Immunomodulating therapeutics

Abstract

Compounds for the prevention and treatment of cancer having one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide and one or more binding peptides from a T cell or NK cell activator, operatively linked to a second immunoglobulin G (IgG) antibody peptide.

Description

IMMUNOMODULATING THERAPEUTICSFIELD OF THE INVENTION

[0001] The invention relates to compounds for the prevention and treatment of cancer.CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims priority from US provisional application no. 63 / 777,297 filed March 25, 2025, the entire contents of which are incorporated herein by reference.CROSS-REFERENCE TO RELATED DOCUMENTS

[0003] This application is filed concurrently with a sequence listing named SEQUENCE LISTING 96717479 encoded to meet the WIPO standards.BACKGROUND

[0004] Angiogenesis is a process wherein new blood vessels are grown to deliver more nutrients. This natural process can be co-opted in many diseases including solid tumor cancers to feed the growth of a tumor. As a result, angiogenesis is an important target for treatment of solid tumors.

[0005] Cancer is a leading cause of death worldwide, second only to cardiovascular disease. For the most part, while leukemias (blood bom cancers) and melanoma (skin cancers) are well served by current treatments, solid tumors have been less well served. As such, the rate of death from solid tumors are a major concern for oncologists and represent a major unmet medical need. Angiogenesis and metastasis are two fundamental processes that are important with respect to solid tumors.

[0006] Cancer metastasis is the major cause of cancer morbidity and mortality and may account for up to 90% of cancer deaths. Although cancer survival rate has been significantly improved over the years, the improvement is primarily due to early diagnosis and cancer growth inhibition. Limited progress has been made in the treatment of cancer metastasis. Current treatments for cancer metastasis are mainly chemotherapy and radiotherapy, though the new generation anti-cancer drugs (predominantly neutralizing antibodies for growth factors and small molecule kinase inhibitors) do have indirect effectson cancer metastasis in addition to their direct effects on cancer growth.

[0007] A critical event that underlies metastasis is the proteolytic degradation of the extracellular matrix (ECM) to promote tumor cell invasion, migration, and homing to distant organs. Urokinase-type plasminogen activator receptor (uPAR) system is a central player in mediating proteolysis during cancer invasion and metastasis. The uPA-mediated degradation of the ECM is also crucial for the initiation of angiogenesis. Antagonists of uPAR can help reduce both angiogenesis and metastasis in solid tumors.

[0008] Cancers can also evade the innate and adaptive immune system. A therapeutic technique is to recruit innate immune cells, such as NK cells. Another strategy is to use adaptive immune cells, such as T-cells to kill tumor cells.

[0009] There are currently limited treatment options comprising medicaments and uses thereof that limit tumor angiogenesis, limit tumor metastasis and stimulate cancer cell lysis by immune cells for solid tumors.SUMMARY

[0010] The present invention includes compounds for the treatment of cancer.

[0011] In an embodiment of the present invention there is provided a compound comprising one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide and one or more binding peptides from a T cell or NK cell activator, operatively linked to a second immunoglobulin G (IgG) antibody peptide. The compound may have the urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist operatively linked to the one or more binding peptides from a T-cell or NK cell activator, which is operatively linked to the one or more IgG antibody peptides.

[0012] In another embodiment of the present invention there is provided a compound comprising one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide, one or more binding peptides from a T cellactivator, operatively linked to a second immunoglobulin G (IgG) antibody peptide and one or more binding peptides from an NK cell activator, operatively linked to a third immunoglobulin (IgG) antibody peptide.

[0013] The first or second IgG antibody peptide may be an Fc fusion peptide and may be a fragment of a human Fc fusion peptide. The Fc fusion peptide may be an IgG protein. The Fc fusion protein may be selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide. The first or second IgG antibody peptide may be a fragment of a human Fc fusion peptide or may be a human kappa constant light (CL) chain.

[0014] Another embodiment of the present invention provides a compound comprising a first heavy chain polypeptide and a second heavy chain polypeptide in which one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, is operatively linked to the first and / or the second heavy chain polypeptide and one or more binding peptides from a T cell or NK cell activator is operatively linked to the first and / or the second heavy chain polypeptide.

[0015] Another embodiment of the present invention provides a compound comprising a first light chain polypeptide, a second light chain polypeptide and a first heavy chain polypeptide and a second heavy chain polypeptide in which, one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to one or more of the light or heavy chain polypeptides and one or more binding peptides from a T cell or NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides.

[0016] Another embodiment of the present invention provides two light chain polypeptides and two heavy chain polypeptides, one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to one or more of the light or heavy chain polypeptides, one or more binding peptides from a T cell activator, operatively linked to one or more of the light or heavy chain polypeptides and one or more binding peptides from an NK cell activator, operatively linked to the one or more of the light or heavy chain polypeptides.

[0017] Another embodiment of the present invention provides a compound comprising a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide and a second heavy chain polypeptide in which one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to the one or more of the light or heavy chain polypeptides, one or more binding peptides from a T cell activator is operatively linked to the one or more of the light or heavy chain polypeptides and one or more binding peptides from an NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides.

[0018] Another embodiment of the present invention provides a compound comprising an antibody capable of binding uPAR, T cells and NK cells and having activity of a uPAR antagonist and a T-cell and NK cell activator.

[0019] The binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist may be an amino terminal fragment (ATF) of uPAR having uPAR binding activity.

[0020] The binding peptide from a T cell activator may be an anti-CD3 single variable domain on a heavy chain (VHH) antibody.

[0021] The binding peptide from a NK-cell activator may be an anti-CD16 VHH antibody, IL-15 or ULBP2.

[0022] The heavy chain polypeptide may be a constant heavy chain peptide of human lgG1. One The first heavy chain polypeptide of the antibody may be a human lgG1 modified with a T366S L368A Y407V hole and the other second heavy chain polypeptide may be a human IgG 1 modified with a T366W knob.

