are composed of the Fc domain of IgG genetically linked to a peptide or protein of interest. Fc-fusion proteins have become valuable reagents for in vivo and in vitro research. Fc fusion binding partners can range from single peptides to ligands that activate upon binding to cell surface receptors, signaling molecules, extracellular domains of receptors that are activated upon dimerization, or as bait proteins used to identify binding partners in protein microarrays. One of the most valuable features of the Fc domain in vivo is that it can dramatically extend the plasma half-life of a protein of interest, an attribute that has led to improved therapeutic efficacy for biotherapeutics and made Fc fusion proteins attractive biotherapeutics. Fc fusion proteins can be part of a pharmaceutical composition that includes the Fc fusion protein and a pharma- ceutically acceptable carrier excipient or carrier. Pharmaceutically acceptable carriers, excipients, and stabilizers are well known in the art (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include the following: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or other suitable polysaccharides. or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0048] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight but non-covalent association. In this structure, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0049] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0050] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81

[1985] ). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries mentioned above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167

[1992] ). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93 / 16185.

[0051] In various aspects, the nucleic acid sequences provided herein encode light and heavy chains that specifically bind to PD-L1 protein.

[0052] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor that plays a key role in downregulating the immune system and promoting self-tolerance by suppressing the inflammatory activity of T cells. PD-1 is an immune checkpoint and guard against autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory inhibitory T cells). PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T and B cells upon TCR and B cell receptor signaling, whereas in resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulators. PD-1 is expressed on activated T cells, B cells, and macrophages, suggesting that PD-1 negatively regulates immune responses more broadly than CTLA-4.

[0053] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells to regulate activation or inhibition. The affinity of PD-L1 to PD-1, as defined by the dissociation constant Kd, is 770 nM. PD-L1 also has significant affinity for the costimulatory molecule CD80 (B7-1), but not for CD86 (B7-2). Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. This mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3ζ. PD-1 signaling attenuates activation of transcription factors NF-κB and AP-1, as well as PKC-θ activation loop phosphorylation (derived from TCR signaling), which is required for IL-2 production. PD-L1 binding to PD-1 also contributes to ligand-induced TCR downregulation during antigen presentation to naive T cells by inducing upregulation of the E3 ubiquitin ligase CBL-b. Upon IFN-γ stimulation, PD-L1 is expressed on T cells, NK cells, macrophages, myeloid DCs, B cells, epithelial cells, and vascular endothelial cells. The PD-L1 gene promoter region has a response element for the interferon regulatory factor IRF-1.

[0054] PD-L1 plays a major role in suppressing the adaptive arm of the immune system during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis. Under normal conditions, the adaptive immune system responds to antigens associated with immune system activation by exogenous or endogenous danger signals. Clonal expansion of antigen-specific CD8+ T cells and / or CD4+ helper cells is then propagated. Binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via the immunoreceptor tyrosine-based switch motif (ITSM). This reduces proliferation of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, inhibitory T cells), further mediated by downregulation of the Bcl-2 gene.

[0055] By overexpressing PD-L1 (either constitutively or by inducing its expression) and / or inhibiting PD-L1 degradation, cancer cells develop immune tolerance mechanisms in response to endogenous immune anti-tumor activity and evade anti-tumor immunity. PD-L1 is overexpressed on tumor cells and on non-transformed cells in the tumor microenvironment, leading to depletion of the tumor microenvironment of cytotoxic T cells, tumor cell survival and proliferation, and cancer progression.

[0056] A "PD-L1 targeting peptide" or "PD-L1 targeting protein" is meant to refer to any peptide or polypeptide (including proteins and fusion proteins) that can specifically bind to PD-L1. A PD-L1 targeting peptide can be an antibody, antibody fragment, etc. that has specific binding to one or more target polypeptides, including PD-L1. In some embodiments, the polypeptide encodes a PD-L1 targeting peptide light chain and a heavy chain. In one embodiment, the nucleic acid sequence of SEQ ID NO: 13 can encode a PD-L1 targeting peptide light chain having the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the nucleic acid sequence of SEQ ID NO: 14 can encode a PD-L1 targeting peptide heavy chain having the amino acid sequence set forth in SEQ ID NO: 7.

[0057] In additional embodiments, the invention provides a fusion protein comprising the amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation.

[0058] The terms "fusion molecule" and "fusion protein" are used interchangeably and are meant to refer to a biologically active polypeptide with or without an additional effector molecule, usually a protein or peptide sequence covalently linked (i.e. fused) by recombinant, chemical or other suitable methods. Optionally, fusion molecules can be used at one or more sites via a peptide linker sequence. Alternatively, peptide linkers may be used to aid in the construction of fusion molecules. In particular, preferred fusion molecules are fusion proteins. In general, fusion molecules can also include conjugate molecules.

[0059] "Operably linked" to each other means that there is a direct or indirect covalent bond between the peptides that make up the fusion protein. Thus, two operably linked domains may be directly covalently linked to each other. Conversely, two operably linked domains may be connected by a mutual covalent bond to an intervening moiety (e.g., a flanking sequence). Two domains may be considered to be operably linked, for example, when they are separated by a third domain, with or without one or more intervening flanking sequences.

[0060] The method for linking two individual elements usually requires the use of a linker.The term "linker" as used herein refers to any bond, small molecule or other vehicle that allows the substrate and the active agent to target the same region, tissue or cell, for example, by physically linking the individual parts of the conjugate.The linker can be any chemical moiety that can link a compound, usually a drug, to the cell-binding agent in a stable covalent manner.

[0061] Fusion proteins provided herein can, for example, comprise the amino acid sequences set forth in SEQ ID NO:6 and 7 operably linked to each other in either orientation. For example, a fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:6 at the C-terminus of the fusion protein and the amino acid sequence set forth in SEQ ID NO:7 at the N-terminus of the fusion protein, or a fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:6 at the N-terminus of the fusion protein and the amino acid sequence set forth in SEQ ID NO:7 at the C-terminus of the fusion protein. The orientation of the amino acid sequences within the fusion protein does not alter the binding specificity of the fusion protein (i.e., a PD-L1 targeted fusion protein) for its target.