[0023] The heavy chain polypeptide may both be Fc fusion peptides and may be fragments of a human Fc fusion peptide. The Fc fusion peptide may be an IgG protein. The Fc fusion protein may be selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide. The first or second IgG antibody peptide may be a fragment of a human Fc fusion peptide or may be a human kappa constant light (CL) chain.

[0024] The light chain polypeptide may be a human kappa constant light (CL) chain.

[0025] The compound may have a linker peptide between the binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist and the first IgG antibody peptide or the one or more heavy or light chain polypeptides, respectively.

[0026] The compound of the present invention may have the first heavy chain polypeptide coded by a first heavy chain sequence and the second heavy chain polypeptide coded by a second heavy chain sequence selected from a group of first heavy chain sequence and second heavy chain sequence combinations consisting of SEQ ID No. 12 and SEQ ID No. 13 or SEQ ID No. 10 and SEQ ID No. 11.

[0027] The compound of the present invention may have the first heavy chain polypeptide and the second heavy chain polypeptide coded by a heavy chain sequence and the first light chain polypeptide and the second light chain polypeptide coded by a light chain sequence selected from a group of heavy chain sequence and light chain sequence combinations consisting of SEQ ID No. 1 and SEQ ID No. 2 or SEQ ID No. 7 and SEQ ID No. 8.

[0028] The compound of the present invention may have the first heavy chain polypeptide coded by a first heavy chain sequence, the second heavy chain polypeptide coded by a second heavy chain sequence and the first light chain polypeptide and the second light chain polypeptide coded by a light chain sequence selected from a group of first heavy chain sequence, second heavy chain sequence and light chain sequence combinations consisting of SEQ ID No. 3, SEQ ID No. 4 and SEQ ID. No. 2; SEQ ID No.5, SEQ ID No. 4 and SEQ ID No. 6 or SEQ ID No. 14, SEQ ID No. 10 and SEQ ID No. 2.

[0029] A compound of the present invention may comprise a sequence consisting of SEQ ID No. 9.

[0030] A compound of the present invention may comprise a first heavy chain encoded by SEQ ID No. 14, a second heavy chain encoded SEQ ID No. 10, and a light chain encoded by SEQ ID No. 2.

[0031] A compound of the present invention may comprise a compound having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the compound of the present invention.

[0032] The compound may be a peptide coded by a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 or SEQ ID No. 14. The compound may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the peptide coded by a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No.6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, or SEQ ID No. 14.

[0033] The compound may be selected from the group consisting of ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16, CD16-Fc1 / CD3-Fc1 / ATF and, ATF-Fc1-K5-ULBP2.

[0034] The compound may comprise and ATF-Fc1-ULBP2-K5.

[0035] The compound may comprise ATF-CD16-K5-IL15T

[0036] The compound may be selected from the group consisting of CD16-IL15-ATF-Arresten and ATF-CD16-CD16-K5.

[0037] The compound may be substantially homologous to the compounds of the example embodiments and having the activity of the compound of the invention.

[0038] Also taught are one or more nucleotides encoding a compound of the invention. Also taught is a vector containing a compound of the invention.

[0039] The compounds may exhibit anti-cancer activity. Thus, also taught are methods of treating cancer comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of cancer. The cancer may be a solid cancer. The methods may inhibit metastasis. The method or use may further comprise one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Reference will now be made, by way of example, to the accompanying drawings which show example embodiments, and in which:

[0041] Figure 1A provides diagram of ATF-Fc1 / CD3-Fc1 / CD16, CD16-Fc1 / CD3-Fc1 / ATF and ATF-Fc1 / CD16, respectively.

[0042] Figure 1B provides a diagram of ATF-CD16-CD16-K5, ATF-CD16-K5-IL15 and CD16-IL15-ATF-Arresten.

[0043] Figure 2A provides a diagram of ATF-Fc-ULBP2-K5.

[0044] Figure 2B provides a diagram of ATF-Fc-K5 / ULBP2.

[0045] Figure 3 shows an SDS-PAGE analysis of ATF-Fc1 / CD16.

[0046] Figure 4 shows an SDS-PAGE analysis of ATF-Fc1 / CD3-Fc1 / CD16.

[0047] Figure 5 shows an SDS-PAGE analysis of CD16-Fc1 / CD3-Fc1 / ATF.

[0048] Figure 6 shows an SDS-PAGE analysis of ATF-Fc1 -K5-ULBP2.

[0049] Figure 7 shows an SDS-PAGE analysis of ATF-Fc1-ULBP2-K5.

[0050] Figure 8 is a graph showing normalized tumor size as effected by various constructs.

[0051] Figure 9 is a graph showing the number of metastases as effected by ATF-Fc1-K5, ATF-Fc1-K5-ULBP2 and ATF-Fc1-ULBP2-K5 constructs.

[0052] Figure 10 is a graph showing the number of metastases as effected by ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16 and CD16-Fc1 / CD3-Fc1 / ATF.

[0053] Figure 11 is a graph showing the number of metastases as effected by various constructs.

[0054] Figure 12 provides a diagram of ATF-CD16-K5-IL15.

[0055] Figure 13 shows an SDS-PAGE analysis of ATF-CD16-K5-IL15.

[0056] Figure 14 provides a diagram of CD16-IL15-ATF-Arresten.

[0057] Figure 15 shows an SDS-PAGE analysis of CD16-IL15-ATF-Arresten.

[0058] Figure 16 provides a diagram of ATF-CD16-CD16-K5.

[0059] Figure 17 shows an SDS-PAGE analysis of ATF-CD16-CD16-K5.DETAILED DESCRIPTION

[0060] Throughout the following description specific details are set forth to provide a more thorough understanding to persons skilled in the art. However, well-known elements may not have been shown or described in detail. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0061] The present invention relates to the combination of a urokinase-type plasminogen receptor (uPAR) antagonist with immune activating motifs in the same compound.

[0062] The present invention uses T-cell or NK-cell activator sequences in combination with uPA-uPAR interaction inhibitors to improve upon the anti-cancer and anti-angiogenic activity of molecules simply inhibiting uPAR activity. The present invention also relates to both T-cell and NK-cell activator sequences in combination with uPA-uPAR interaction inhibitors to improve upon the anti-cancer and anti-angiogenic activity of molecules simply inhibiting uPAR activity.

[0063] The present invention also uses the fifth kringle domains of plasminogen fused to the C-terminus of a compound or antibody to enhance the anti-angiogenic effects of the uPAR antagonist and T-cell and / or NK-cell activator compounds.

[0064] In an embodiment of the present invention there is provided a compound comprising one or more binding peptides from uPAR antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide and one or more binding peptides from a T cell or NK cell activator, operatively linked to a second IgG antibody peptide. An example embodiment may comprise binding peptides of a uPAR antagonist, a T-cell activator and an NK cell activator. The compound may have the uPAR antagonist having activity of a uPAR antagonist operatively linked to the one or more binding peptides from a T-cell or NK cell activator, which is operatively linked to the one or IgG antibody peptides.

[0065] Another example embodiment includes a compound comprising one or more uPAR antagonists having activity of a uPAR antagonist, operatively linked to a first IgG antibody peptide, one or more binding peptides from a T cell activator operatively linked to a IgG antibody peptide and one or more binding peptides from an NK cell activator, operatively linked to a third IgG antibody peptide.

[0066] The first or second IgG antibody peptide may be an Fc fusion peptide and may be a fragment of a human Fc fusion peptide. The Fc fusion peptide may be an IgG protein or the Fc fusion protein may be selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide. The first or second IgG antibody peptide may be a fragment of a human Fc fusion peptide, a heavy chain constant region (CH) of human IgG 1 or may be a human kappa constant light (CL) chain.

[0067] The isotype of the Fc domain (i.e. IgG 1, 2, 3 or 4) can be selected pending a preference of improved half-life and / or ADCC and complement activity is desired. For example, in some situations, an IgG 1 isotype may be preferred to increase the anti-cancer activity of the antibody by increasing ADCC. In other situations, an lgG4 isotype may be preferred for example to reduce ADCC which may allow for less toxicity and thereby permit higher doses. In some cases, an lgG3 Fc which is intermediate in ADCC activity to either lgG1 or lgG4 may be preferred. Note the Fc domain forms a natural homodimer.

[0068] Embodiments of the present invention include multi-specific antibodies that comprise two heavy chain polypeptide sequences with or without one or more light chain polypeptide sequences. An embodiment of the present invention provides a first heavy chain polypeptide and a second heavy chain polypeptide in which one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to the first and / or the second heavy chain polypeptide and one or more binding peptides from a T cell or NK cell activator is operatively linked to the first and / or the second heavy chain polypeptide. Another embodiment of the present invention provides a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide and a second heavy chain polypeptide in which, one or more binding peptides from a urokinase-type plasminogenreceptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to one or more of the light or heavy chain polypeptides and one or more binding peptides from a T cell or NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides.

[0069] Another embodiment of the present invention provides a first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide and a second heavy chain polypeptide in which one or more binding peptides from a urokinasetype plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to the one or more of the light or heavy chain polypeptides, one or more binding peptides from a T cell activator is operatively linked to the one or more of the light or heavy chain polypeptides and one or more binding peptides from an NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides. An embodiment of the present invention may provide a compound comprising an antibody capable of binding uPAR, T cells and NK cells and having activity of a uPAR antagonist and a T-cell and NK cell activator.

[0070] There are known methods in the art to develop bispecific antibodies including the use of knobs-in-holes technology. Knobs-in-holes technology uses mutated constant heavy chain 3 (CH3) domains of the Fc region to promote the formation of stable heterodimers. These mutations include, a first heavy chain specific to a first antigen with a hole created by the mutations T366'S:L368'A:Y407'V in the CH3 domain and a second heavy chain with a second antigen specificity that has a knob created by a mutation in the CH3 domain of T366W. (Atwell et.al. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. JMB Volume 270, Issue 1, 4 July 1997, 26-35). The mutations allow the two heavy chains with different antigen specificities to assemble due to the association of the knob of the second heavy chain into the hole of the first heavy chain. (Von Kreudenstein et.al. Improving biophysical properties of a bispecific antibody scaffold to aid developability. mAbs 5:5, 646-654 (2013))

[0071] The first and second heavy chain polypeptides of some embodiments of the invention may be heavy chain polypeptides comprising a constant heavy chain peptide of human lgG1. The first heavy chain polypeptide may be a human lgG1 modified with aT366S L368A Y407V hole and the second heavy chain polypeptide may be a human IgG 1 modified with a T366W knob.

[0072] The light chain polypeptides of the antibody embodiments may be a human kappa constant light (CL) chain.

[0073] Another embodiment of the present invention provides a compound comprising an antibody capable of binding uPAR, T cells and NK cells and having activity of a uPAR antagonist and a T-cell and NK cell activator.

[0074] Some proteins used in this invention are entirely endogenous sequences, (specifically ATF-Fc1-K5-ULBP2 and ATF-Fc1-ULBP2-K5) thus there is little chance of unwanted immunogenicity against the therapeutics, which means the proteins should be able to be administered repeatedly, even as an mRNA.

[0075] Accordingly, the present invention also provides DNA, RNA and mRNA treatments. DNA may be administered, reach the damaged cells, enter the cell and express the proteins of the invention. Multiple delivery techniques can be used. For example, DNA may be entered into an engineered virus to deliver the DNA into a chromosome. Naked DNA approaches can also be used. Alternatively, one can administer a gene that causes a needed protein to be expressed.

[0076] In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease.

[0077] In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations.

[0078] In in vivo gene therapy, a vector (typically, a virus) is introduced to the patient, which then achieves the desired biological effect by passing the genetic material (e.g. for the therapeutic protein) into the patient's cells. In ex vivo gene therapies, such as CAR-T therapeutics, the patient's own cells (autologous) or healthy donor cells (allogeneic) aremodified outside the body (hence, ex vivo) using a vector to express a particular protein, such as a chimeric antigen receptor.