[0062] The light and heavy chains of the B7-H3 targeting peptide can be operably linked to each other in either orientation without affecting the binding specificity or sensitivity of the targeting peptide. In one aspect, the protein comprises SEQ ID NOs:6 and 7, with the C-terminus of SEQ ID NO:6 directly linked to the N-terminus of SEQ ID NO:7. In another aspect, the protein comprises SEQ ID NOs:7 and 6, with the C-terminus of SEQ ID NO:7 directly linked to the N-terminus of SEQ ID NO:6.

[0063] The fusion proteins provided herein can include additional protein domains, such as additional targeting domains to provide the fusion protein with specific binding to one or more target polypeptides. For example, the fusion protein can be a trispecific killer engager (TriKE) molecule, which includes a PD-L1 targeting peptide as a targeting domain.

[0064] NK cells are cytotoxic lymphocytes of the innate immune system that enable immune surveillance. Like cytotoxic T cells, NK cells deliver reservoirs of membrane-permeable and apoptosis-inducing granzymes and perforin granules. Unlike T cells, NK cells recognize targets without the need for antigen priming by engaging activating receptors in the absence of MHC recognition. NK cells express CD16, an activating receptor that binds the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killer activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL-15 activating functions contribute to improved cancer defense.

[0065] Therapeutically, adoptive transfer of NK cells can induce remission in patients with refractory acute myeloid leukemia (AML) when combined with, for example, lymphodepleting chemotherapy and IL-2 to stimulate NK cell survival and in vivo expansion. This therapy can be limited by lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function. NK cell-based immunotherapy can be enhanced by generating reagents that drive NK cell antigen specificity, proliferation, and / or persistence while bypassing the adverse effects of Treg inhibition.

[0066] Trispecific killer engager molecules are targeting fusion proteins that contain two domains capable of driving NK cell-mediated killing of tumor cells (e.g., CD33+ tumor cells and / or EpCAM+ tumor cells) and an intracellular NK activation domain capable of generating an NK cell-independent signal to drive NK cell proliferation and / or enhance NK cell-driven cytotoxicity against, for example, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

[0067] NK cells respond to a variety of cytokines, including IL-15, which are involved in, for example, NK cell homeostasis, proliferation, survival, activation, and / or development. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing NK cell activation while bypassing Treg inhibition of the immune response. In addition to promoting NK cell homeostasis and proliferation, IL-15 can rescue functional defects of NK cells that may arise in the post-transplant environment. IL-15 can also stimulate CD8+ T cell function, further enhancing its immunotherapeutic potential. Furthermore, based on preclinical studies, the toxicity profile of IL-15 may be more favorable than IL-2 at low doses. IL-15 plays a role in NK cell developmental homeostasis, proliferation, survival, and activation. IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and the common gamma chain (CD132). IL-15 can also activate NK cells and reverse the functional defect in NK cell engraftment after hematopoietic stem cell transplantation (HSCT).

[0068] The fusion proteins provided herein can be TriKE molecules that include one or more NK cell engager domains (e.g., CD16, CD16+CD2, CD16+DNAM, CD16+NKp46), one or more targeting domains (which target, e.g., tumor cells or virally infected cells, such as PD-L1 targeting peptides described herein), and one or more cytokine NK activation domains (e.g., IL-15, IL-12, IL-18, IL-21, or other NK cell enhancing cytokines, chemokines, and / or activating molecules), each operably linked to the other domains.

[0069] For example, a fusion protein described herein can be a TriKE molecule comprising a CD16 NK cell engager domain, such as a CD16 domain having the amino acid sequence set forth in SEQ ID NO: 2 or 23, a PD-L1 targeting fusion protein domain, such as a PD-L1 fusion protein having the amino acid sequence set forth in SEQ ID NO: 6 and 7, and an IL-15 cytokine NK activation domain, such as IL-15 having the amino acid sequence set forth in SEQ ID NO: 4, 21 or 22.

[0070] The different protein domains of a TriKE molecule can be in operable linkage to one another, for example, a linker can be used to covalently link the protein domains of a TriKE molecule to one another.

[0071] The elements of the fusion protein may be assembled and operably linked to one another using one or more linkers. The linker may be susceptible to or substantially resistant to acid-induced, photo-induced, peptidase-induced, esterase-induced, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Linkers are classified based on chemical motifs known in the art, including disulfide groups, hydrazine or peptide (cleavable), or thioester groups (non-cleavable). Linkers also include charged linkers and hydrophilic forms thereof known in the art.

[0072] Linkers suitable for fusing two or more proteins or protein domains include natural linkers and empirical linkers.Natural linkers are derived from multi-domain proteins that naturally occur between protein domains.Natural linkers can have several properties, such as length, hydrophobicity, amino acid residues, and secondary structure, that can affect fusion protein in different ways.

[0073] The study of linkers in natural multidomain proteins has led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers can be classified into three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide some degree of movement or interaction with the linked domains. They are generally composed of small non-polar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr) that provide flexibility and allow mobility of the connecting functional domains. Rigid linkers can successfully maintain a fixed distance between the domains to maintain independent functions, which can provide efficient separation of protein domains or sufficient reduction of their interference with each other. Cleavable linkers can allow the release of functional domains in vivo. By utilizing unique in vivo processes, they can be cleaved under specific conditions, such as the presence of reducing agents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, or achieve independent action / metabolism of individual domains of recombinant fusion proteins after linker cleavage.

[0074] Non-limiting examples of linkers include linkers having the amino acid sequences set forth in SEQ ID NOs: 3, 5, 16 and 17.

[0075] In one aspect, SEQ ID NO:2 or 23 and SEQ ID NO:4, 21 or 22 are linked by SEQ ID NO:3 or SEQ ID NO:16. In another aspect, SEQ ID NO:4, 17 or 18 and SEQ ID NO:6 or 7 are linked by SEQ ID NO:5 or SEQ ID NO:17. In other aspects, SEQ ID NOs:6 and 7 are in operable linkage, in either orientation.

[0076] In a further embodiment, the present invention provides a fusion protein comprising a sequence as set forth in SEQ ID NO:1 or 15, and a sequence having 90% or greater identity to SEQ ID NO:1 or 15.