[0079] One of the major events that underlie metastasis is the proteolytic degradation of the extracellular matrix (ECM) to promote tumor cell invasion, migration, and homing to distant organs. Even though several protease systems are implicated in this process, a large body of evidence identified the urokinase-type plasminogen activator receptor (uPAR) system as a central player in mediating proteolysis during cancer invasion and metastasis. The uPA-mediated degradation of the ECM is also crucial for the initiation of angiogenesis.

[0080] uPAR is a is a GPI-anchored cell membrane receptor that localizes urokinase (uPA) proteolytic activity on the cell surface. Its main function is focusing of urokinase (uPA) proteolytic activity, responsible for degradation of ECM components, on the cell surface. uPAR expression is increased in many human cancers and correlates with a poor prognosis and early invasion and metastasis and has been reported on as many as 80% of tumors. At some point, the uPA-UPAR complex is often internalized to break apart the complex, and unoccupied UPAR may be recycled to the surface.

[0081] There are two broad mechanisms to inhibit the function of UPAR; (1) binding of uPA with uPAR, and (2) preventing the cell signaling that can occur by UPAR which involves both uPA binding and its subsequent binding with co-receptors such as integrins.

[0082] The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby cause tumor cell migration from the primary site of origin to a distant secondary organ.

[0083] uPA is often separated into two regions. The first region which is responsible for binding of uPA to UPAR is the amino terminal fragment (ATF). The ATF can be further divided into two domains: an epidermal growth factor (EGF)-like domain and a kringle domain. The “G” domain is the EGF-like region and the “K” is the kringle domain. The majority of the binding occurs with just the EGF-like domain alone, but the entire ATF region including both the EGF-like domain and the kringle domain does have even greater binding the just the EGF-like domain alone.

[0084] An ATF-Fc fusion (Chinese patent No. CN101050237) has been shown to reduce tumor growth and metastases (HuXW, Duan HF, Gao LH, etal. Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor. Cancer Biol Ther. 2008;7(5):651-659. doi:10.4161 / cbt.7.5.5643). Further, an ATF conjugated to the Fc fragment of human immunoglobulin G1 in combination with trastuzumab, a drug that targets the HER2 oncogene, synergistically inhibited HER2-positive breast cancer growth and metastasis in a mouse xenograft model. (Zhou H, Wang H, Yu G, Wang Z, Zheng X, Duan H and Sun J. Synergistic inhibitory effects of an engineered antibody-like molecule ATF-Fc and trastuzumab on tumor growth and invasion in a human breast cancer xenograft mouse model. Oncology Letters 14:5189-5196).

[0085] In the present invention, the binding peptide from a uPAR antagonist having activity of a uPAR antagonist may be an ATF of uPAR having uPAR binding activity.

[0086] An antibody (2G10) was identified from a phage display library that prevented uPA association with uPAR, been reported to prevent uPAR and uPA binding(Duriseti S, Goetz DH, Hostetter DR, LeBeau AM, Wei Y, Craik CS. Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration. J Biol Chem. 2010;285(35):26878-26888. doi:10.1074 / jbc. M109.077677). Non-natural smaller peptides which inhibit the uPA / uPAR interaction have been identified from combinatorial libraries as well.

[0087] There are reports of combining inhibitors of uPA-uPAR (e.g. ATF) with inhibitors such as protease inhibitor domains as well as the anti-angiogenic functional domains of vasostatin. Similarly, there have been reports of Endostatin (P125A) fused to the C-terminus of other antibodies such as Herceptin. The present Applicant’s prior publication WO2023168531 A1 discloses compounds that combined a uPAR antagonist with additional anti-angiogenic activity conferred by endostatin and / or plasminogen derived sequences in the same Fc fusion protein. These compounds reduced tumor formation and metastatic activity in a zebrafish xenograft model. However, there is a need in the art to combine the anti-angiogenic and anti-metastatic activity of uPAR with immune activators to enhance anti-cancer activity.

[0088] Cancer immunotherapy treatments activate the immune system to recognize and selectively kill cancer cells. (Waldman AD, Fritz JM and Lernardo MJ A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nature Reviews Immunology volume 20, 651–668 (2020)) For example, adoptive T cell therapy comprises administering autologous or allogenic T cells to patients with cancer. The National Cancer Center began using ATC therapy with tumor-infiltrating lymphocytes from melanomas at least as early as 1988 (Rosenberg S et.al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 1988 Dec 22;319(25):1676-80). T-cells can also be engineered to express chimeric antigen receptors (CARs), which specifically target tumor cell antigens, to kill cancer cells. The FDA approved CAR T-cell therapy to treat adults with certain types of large B-cell lymphoma in 2017. (Labanieh L, Majzner RG and Mackall CL Programming CAR-T cells to kill cancer. Nature Biomedical Engineering volume 2, 377–391 (2018)). Another promising cancer immunotherapy uses NK cells to attack tumor cells. NK cells are innate immune cells that can directly kill cancer cells, release cytokines to directly lyse cancer cells, and / or activate T-cell adaptive anti-tumor responses. (Chu J, Gao F, Yan M, Zhao S, Yan Z, Shi B & Liu Y Natural killer cells: a promising immunotherapy for cancer. Journal of Translational Medicine volume 20, Article number: 240 (2022)).