[0077] In one embodiment, the invention provides a fusion protein comprising, in operably linked form, SEQ ID NO: 2 or 23; SEQ ID NO: 4, 21 or 22; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

[0078] The fusion proteins described herein can include wild-type (wt) IL-15 or mutant IL-15 cytokine NK activation domains. Mutant IL-15 can include, for example, IL-15 that includes a substitution of the N72 amino acid. Non-limiting examples of N72 substitutions include N72A and N72D mutations.

[0079] In some embodiments, SEQ ID NO: 4 has an N72 substitution. In various embodiments, the N72 mutation is N72A or N72D, and the protein is set forth in SEQ ID NO: 21 or 22, respectively.

[0080] In one embodiment, the invention provides a fusion protein comprising SEQ ID NO:23, SEQ ID NO:21 or 22, and, in either orientation, SEQ ID NO:6 and 7. In one aspect, SEQ ID NO:23 is operably linked to SEQ ID NO:21 or 22 by a linker of SEQ ID NO:3 or 16. In another aspect, SEQ ID NO:21 or 22 is operably linked to sequences 6 and 7 in either orientation by a linker of SEQ ID NO:5 or 17.

[0081] The fusion protein may comprise, in operative linkage, a camelid or human CD16 NK cell engager domain (SEQ ID NO: 2 or 23, respectively), a wt or variant IL-15 cytokine NK activation domain (SEQ ID NO: 4, 21 or 22), and a PD-L1 targeting peptide light chain and heavy chain (SEQ ID NO: 6 and 7, respectively). The CD16 NK cell engager domain may be linked to the IL-15 cytokine NK activation domain by a linker having the amino acid sequence set forth in SEQ ID NO: 3 or 16. The IL-15 cytokine NK activation domain may be linked to the PD-L1 targeting peptide by a linker having the amino acid sequence set forth in SEQ ID NO: 5 or 17. The IL-15 cytokine NK activation domain may be linked to the heavy chain (linked to the light chain) of the PD-L1 targeting peptide, or the light chain (linked to the heavy chain) of the B7-H3 targeting peptide.

[0082] For example, a fusion protein may comprise, in operative linkage, from the N-terminus to the C-terminus: SEQ ID NOs: 2, 4, 6 and 7; SEQ ID NOs: 2, 4, 7 and 6; SEQ ID NOs: 23, 21, 6 and 7, SEQ ID NOs: 23, 21, 7 and 6, SEQ ID NOs: 23, 22, 6 and 7, or SEQ ID NOs: 23, 22, 7 and 6 may include.

[0083] Specifically, the fusion protein comprises, in operable linkage, from the N-terminus to the C-terminus: SEQ ID NOs: 2, 3, 4, 5, 6 and 7; SEQ ID NOs: 2, 3, 4, 17, 6 and 7; SEQ ID NOs: 2, 16, 4, 5, 6 and 7; SEQ ID NOs: 2, 16, 4, 17, 6 and 7; SEQ ID NOs: 2, 3, 4, 5, 7 and 6; SEQ ID NOs: 2, 3, 4, 17, 7 and 6; SEQ ID NOs: 2, 16, 4, 5, 7 and 6, or SEQ ID NOs: 2, 16, 4, 17, 7 and 6 may include.

[0084] In other embodiments, the fusion protein comprises, in operable linkage, N-terminus to C-terminus: SEQ ID NOs: 23, 3, 21, 5, 6 and 7; SEQ ID NOs: 23, 3, 21, 17, 6 and 7; SEQ ID NOs: 23, 16, 21, 5, 6 and 7; SEQ ID NOs: 23, 16, 21, 17, 6 and 7; SEQ ID NOs: 23, 3, 21, 5, 7 and 6; SEQ ID NOs: 23, 3, 21, 17, 7 and 6; SEQ ID NOs: 23, 16, 21, 5, 7 and 6; SEQ ID NOs: 23, 16, 21, 17, 7 and 6; SEQ ID NOs: 23, 3, 22, 5, 6 and 7; SEQ ID NOs: 23, 3, 22, 17, 6 and 7; SEQ ID NOs: 23, 16, 22, 5, 6 and 7; SEQ ID NOs: 23, 16, 22, 17, 6 and 7; SEQ ID NOs: 23, 3, 22, 5, 7 and 6; SEQ ID NOs: 23, 3, 22, 17, 7 and 6; SEQ ID NOs: 23, 16, 22, 5, 7 and 6, or SEQ ID NOs: 23, 16, 22, 17, 7 and 6 may include.

[0085] In some embodiments, the fusion protein further comprises a half-life extension (HLE) molecule.

[0086] The circulating half-life of targeting proteins such as IgG immunoglobulins can be regulated by the affinity of the Fc region for the neonatal Fc receptor (FcRn). A second general category of effector functions includes those that operate after the immunoglobulin binds an antigen. In the case of IgG, these functions involve the participation of the complement cascade or cells that bear Fc gamma receptors (FcγR). Binding of the Fc region to FcγR triggers certain immune effects, such as endocytosis of immune complexes, phagocytosis and destruction of immunoglobulin-coated particles or microorganisms (also called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of immunoglobulin-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, modulation of immune system cell activation, and modulation of immunoglobulin production. While certain engineered binding polypeptides (e.g., antibody variants (e.g., scFv) or antibody fragments (e.g., Fab fragments)) benefit from their smaller molecular size and / or monovalency, they suffer from several disadvantages due to the absence of a functional Fc region. For example, Fab fragments lack the Fc region necessary for FcRn binding and have a short half-life in vivo because their small size causes them to be rapidly filtered from the blood by the kidneys.

[0087] Engineered targeting polypeptides such as the fusion proteins described herein may exhibit reduced binding to FcRn when compared to native binding polypeptides, and therefore have reduced serum half-life. Fc variants with improved affinity for FcRn are expected to have longer serum half-life, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered polypeptide is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-life, and such molecules are also useful, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting polypeptide has toxic side effects when present in the circulation for an extended period of time.

[0088] In some embodiments, the fusion protein further comprises a half-life extension (HLE) molecule.

[0089] The circulating half-life of targeting proteins such as IgG immunoglobulins can be regulated by the affinity of the Fc region for the neonatal Fc receptor (FcRn). A second general category of effector functions includes those that operate after the immunoglobulin binds an antigen. In the case of IgG, these functions involve the participation of the complement cascade or cells that bear Fc gamma receptors (FcγR). Binding of the Fc region to FcγR triggers certain immune effects, such as endocytosis of immune complexes, phagocytosis and destruction of immunoglobulin-coated particles or microorganisms (also called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of immunoglobulin-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, modulation of immune system cell activation, and modulation of immunoglobulin production. Certain engineered binding polypeptides (e.g., antibody variants (e.g., scFv) or antibody fragments (e.g., Fab fragments)), while benefiting from their smaller molecular size and / or monovalency, also suffer from several disadvantages due to the absence of a functional Fc region. For example, Fab fragments lack the Fc region necessary for FcRn binding and have a short half-life in vivo because their small size causes them to be rapidly filtered from the blood by the kidneys.

[0090] Engineered targeting polypeptides such as the fusion proteins described herein may exhibit reduced binding to FcRn when compared to native binding polypeptides, and therefore have reduced half-life in vivo. Fc variants with improved affinity for FcRn may have longer serum half-life, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered polypeptide is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity have shorter half-life, and such molecules are also useful, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting polypeptide has toxic side effects when present in the circulation for an extended period of time.

[0091] The fusion proteins described herein can include a half-life extension (HLE) molecule to extend their half-life in vivo upon administration to a subject.

[0092] As used herein, the term "half-life" refers to the biological half-life of a particular targeting polypeptide in vivo. Half-life can be represented by the time required for half of the amount administered to a subject to be removed from the circulation and / or other tissues of the animal. When a clearance curve of a targeting polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibration of the administered targeting polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The β-phase typically represents the catabolism of the targeting polypeptide in the intravascular space. Thus, the term half-life as used herein preferably refers to the half-life of a targeting polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

[0093] Increased half-life is generally useful in in vivo applications of immunoglobulins, particularly antibodies, and most particularly antibody fragments of small size. Approaches described in the art to achieve such an effect include fusing small bispecific antibody constructs to larger proteins, which preferably do not hinder the therapeutic effect of the protein construct. An example of such further development of bispecific T cell engagers is described in US2017 / 0218078A1, which provides a half-life extended format of bispecific T cell engaging molecules (HLE format) comprising a first domain that binds to a target cell surface antigen, a second domain that binds to an extracellular epitope of the human and / or macaque CD3 epsilon chain, and a third domain that is a specific Fc modality (HLE molecule).

[0094] As used herein, the terms "half-life extender," "HLE sequence," and the like, are meant to refer to any molecule, such as a protein or polypeptide, that can be conjugated or fused to a polypeptide of interest to increase or extend its half-life in vivo. Specifically, an HLE sequence generally comprises the Fc or scFc region of an immunoglobulin.

[0095] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of its two heavy chains. A native Fc region is a homodimer. In contrast, as used herein, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) refers to a synthetic Fc region consisting of Fc domains (or Fc portions) genetically linked (i.e., encoded in a single contiguous gene sequence) within a single polypeptide chain. Thus, a genetically fused Fc region (i.e., a scFc region) is a monomer.

[0096] The term "Fc domain" refers to that portion of a single immunoglobulin heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, given that the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain.

[0097] An scFc region as described herein comprises at least two Fc domains genetically fused via an intervening linker polypeptide (e.g., an Fc connecting peptide) between the Fc moieties. An scFc region can comprise two identical Fc moieties or can comprise two non-identical Fc moieties.

[0098] Non-limiting examples of Fc domains that can be used to prepare HLE molecules that can be incorporated into any of the fusion proteins described herein (either alone or in combination with another Fc domain via a linker polypeptide) include any of the polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 30-37.

[0099] Non-limiting examples of linker polypeptides that can be used in the preparation of scFc regions that can be used in the preparation of HLE molecules include any of the polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 38 to 39.

[0100] The HLE molecules described herein can comprise an Fc domain having amino acids comprising any one of SEQ ID NOs: 30 to 37, or an scFc region having a first Fc domain having amino acids comprising any one of SEQ ID NOs: 30 to 37 fused to a second Fc domain having amino acids comprising any one of SEQ ID NOs: 30 to 37 via a linker having amino acids comprising any one of SEQ ID NOs: 38 to 39. For example, the HLE molecule can comprise any one of SEQ ID NOs: 25 to 29.

[0101] In additional embodiments, the invention provides an isolated nucleic acid sequence encoding any of the fusion proteins described herein.

[0102] A fusion protein as described herein, for example a TriKE fusion protein comprising in operative linkage a CD16 NK cell engager domain, such as a CD16 domain having the amino acid sequence set forth in SEQ ID NO:2, a PD-L1 targeting fusion protein domain, such as PD-L1 having the amino acid sequence set forth in SEQ ID NO:6 and 7, and an IL-15 cytokine NK activation domain, such as IL-15 having the amino acid sequence set forth in SEQ ID NO:4, and as set forth in SEQ ID NO:1, may be encoded by a nucleic acid sequence. In one aspect, the sequence is SEQ ID NO:8 or 18, or a sequence having 90% or greater sequence identity thereto.

[0103] In another embodiment, the invention provides a method of treating cancer in a subject, comprising administering to the subject any of the fusion proteins described herein, thereby treating the cancer.

[0104] The term "subject" as used herein refers to any individual or patient on whom the method of the present invention is carried out. Generally, the subject is a human being, but as will be understood by those skilled in the art, the subject can be an animal. Thus, other animals are included in the definition of subject, including domestic animals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, chickens, and vertebrates such as primates (including monkeys, chimpanzees, orangutans, and gorillas).

[0105] The term "treatment" is used interchangeably herein with the term "therapy" and refers to both 1) therapeutic procedures or measures that cure, slow, reduce symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and 2) preventative / prophylactic measures. Those in need of treatment can include individuals who already have a particular medical disorder, as well as those who may eventually acquire the disorder (i.e., those in need of preventative measures).