[0089] Antibodies have been developed that engage T-cells and cancer cells. These antibodies bind both cancer cell antigens and a T-cell receptor on T cells. This simultaneous binding triggers cytolytic synapse formation and tumor cell lysis. For example, Tri specific T-cell Activating Constructs (TriTACs), have a tumor antigen binding domain, a human serum albumin binding domain (ABD) and a T-cell binding domain comprising T-cell receptor subunit CD3. The tumor antigen binding domain and the ABD are comprised of humanized single-domain antibodies (VHHs) from the variable regions of camelid heavy-chain–only antibodies. In this study, the configuration anti-tumor target domain, anti-human serum albumin (ABD) and anti-CD3 domain had the most potent T-cell dependent cellular cytotoxicity. TriTAC constructs inhibited tumor growth in a mouse model. (Austin R et.al. TriTACs, a Novel Class of T-Cell–Engaging Protein Constructs Designed for the Treatment of Solid Tumors Mol Cancer Ther (2021 ) 20 (1 ): 109–120 and US9708412B2 and US2016340444A1) Similar constructs have been developed withdomains to recognize innate immune cells, ABD and a domain to recognize a target antigen (US2019225702A1). CD3-targeting bispecific antibodies kill cancer cells through binding the CD3 receptor on T-cells, which activates the T-cell independently of MHC, and an antigen on tumor cells bring the activated T cell and tumour cell in close proximity. Blinatumomab, a CD3-targeting bispecific antibody, is approved by the FDA to treat B-cell precursor acute lymphoblastic leukemia. (Benonisson et.al. CD3-Bispecific Antibody Therapy Turns Solid Tumors into Inflammatory Sites but Does Not Install Protective Memory. Mol Cancer Ther (2019) 18 (2): 312–322.).

[0090] The binding peptide from a T cell activator in a compound of the present invention may be an anti-CD3 a single variable domain on a heavy chain (VHH) antibody.

[0091] NK cells can be activated by CD16, IL15 and ULBP2. CD16 is an NK cell activating receptor that binds the Fc domain of IgGs and drives antibody-dependent cell-mediated cytotoxicity (ADCC). After activation, NK cells release cytotoxic granules that directly lyse tumor cells. (Meng et.al. Leveraging CD16 fusion receptors to remodel the immune response for enhancing anti-tumor immunotherapy in iPSC-derived NK cells. Journal of Hematology & Oncology volume 16, Article number: 62 (2023)). Bispecific antibodies with a first domain of CD16 vhh and a second binding domain of a vhh to a tumor antigen have been used to reduce tumor growth in a mouse model (CN112679609A). CD16-mediated activation of NK cells is a potent signal for inducing ADCC when using these antibodies.

[0092] IL-15 is a cytokine that activates NK cells. IL-15 therapies have been tested in several clinical trials in solid cancer, hematologic malignancies and metastasis. (Chu J, Gao F, Yan M, Zhao S, Yan Z, Shi B & Liu Y Natural killer cells: a promising immunotherapy for cancer. Journal of Translational Medicine volume 20, Article number: 240 (2022)). Finally, ULBP2 is a ligand of the NKG2D receptor which is an activating receptor for innate immune responses. In mice, ectopic expression of ULBP2 exhibits an anti-tumor response by recruiting NK cells and T-cells to the tumor. Further, IL15 may help to enhance the immune response against ULBP-expressing tumors by preventing the downregulation of NKG2D. (Sutherland s, et.al. ULBPs, human ligands ofthe NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood 108:4, 1313-1319(2006)). The IL15 N72D mutant exhibits superagonist activity which is preserved when linked to a single-chain T-cell receptor domain to generate a tumor-specific fusion protein. (Zhu et.al. Novel Human Interleukin-15 Agonists J. Immunol 183:6, 3598 (2009)).

[0093] The binding peptide from a NK-cell activator in a compound of the present invention may be an anti-CD16 VHH antibody, IL-15 or ULBP2.

[0094] Single domain antibody fragments (VHH) comprise an antigen binding domain of the heavy chain without associated light chains. The first known VHH was isolated from camelids (Hamers-Casterman et al., Naturally occurring antibodies devoid of light chains. Nature 363:446-8 (1993)). CD3-specific VHH antibody sequences were disclosed in US 2016 / 0280795 A1. CD16-specific VHH antibody sequences were disclosed in CN112679609A and US 9,598.499 B2.

[0095] The compound may have a linker peptide between the binding peptide from a uPAR antagonist having activity of a uPAR antagonist and the first IgG antibody peptide or the one or more heavy or light chains polypeptides, respectively.

[0096] The compound may have a linker peptide in between the one or more binding peptides from a T-cell or NK-cell activator and the first IgG antibody peptide or one or more heavy or light chains polypeptides, respectively.

[0097] The compound may further comprise a kringle domain 5 from a plasminogen operatively linked to the C terminal of one or more IgG antibody peptides or one or more heavy or light chain polypeptides, respectively.

[0098] Molecules that inhibit angiogenesis can inhibit cancer growth and metastasis. Plasminogen is an endogenous protein that can be cleaved by urokinase activated plasmin and contains five kringle (K) domains of ~80 residues each. Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen, and studies using recombinant angiostatin demonstrated tumor inhibitory activity resides in domains K1 -38. Angiostatin was originally isolated from tumor bearing mice, 6 and has both potent antiangiogenic activity and antiproliferative activity toward endothelial and cancer cells.7 Larger fragments of plasminogen that contain all 5 kringle domains (K1-5) may be even more active than angiostatin alone (K1-3) (Cao R, Wu HL, Veitonmäki N, et al.Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc Natl Acad Sci U S A. 1999;96(10):5728-5733. doi: 10.1073 / pnas.96.10.5728).

[0099] Recent evidence supports dual antitumor mechanisms for plasminogen derivatives, one affecting angiogenesis and another targeting tumor cells directly. (Sun Q, Xu Q, Dong X, et al. A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy. Int J cancer.2008; 123(4):942-950. doi: 10.1002 / I JC.23537).

[0100] Kringle 5 (K5), like angiostatin, is a byproduct of the proteolytic cleavage of plasminogen. In a recent study, Ansell et al., demonstrated K5 functions as a competitive antagonist of hepatocyte growth factor (HGF). (Cho HM, Rosenblatt JD, Kang YS, et al. Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein. Mol Cancer Ther. 2005;4(6):956-967. doi:10.1158 / 1535-7163.MCT-04-0321).

[0101] HGF contains kringle motifs and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiostatic role, K5 can direct a potent antitumor response with its ability to recruit tumor-associated neutrophils and Natural Killer T cells.