[0106] The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", "effective amount" and the like refer to an amount of a subject compound that elicits a biological or medical response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician. Generally, the response is either an alleviation of symptoms in the patient or a desired biological outcome. Such an amount should be sufficient to treat cancer. An effective amount can be determined as described herein.

[0107] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal, ocular administration, as well as injection, inhalation, and spray. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to forms of administration other than enteral and topical administration.

[0108] The fusion proteins described herein can be formulated into pharmaceutical compositions comprising the fusion protein and a pharma- ceutically acceptable carrier. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels. Examples of excipients include, but are not limited to, anti-adhesives such as magnesium stearate, binders such as sugars and their derivatives (such as sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0109] Pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration.Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained-release preparations, lipid complexes, etc.

[0110] The methods described herein are directed to the treatment of cancer. The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth beginning at one site (primary site) with the potential to invade and metastasize to other sites (secondary sites, metastases), which distinguishes cancer (malignant tumors) from benign tumors. Virtually every organ can be affected, leading to over 100 types of cancer that can affect humans. Cancer can result from many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, "neoplasm" or "tumor," including grammatical variations thereof, means a new and abnormal growth of tissue that may be benign or cancerous. In related aspects, neoplasm refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, brain cancer, head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0111] Exemplary cancers as described by the National Cancer Institute include: acute lymphocytic leukemia, adult; acute lymphocytic leukemia, pediatric; acute myeloid leukemia, adult; adrenal cortical carcinoma; adrenal cortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma, pediatric; cerebral astrocytoma, pediatric; extrahepatic bile duct cancer; bladder cancer; bladder cancer, pediatric; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, pediatric; brain tumor, adult; brain tumor, brain stem glioma, pediatric; brain tumor, cerebellar astrocytoma, pediatric; brain tumor, brain astrocytoma / malignant glioma, pediatric; brain tumor, ependymoma, pediatric; brain tumor, medulloblastoma, pediatric; brain tumor, supratentorial primitive neuroectodermal tumor Tumor), Children;Brain Tumors, Visual Pathway and Hypothalamic Glioma, Children;Brain Tumors, Children (Other);Breast Cancer;Breast Cancer and Pregnancy;Breast Cancer, Children;Breast Cancer, Male;Bronchial Adenoma / Carcinoid, Children:Carcinoid Tumors, Children;Gastrointestinal Carcinoid Tumors;Adrenal Cortical Carcinoma;Pancreatic Islet Cell Carcinoma;Carcinoma of Unknown Primary;Primary Central Nervous System Lymphoma;Cerebellar Astrocytoma, Children;Cerebral Astrocytoma / Malignant Glioma, Children;Cervical Cancer;Childhood Cancer;Chronic Lymphocytic Leukemia;Chronic Myeloid Leukemia;Chronic Myeloproliferative Disorders;Clear Cell Sarcoma of Tendon Sheath;Colon Cancer;Colorectal cancer, children;Cutaneous T-cell lymphoma;Endometrial cancer;Ependymoma, children;Ovarian epithelial cancer;Esophageal cancer;Esophageal cancer, children;Ewing's sarcoma family of tumors;Extracranial germ cell tumors, children;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (Stomach) cancer;Gastric (stomach) cancer, children;Gastrointestinal carcinoid tumors;Extracranial germ cell tumors, children;Extragonadal germ cell tumors;Ovarian germ cell tumors;Gestational trophoblastic tumors;Glioma.Childhood brain stem;Glioma. Pediatric visual pathway and hypothalamus;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) cancer, adult (primary);Hepatocellular (liver) cancer, childhood (primary);Hodgkin's lymphoma, adult;Hodgkin's lymphoma, childhood;Hodgkin's lymphoma in pregnancy;Hypopharyngeal cancer;Hypothalamus and visual pathway glioma, childhood;Intraocular melanoma;Islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney cancer;Laryngeal cancer;Laryngeal cancer, childhood;Leukemia, acute lymphoblastic, adult;Acute lymphoblastic leukemia, childhood;Acute myeloid leukemia, adult;Acute myeloid leukemia, childhood;Leukemia, Chronic Myelogenous;Hairy Cell Leukemia;Lip and Oral Cavity Cancer;Liver Cancer, Adult (Primary);Liver Cancer, Childhood (Primary);Non-Small Cell Lung Cancer;Small Cell Lung Cancer;Lymphoblastic Leukemia, Adult Acute;Lymphoblastic Leukemia, Childhood Acute;Chronic Lymphocytic Leukemia;AIDS-Related Lymphoma;Central Nervous System Lymphoma (Primary);Cutaneous T-Cell Lymphoma;Hodgkin's Lymphoma, Adult;Hodgkin's Lymphoma;Childhood;Hodgkin's Lymphoma in Pregnancy;Non-Hodgkin's Lymphoma, Adult ;Non-Hodgkin's lymphoma, children;Non-Hodgkin's lymphoma in pregnancy;Primary central nervous system lymphoma;Waldenström's macroglobulinemia;Male breast cancer;Malignant mesothelioma, adults;Malignant mesothelioma, children;Malignant thymoma;Medulloblastoma, children;Melanoma;Intraocular melanoma;Merkel cell carcinoma;Malignant mesothelioma;Metastatic squamous cell carcinoma of the neck of unknown primary;Multiple endocrine neoplasia syndrome, children;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Chronic myeloid leukemia;Myeloid leukemia Hematologic malignancies, acute, childhood;multiple myeloma;chronic myeloproliferative disorders;nasal cavity and paranasal sinus cancer;nasopharyngeal cancer;nasopharyngeal cancer, childhood;neuroblastoma;non-Hodgkin's lymphoma, adult;non-Hodgkin's lymphoma, childhood;non-Hodgkin's lymphoma in pregnancy;non-small cell lung cancer;oral cancer, childhood;cancer of the oral cavity and lip;oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;ovarian cancer, childhood;ovarian epithelial cancer;ovarian germ cell tumor;ovarian low malignant potential tumor;pancreatic cancer;pancreatic cancer, childhood, islet cell pancreatic ;Nasal sinus and nasal cavity cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Pineal and supratentorial primitive neuroectodermal tumors, pediatric;Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuroblastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Primary liver cancer, adult;Primary liver cancer, pediatric;Prostate cancer;Rectal cancer;Kidney cancer;Renal cell (kidney) cancer;Renal cell carcinoma, pediatric;Renal pelvis and ureter transitional cell carcinoma (Renal Pelvis and Ureter,Transitional Cell Cancer);Retinoblastoma;Rhabdomyosarcoma, childhood;Salivary gland cancer;Salivary gland cancer, childhood;Sarcoma, Ewing's tumor family;Kaposi's sarcoma;Sarcoma (osteosarcoma) Malignant fibrous histiocytoma of bone;Sarcoma, rhabdomyosarcoma, childhood;Soft tissue sarcoma, adult;Soft tissue sarcoma, childhood;Sezary syndrome;Skin cancer;Skin cancer, childhood;Skin cancer (melanoma);Merkel cell skin cancer;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma, adult;Soft tissue sarcoma, pediatric;Metastatic squamous cell carcinoma of the neck, unknown primary site;Stomach (Gastric) cancer;Stomach (Gastric) cancer, pediatric;Supratentorial primitive neuroectodermal tumor, pediatric;Cutaneous T-cell lymphoma;Testicular cancer;Thymoma, pediatric;Malignant thymoma;Thyroid cancer;Thyroid cancer, pediatric;Renal pelvic transitional cell carcinoma;Gestational trophoblastic tumor, pediatric;Carcinoma of unknown primary site, pediatric;Rare childhood cancers;Ureteral pelvic transitional cell carcinoma;Urethral cancer;Uterine sarcoma;Vaginal cancer;Optical pathway and hypothalamic glioma, pediatric;Vulvar cancer;Waldenstrom's macroglobulinemia;and Wilms' tumor. ;

[0112] In one aspect, the cancer is selected from non-small lung cancer, squamous cell carcinoma of the skin, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal carcinoma, prostate cancer, cervical cancer, ovarian cancer, melanoma, brain cancer, leukemia, lymphoma, myeloma, head and neck cancer, or breast cancer.

[0113] In some embodiments, administration of the fusion proteins described herein can be combined with one or more additional therapeutic agents. The phrases "combination therapy," "combined with," and the like refer to the use of two or more drugs or treatments at the same time to increase response. The fusion proteins of the present invention and pharmaceutical compositions thereof can be used in combination with other drugs or treatments that are used to treat, for example, cancer. In particular, administration of the fusion proteins to a subject can be combined with a chemotherapeutic agent, surgery, radiation therapy, or a combination thereof. Such therapies can be administered prior to, simultaneously with, or after administration of the compositions of the present invention.

[0114] The term "chemotherapeutic agent" as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, rifapril ... Vin, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, Erbitux (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), rituximab, Synagis (palivizumab), Mylotarg (gemtuzumab ozogamicin), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (dacliximab), NeutroSpec (technetium (99mTc)-fanolesomab), tocilizumab, ProstaScint (indium-III-labeled caproate) Mabpendetide), Bexxar (tositumomab), Zevalin (britumomab tiuxetan conjugated to yttrium-90 (IDEC-Y2B8)), Xolair (omalizumab), MabThera (rituximab), ReoPro (abciximab), MabCampath (alemtuzumab), Simulect (basiliximab), LeukoScan (sulesomab), CEA-Scan (arcitumomab), Verluma (nofetumomab), Panorex (edrecolomab), alemtuzumab, CDP870, natalizumabGilotrif (afatinib), Lynparza (olaparib), Perjeta (pertuzumab), Otdivo (nivolumab), Bosulif (bosutinib), Cabometyx (cabozantinib), Ogivri (trastuzumab-dkst), Sutent (sunitinib malate), Adcetris (brentuximab vedotin), Alecensa (alectinib), C alquence (acalabrutinib), Yescarta (ciloleucel), Verzenio (abemaciclib), Keytruda (pembrolizumab), Aliqopa (copanlisib), Nerlynx (neratinib), Imfinzi (durvalumab), Darzalex (daratumumab), Tecentriq (atezolizumab), and Tarceva (erlotinib). Examples of immunotherapeutic agents include, but are not limited to, interleukins (Il-2, Il-7, Il-12), cytokines (interferon, G-CSF, imiquimod), chemokines (CCL3, CCl26, CXCL7), immunomodulatory imids (thalidomide and its analogs).

[0115] In some aspects, an immune checkpoint inhibitor is further administered to the subject.

[0116] Immune checkpoints are regulators of the immune system that are essential for self-tolerance to prevent the immune system from indiscriminately attacking cells. Immune checkpoints can be inhibitory checkpoint molecules (e.g., that promote or induce immune tolerance) or stimulatory checkpoint molecules (e.g., that promote or induce an immune response).

[0117] Throughout tumor progression, the immune system exerts strong selective pressures, leading to immune tumor editing. As a result, malignant tumors often choose immune suppression and tolerance mechanisms to avoid immune destruction. Immune checkpoint blockade inhibits T cell negative costimulation to unleash antitumor T cell responses that recognize tumor antigens. Inhibitory checkpoint molecules are therefore targets for cancer immunotherapy due to their potential use in multiple cancer types.

[0118] Immune checkpoints of inhibitory pathways are fundamental in the immune system to maintain self-tolerance and regulate immune responses. Different immune cells are present in the tumor microenvironment. The expression of immune cell ligands (by cancer cells) and immune cell ligand-receptor interactions, as well as the secretion of stimulatory growth factors, chemokines, and cytokines, are important in evading immune recognition or immobilizing effector T cells. The expression of these ligands and receptors by cancer cells provides some cancers with protection from attack by stimulating immune checkpoint targets.

[0119] Inhibitory checkpoint molecules include adenosine A2A receptor (A2AR); B7-H3 and B7-H4; B and T lymphocyte attenuator (BTLA); cytotoxic T lymphocyte-associated protein 4 (CTLA-4); indoleamine 2,3-dioxygenase (IDO); killer cell immunoglobulin-like receptor (KIR); lymphocyte activation gene-3 (LAG3); nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2); programmed cell death 1 protein (PD-1) and its ligands PD-1 ligand 1 (PD-L1) and PD-L2; sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7); SIGLEC9; T cell immunoglobulin domain and mucin domain 3 (TIM-3); and V domain Ig suppressor of T cell activation (VISTA).