[0102] Thus plasminogen has multiple kringle domains which appear to be active alone (angiostatin (K1-4) and K5) and in combination. Thus use of plasminogen derived sequences, in particular the K5 domain with other anti-cancer proteins has been reported (Wang H, Yang Z, Gu J. Therapeutic targeting of angiogenesis with a recombinant CTT peptide-endostatin mimic-kringle 5 protein. Mol Cancer Ther 2014;13(11):2674-2687. doi:10.1158 / 1535-7163.MCT-14-0266).

[0103] Arrestins (or arrestens, abbreviated Arr) have anti-angiogenesis effects. They are c-terminal fragments of the a chain of type IV collagen. Arrestin is an angiogenesis inhibitor. Aikio et.al. demonstrated that arresten inhibited migration and tumor growth in a mouse tumor xenograft mode. This study also found a novel role for arresten in oral squamous carcinoma cell proliferation, survival, motility and invasion. (Aikio et.al. Arresten, a collagen-derived angiogenesis inhibitor, suppresses invasion ofsquamous cell carcinoma. PLoS One 2012;7(12):e51044).

[0104] The compounds may exhibit anti-cancer activity. Thus, also taught are methods of treating cancer comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of cancer. The cancer may be a solid cancer. The methods may inhibit metastasis. The method or use may further comprise one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

[0105] The compound may be selected from the group consisting of ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16, CD16-Fc1 / CD3-Fc1 / ATF and ATF-Fc1-K5-ULBP2.

[0106] The compound may comprise ATF-Fc1-ULBP2-K5.

[0107] Figure 1A depicts a diagram of ATF-Fc1 / CD3-Fc1 / CD16, CD16-Fc1 / CD3-Fc1 / ATF and ATF-Fc1 / CD16, respectively.

[0108] The compound may comprise ATF-CD16-K5-IL15.

[0109] The compound may be selected from the group consisting of CD16-IL15-ATF-Arresten and ATF-CD16-CD16-K5.

[0110] Figure 1B provides a diagram of ATF-CD16-CD16-K5, ATF-CD16-K5-IL15 and CD16-IL15-ATF-Arresten, respectively.

[0111] The terms “polypeptide”, “peptide”, and “protein” are typically used interchangeably herein to refer to a polymer of amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Each protein or polypeptide will have a unique function. The invention includes polypeptides and functional fragments thereof, as well as mutants and variants having the same biological function or activity.

[0112] In some embodiments, polymeric molecules (e.g., a polypeptide sequence or nucleic acid sequence) are considered to be “homologous” to one another if their sequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.

[0113] In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.

[0114] The term “nucleic acid fragment” as used herein refers to a nucleic acid sequence that has a deletion. In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.

[0115] The term "construct" refers to a nucleic acid sequence encoding a protein, operably operatively linked to a promoter and / or other regulatory sequences.

[0116] The term "genomic sequence" refers to a sequence having non-contiguous open reading frames, where introns interrupt the protein coding regions.

[0117] As used herein, the terms "encoding", "coding", or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid may be the requisite information to guide translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).

[0118] The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. For instance, polynucleotide sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993).

[0119] The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 80%, 85%, or at least about90%, or at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as BLAST, as discussed above.

[0120] As used herein, “heterologous nucleic acid sequence” is any sequence placed at a location in the genome where it does not normally occur.

[0121] A particular nucleic acid sequence also encompasses conservatively modified variants thereof (such as degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Thus, a nucleic acid sequence encoding a protein sequence disclosed herein also encompasses modified variants thereof as described herein. Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.

[0122] “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with coding sequences of interest to control expression of the coding sequences of interest, as well as expression control sequences that act in trans or at a distance to control expression of the coding sequence.

[0123] A “coding sequence” or “open reading frame” is a sequence of nucleotides that encodes a polypeptide or protein. The termini of the coding sequence are a start codon and a stop codon. The disclosure also includes native, isolated, or recombinant nucleic acid sequences encoding a protein, as well as vectors and / or (host) cells containing the coding sequences for the protein.

[0124] The terms " Fc domain", “Fc peptide” or " Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-term inus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to theEU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable selfassociation. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0125] Fused or linked means that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0126] Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment' a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby is intended. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein. Accordingly, the present disclosure relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences encoded thereby.

[0127] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0128] The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.ExamplesExample 1 - Constructing ATF-Fc1 / CD16

[0129] This Fc fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide (underlined) bound to ATF (italicized) bound to a constant heavy (CH) chain region of human lgG1 (bold) and the light chain sequence has a signal peptide (underlined) bound to a CD16 single variable domain on a heavy chain (VHH) antibody (italicized) bound to a human kappa constant light (CL) chain (bold). The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 1 and the light chain sequences is set out in SEQ ID. No. 2, below.

[0130] Heavy chain: ATF-Fc1MHSSALLCCLVLLTGVRAS / VELHQ VPSNCDCLNGGTC VSNKYFSNIHWCNCPKKFG GQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQL GLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0131] Light chain: CD16-CLMHSSALLCCLVLLTGVRAQ VQLQESGGGS VQ TGGSLRLSCAASGD TTSEYWGA WFR QAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYC AAARRGTNAFL THDKYGYWGQGTQVTVSSRWAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

[0132] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >71% as determined by SEC-HPLC at 280 nm. Endotoxin: < 1 EU / mg. Concentration was 1.15mg / mL. The formulation buffer is PBS.

[0133] As shown in Figure 3, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1 ) and non-reduced (Lane 2) conditions. Gel stained with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 2 - Constructing ATF-Fc1 / CD3-Fc1 / CD16

[0134] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The first heavy chain sequence has a signal peptide (underlined) bound to ATF (italicized) bound to a constant heavy (CH) chain region (bold) of human lgG1 modified with a T366S L368A Y407V hole (not-bolded), the second heavy chain sequence has a signal peptide (underlined) bound to an anti-CD3 single variable domain on a heavy chain (VHH) antibody (italicized) bound to a CH region of human IgG 1 (bold) modified with a T366W knob (not bold) and the light chain sequence has a signal peptide (underlined) bound to an anti-CD16 single variable domain on a heavy chain (VHH) antibody (italicized) bound to a human kappa constant light (CL) chain (bold). The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 3, the second heavy chain sequence is set out in SEQ ID No. 4 and the light chain sequence is set out in SEQ ID No. 2, below.

[0135] Heavy chain 1: ATF-Fc1 (mutation) MHSSALLCCLVLLTGVRAMSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCE IDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNP DNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSASTKGPSVFPLAPSSKSTSGG'TAM_G CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0136] Heavy chain 2: CD3-Fc1 MHSSALLCCL\ / LLTG\ / RAQVQLQESGGGLVQAGGSLRLSCAASGRTFSNYHMGWFRQAPGK ERELVAAISGSGGSTYYTDSVKGRFTISRNNAKNTMSLQMSNLKPEDTGVYYCTTPTEKGSSID YI / I / GQGTQVT\ / SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0137] Light chain: CD16-CL MHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFRQAPGK EREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFL THD YG YI / I / GQG TQ t / T\ / SSRTVAAPSVFI FPPSDEQLKSGTASWCLLN N FYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

[0138] Purity was > 85% as determined by reducing SDS-PAGE. Purity was >75% as determined by SEC-HPLC at 280 nm. Endotoxin: < 1 Ell / rng. Concentration was 2.33 mg / mL. The formulation buffer was PBS.

[0139] As shown in Figure 4, SDS-PAGE analysis of the construct in β-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 3 - Constructing CD16-Fc1 / CD3-Fc1 / ATF

[0140] This fusion protein has two heavy chain polypeptides and two light chainpolypeptides. The first heavy chain sequence has a signal peptide (underlined) bound to an anti-CD16 single variable domain on a heavy chain (VHH) antibody (italicized) bound to a constant heavy (CH) chain region (bold) of human lgG1 modified with a T366S L368A Y407V hole (not-bolded), the second heavy chain sequence has a signal peptide (underlined) bound to an anti-CD3 single variable domain on a heavy chain (VHH) antibody (italicized) bound to a CH region of human lgG1 (bold) modified with a T366W knob (not bold). The light chain sequence has a signal peptide bound to ATF bound to a human kappa constant light (CL) chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 5, the second heavy chain sequences is set out in SEQ ID No. 4 and the light chain sequence is set out in SEQ ID No. 6 below.

[0141] Heavy chain 1: CD16-Fc1 MHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFRQAPGK EREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFL THD YGYI / I / GQGTQVTV'SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0142] Heavy chain 2: CD3-Fc1 MHSSALLCCLVLLTGVRAQVQLQESGGGLVQAGGSLRLSCAASGRTFSNYHMGWFRQAPGK ERELVAAISGSGGSTYYTDSVKGRFTISRNNAKNTMSLQMSNLKPEDTGVYYCTTPTEKGSSID YI / I / GQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0143] Light chain: ATF MHSSALLCCLVLLTGVRAMPIFLDHILNKFWILHYARTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0144] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >66% as determined by SEC-HPLC at 280 nm. Endotoxin: < 1 Ell / rng. Concentration was 1.20 mg / mL. The formulation buffer is PBS.

[0145] As shown in Figure 5, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1 ) and non-reduced (Lane 2) conditions. Gel stained with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 4 - Constructing ATF-Fc1-K5-ULBP2

[0146] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide (underlined) bound to ATF (italics) bound to Fc1 bound to an IgG heavy chain sequence bound (bold) to K5 (italics and underlined) and the light chain sequence has a signal peptide (underlined) bound to ULBP2 (italics) bound to a human kappa constant light (CL) chain (bold). The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 7 and the light chain sequence is set out in SEQ ID No. 8 below.

[0147] Heavy chain: ATF-Fc1 -K5MEWSWVFLFFLSVTTGVHSSNELHQ VPSNCDCLNGGTC VSNKYFSNIHWCNCPKKF GGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQ LGLGKHNYCRNPDNRRRPWCYVQ VGLKPL VQECMVHDCADGSGSGSASTKGPSX / F PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEE DCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPD GDVGGPWCYTANPRKLYDYCDVPQCAA

[0148] Light chain: ULBP2M E WS WVF LF F LSVTTGVH S GRADPHSLCYDITVIPKFRPGPRWCA VQGQ VDEKTFLH YDCGNKTVTPVSPLGKKLNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQ ARMSCEQKAEGHSSGSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKWENDKV VAMSFHYFSMGDCIGWLEDFLMGMDSTLEPSAGAPLAMRTV AAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0149] Purity was > 90% as determined by reducing SDS-PAGE. Concentration was 1 mg / mL. The buffer formulation was PBS, pH 7.4. No endotoxins were detected.

[0150] As shown in Figure 6, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Sample 1, lane 2) and non-reduced (Sample 2, lane 3) conditions. Gel stained with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50-75 kDa and 45-70 kDa, respectively. Non-reduced protein migrates about 116-235 kDa.Example 5- Constructing ATF-Fc1-ULBP2-K5

[0151] This fusion protein uses ATF bound to Fc1 bound to ULBP2 bound to K5. There are linkers between Fc1 and ULBP2 and between ULBP2 and K5. The moleculewas produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The sequence is set out in SEQ ID No. 9, below.ATF-Fc1-ULBP2-K5 MEWSWVFLFFLSVTTGVHSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKF GGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQ LGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSPPCPAPELL G G P S VF L F P P KP KDTLM I S RTP E VTC VWD VS H E D P E VKF N WYVD G VE VH N AKTKP R EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGRADPHSL CYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTTAWKAQ NPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSFDGQIF LLFDSEKRMWTTVHPGARKMKEKWENDKWAMSFHYFSMGDCIGWLEDFLMGMDS TLEPSAGAPLAMGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQD WAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRKLYDYCDVPQC AA

[0152] Purity was > 90% as determined by reducing SDS-PAGE. Concentration was 1 mg / mL. The buffer formulation was PBS, pH 7.4. No endotoxins were detected.