[0120] The adenosine A2A receptor (A2AR) is a critical checkpoint in cancer therapy because adenosine in the immune microenvironment leads to activation of the A2a receptor, inducing a negative immune feedback loop, and the tumor microenvironment has a relatively high concentration of adenosine.

[0121] B7 homolog 3 (B7-H3), also known as cluster of differentiation 276 (CD276), is a human protein encoded by the CD276 gene. The B7-H3 protein is a 316 amino acid long type I transmembrane protein that exists in two isoform states determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans it consists of one pair (2Ig-B7-H3) or two identical pairs (4Ig-B7-H3) due to exon duplication. B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein has very limited expression on normal tissues due to post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed at high frequency in many different cancer types (60% of all cancers). In non-malignant tissues, B7-H3 has a primarily inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. In malignant tissues, B7-H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7-H3 also has non-immunological pro-tumorigenic functions, such as influencing migration, invasion, angiogenesis, chemoresistance, epithelial to mesenchymal transition, and tumor cell metabolism. Due to its selective expression in solid tumors and its pro-tumorigenic functions, B7H3 is the target of several anti-cancer drugs, including enoblituzumab, ombultamab, MGD009, MGC018, DS-7300a, and CAR T cells.

[0122] B7-H4, also called VTCN1 (V-set domain-containing T-cell activation inhibitor 1), belongs to the B7 family of costimulatory proteins. B7-H4 is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion by interacting with ligands expressed by T lymphocytes.

[0123] B and T lymphocyte attenuation factor (BTLA), also known as CD272, is a surface protein whose expression is induced during T cell activation and remains on Th1 cells but not on Th2 cells. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from naive to effector cell phenotype, but tumor-specific human CD8+ T cells express high levels of BTLA. Similar to programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4), BTLA activates inhibitory pathways and regulates T cell activation. However, unlike PD-1 and CTLA-4, BTLA exerts T cell inhibition through interaction with tumor necrosis family receptor (TNF-R) rather than the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses.

[0124] CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is only increased in conventional T cells after activation - a notable phenomenon, especially in cancer. CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 binds CD80 and CD86 with greater affinity and avidity than CD28, and is therefore able to outcompete CD28 for its ligand. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA4 is also found on regulatory T cells, where it contributes to their suppressive function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4.

[0125] Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. IDO is an immunomodulatory enzyme produced by alternatively activated macrophages and other immunomodulatory cells. IDO is an immune checkpoint molecule since it suppresses T cells and NK cells, generates Tregs and myeloid-derived suppressor cells, and supports angiogenesis. IDO allows tumor cells to escape the immune system by two main mechanisms. The first mechanism is based on tryptophan depletion from the tumor microenvironment, which leads to immunosuppression. The second mechanism is based on the production of a catabolic product called kynurenine, which is cytotoxic to T lymphocytes and NK cells. Overexpression of human IDO (hIDO) has been described for various human tumor cell lines and is often associated with poor prognosis. Tumors with increased production of IDO include prostate, ovarian, lung, or pancreatic cancer, or acute myeloid leukemia.

[0126] Killer cell immunoglobulin-like receptors (KIRs) are a family of type I transmembrane glycoproteins expressed on the plasma membrane of NK cells and a small number of T cells. KIRs regulate the killing function of these cells by interacting with major histocompatibility (MHC) class I molecules expressed on all nucleated cell types. KIR receptors can distinguish between major histocompatibility (MHC) class I allelic variants, allowing them to detect virally infected or transformed cells. Most KIRs are inhibitory, meaning that recognition of MHC molecules suppresses the cytotoxic activity of NK cells.

[0127] Lymphocyte activation gene-3 (LAG3), also known as CD223, is a cell surface molecule with diverse biological effects on T cell function. The primary ligand for LAG3 is MHC class II, to which it binds with higher affinity than CD4. The protein has been reported to negatively regulate T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1, and to play a role in Treg suppressive function. LAG3 also helps maintain CD8+ T cells in a tolerogenic state and, in conjunction with PD-1, helps maintain CD8 exhaustion during chronic viral infections. LAG3 is known to be involved in dendritic cell maturation and activation.

[0128] Nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2, also known as cytochrome b(558) subunit beta or cytochrome b-245 heavy chain, is an enzyme in myeloid cells that generates immunosuppressive reactive oxygen species. Genetic and pharmacological inhibition of NOX2 in myeloid cells improves the antitumor function of nearby NK and T cells and induces autoimmunity in humans and experimental animals.

[0129] The programmed death 1 receptor or (PD-1) is an immune checkpoint that guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 signaling depends on the interaction with one of its two ligands, PD-L1 and PD-L2. The advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.

[0130] T-cell immunoglobulin and mucin domain 3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a cell surface molecule expressed on IFNγ producing CD4+Th1 and CD8+Tc1 cells, Th17 cells, regulatory T cells, and innate immune cells (dendritic cells, NK cells, monocytes). TIM-3 functions as a negative regulator of Th1 / Tc1 function by inducing cell death upon interaction with its ligand, galectin-9.

[0131] TIM-3 is an immune checkpoint that, together with other inhibitory receptors including PD-1 and LAG3, mediates the exhaustion of CD8+ T cells. TIM-3 has also been shown to regulate macrophage activation and as a CD4+ Th1-specific cell surface protein that enhances the severity of experimental autoimmune encephalomyelitis in mice. Expression of TIM-3 is upregulated in tumor-infiltrating lymphocytes in lung, gastric, head and neck cancer, schwannoma, melanoma, and follicular B-cell non-Hodgkin's lymphoma.

[0132] V-domain Ig suppressor of T-cell activation (VISTA) is a type I transmembrane protein that functions as an immune checkpoint. VISTA is produced at high levels in tumor-infiltrating lymphocytes, such as myeloid-derived suppressor cells and regulatory T cells, and its blockade with antibodies results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. Because VISTA is expressed primarily on hematopoietic cells, the consistent expression of VISTA on leukocytes within tumors means that VISTA blockade may be effective across a broad range of solid tumors.

[0133] Sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7), also known as CD328 and SIGLEC9 (also known as CD329), is a protein found on the surface of various immune cells, including natural killer cells and macrophages (SIGLEC7), as well as neutrophils, macrophages, dendritic cells, and activated T cells (SIGLEC9). SIGLEC7 and 9 suppress the immune function of these cells by binding to terminal sialic acids on glycans that cover the surface of the cells.

[0134] "Immune checkpoint inhibitors" or "checkpoint inhibitor therapy" is a form of cancer treatment that uses immune checkpoints to affect the function of the immune system. Immune checkpoints can be stimulatory or inhibitory. Tumors can use these checkpoints to defend themselves against immune system attack. Checkpoint therapy can block inhibitory checkpoints and restore immune system function.

[0135] In various aspects, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, an A2AR inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, BTLA, an IDO inhibitor, a KIR inhibitor, a LAG3 inhibitor, a NOX2 inhibitor, a SIGLEC7 inhibitor, a SIGLEC9 inhibitor, a TIM-3 inhibitor, and a VISTA inhibitor.

[0136] Currently, several checkpoint inhibitors are used to treat cancer. PD-1 inhibitors include pembrolizumab (Keytruda) and nivolumab (Opdivo). PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). CTLA-4 inhibitors include iprimumab (Yervoy). Several other checkpoint inhibitors are being developed, including anti-B7-H3 antibody (MGA271), anti-KIR antibody (lirilumab), and anti-LAG3 antibody (BMS-986016).

[0137] array: TIFF2024534467000001.tif211146TIFF2024534467000002.tif195146TIFF2024534467000003.tif213146TIFF2024534467 000004.tif224146TIFF2024534467000005.tif206145TIFF2024534467000006.tif202145TIFF2024534467000007.tif63144

[0138] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. An isolated nucleic acid sequence set forth in SEQ ID NO: 13 or 14, or a sequence having 90% identity thereto.

2. A protein encoded by the nucleic acid sequence of claim 1.

3. The protein of claim 2, wherein the amino acid sequence is selected from SEQ ID NO: 6 or 7.

4. The amino acid sequences set forth in SEQ ID NOs: 6 and 7 operably linked to each other in either orientation A fusion protein comprising:

5. The fusion protein of claim 4, wherein the protein comprises a) SEQ ID NOs: 6 and 7, with the C-terminus of SEQ ID NO: 6 directly linked to the N-terminus of SEQ ID NO: 7, or b) SEQ ID NOs: 7 and 6, with the C-terminus of SEQ ID NO: 7 directly linked to the N-terminus of SEQ ID NO:

6.

6. A fusion protein comprising a sequence set forth in SEQ ID NO: 1 or 15, and a sequence having 90% or more identity to SEQ ID NO: 1 or 15.

7. A fusion protein comprising, in operable linkage, SEQ ID NO: 2 or 23; SEQ ID NO: 4, 21 or 22; SEQ ID NO: 6 and 7, or SEQ ID NO: 7 and 6.

8. a) SEQ ID NO: 2 or 23 and SEQ ID NO: 4, 21 or 22 are linked by SEQ ID NO: 3 or SEQ ID NO: 16; b) SEQ ID NO: 4, 17 or 18 and SEQ ID NO: 6 or 7 are linked by SEQ ID NO: 5 or SEQ ID NO: 17, and / or c) SEQ ID NOs: 6 and 7 are operably linked in either orientation; The fusion protein of claim 7.

9. The fusion protein of claim 7 further comprising a half-life extension (HLE) molecule.

10. 10. The fusion protein of claim 9, wherein the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs: 25-29.

11. SEQ ID NO:4 has an N72 substitution; The fusion protein of claim 7, wherein the N72 mutation is N72A or N72D.

12. The fusion protein of claim 11, wherein the protein is set forth in SEQ ID NO: 21 or 22.

13. An isolated nucleic acid sequence encoding the fusion protein of claim 6.

14. 14. The isolated nucleic acid sequence of claim 13, which is SEQ ID NO: 8 or SEQ ID NO:

18.

15. 10. The fusion protein of claim 6, for use in a method for treating cancer in a subject, the method comprising administering the fusion protein to the subject, thereby treating the cancer.

16. a) the method further comprises administering to the subject an immune checkpoint inhibitor; wherein the immune checkpoint inhibitor is a programmed cell death 1 protein (PD-1) inhibitor, a PD-1 ligand 1 (PD-L1) inhibitor, a PDD-L2 inhibitor, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, an adenosine A2A receptor (A2AR) inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a B and T lymphocyte attenuation factor (BTLA) inhibitor, an indoleamine 2,3-dioxygenase (IDO) inhibitor, or a killer cell immunoglobulin-like receptor (KCR) inhibitor. (KIR) inhibitors, lymphocyte activation gene-3 (LAG3) inhibitors, nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2) inhibitors, sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7) inhibitors, SIGLEC9 inhibitors, T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitors, and T-cell V-domain Ig suppressor of activation (VISTA) inhibitors; and / or b) the cancer is selected from non-small lung cancer, squamous cell carcinoma of the skin, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal carcinoma, prostate cancer, cervical cancer, ovarian cancer, melanoma, brain cancer, leukemia, lymphoma, myeloma, head and neck cancer, or breast cancer; The fusion protein of claim 15.

17. A fusion protein comprising SEQ ID NO:23, SEQ ID NO:21 or 22, and SEQ ID NO:6 and 7 in either orientation.

18. a) SEQ ID NO: 23 is operably linked to SEQ ID NO: 21 or 22 by a linker of SEQ ID NO: 3 or 16; b) SEQ ID NO: 21 or 22 is operably linked to SEQ ID NO: 6 and 7 in either orientation by a linker of SEQ ID NO: 5 or 17; and / or c) further comprising a half-life extension (HLE) molecule; 18. The fusion protein of claim 17.

19. 19. The fusion protein of claim 18, wherein the HLE molecule is an Fc or scFc antibody fragment comprising any one of SEQ ID NOs: 25-29.