[0153] As shown in Figure 7, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Sample 1, lane 2) and non-reduced (Sample 2, lane 3) conditions. Gel stained with Coomassie Blue. As a result, the reduced protein migrates at about 66-116 kDa, respectively. Non-reduced protein migrates about 116-235 kDa.Example 6- In Vivo Anti-Cancer Testing Using Zebrafish ModelZebrafish tumor xenograft (ZTX) model

[0154] Transgenic Tg(fli1: EGFP)y1zebrafish embryos were raised at 28°C for 48 hours in E3 embryo medium with 0.2 mM 1-Phenyl-2-Thiourea. Unfertilized eggs or larvae that did not appear healthy or exhibited any obvious developmental defects were excluded before experimental onset.

[0155] The zebrafish model is a remarkable short but predictive model to screen anticancer agents that has gained wide acceptance as a useful in vivo model. The purpose of this study was to evaluate the effects of the constructs on tumor size and metastasis using a lung patient derived xenograft (PDX) model. The LXFL 1176 PDX model of nonsmall cell lung cancer was chosen due to the close balance between uPAR expression and PD-L1 (aka CD274) as well as expression levels of uPAR (CD87) as DU-145 cells (see Table 1).Table 1. Comparative expression of uPAR and PD-L1Gene expression Ico2 GCRL1 Model Indication CD87 CD274 □ 5 <nc expression (210845 s at*) (227458 at*)LXFL 1176 PDX Non-small cell lung 10,297 9,338 8cancer (large cell □ 9subtype) □ - ‘IDU 145 Prostate cancer cell 10 7,0 □ '2 ver; high expression )line

[0156] The LXFL 1176 tumor tissue sample was enzymatically and mechanically dissociated to generate a single cell suspension of tumor cells and tumor-associated cells. This tumor cell suspension was labeled with Dil red fluorescent dye. Approximately 700 Dil-labeled tumor cells were subcutaneously co-implanted into the perivitelline space of 2 days old zebrafish larvae with the different constructs at 0.25 or 0.5mg / mL.

[0157] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat collected from a healthy donor. PBMCs were isolated by density gradient centrifugation using Lymphoprep medium following the provider’s instructions and administered i.v. after tumor implantation at 4:1 PBMC:tumor cells ratio.

[0158] The PBMCs were from healthy donors which have T cells (40-50%), NK (5-20%) cells and monocytes (10-20%). The K5 domain is thought to recruit NKT cells but block tumour associated macrophage migration. Hence the inclusion of an Fc4 and Fc1for the K5 C-terminal fusions - which would be independent of CDC and ADCC activity. The immune cell populations were not enriched so as to better mirror cell populations that occur in patients and allow for activities that would not be observed easily with purified NK or T cells. In similar short-term experiments, T cells are normally activated for several days prior to transplantation or to assess cancer cell killing activity. However, two of the tri-specific constructs have anti-CD3 activity thus T cells were not be preactivated. Therefore, this study likely underestimates the activity of the two test articles and the positive control. Therefore, a higher effector: target cell ratio, 4:1 ratio of PBMCs: PDX cell, was used. It should be noted that NK cells do not need stimulation and thus will likely exhibit faster cell killing kinetics in this model.

[0159] Larvae in which tumor cells had been inadvertently injected into circulation or larvae with erroneous implantation of the tumor in the yolk rather than the perivitelline space were excluded from the study.

[0160] Selected tumor-bearing embryos were sorted into experimental groups (20 embryos / group), and pictures of primary tumors were taken after injection. Tumor-bearing embryos were incubated in E3 / PTU medium for 72 hours at 35.5°C. After incubation, pictures of the primary tumors and the CVPs were taken using a red fluorescent filter. Larvae that died or were lost by other means during the study were excluded from the final analysis.Choice of dose of test articles

[0161] In a previous study there was successful dosing of ATF-Fc4-K5 and ATF-Fc4-Endo at 1 mg / mL which gave a 70% reduction (p<0.001 ) in tumour volume using DU-145 cells. Those experiments did not involve any immune attack; therefore a lower dose of fusion proteins was thought to be preferable in the current experiment in order to be able to observe the effect of the immune system on top of the direct anti-tumour effects. Choice and Dosing of Tecentriq as a positive control

[0162] Tecentriq, also known as Atezolizumab, is an anti-PDL1 monoclonal antibody used to treat cancer including non-small cell lung cancer. It was approved for clinical use in the United States in 2021. Tecentriq was tested in a model using melanoma cells andmatched tumor-infiltrating lymphocytes (TILs) isolated from patients with metastatic disease. In these experiments, Tecentriq was co-injected with melanoma cells subcutaneously into 2-day-old zebrafish embryos at 0.2 mg / mL in presence of TILs administered intravenously. Results showed that Tecentriq significantly reduced primary tumor size compared to a control group including TILs intravenously in absence of Tecentriq. In this study, tecentriq will be used at 0.5 mg / mL to match the concentrations of the constructs.Analysis of tumor growth inhibition and metastasis

[0163] Anti-cancer efficacy of the constructs was determined as the change in primary tumor size (i.e. tumor growth or reduction) and the number of tumor cells disseminated to the distal caudal venous plexus (CVP) three days after implantation.

[0164] Images obtained right after implantation (day 0) and after 72 hours incubation (day 3) were analysed by using in-house developed software, and tumor growth regression was calculated and normalized to the negative control group. In addition, the number of metastasized tumor cells to the CVP was counted manually.Statistical analysis

[0165] Data are shown as mean ± SEM, D’Agostino-Pearson omnibus normality test was performed followed by a One-way ANOVA, Kruskal-Wallis test, two-tailed Student’s t-test or Mann-Whitney test where appropriate. Graphs and experimental data were obtained / analysed by using GraphPad Prism v9.0.2.Constructs tested in the zebrafish tumor xenograft modelTable 2. Description of experimental groups for efficacy analysis of compounds using the LXFL 1176 PDX model in ZTX.Treatment Cone. Group size Administration Administration Evaluation of Evaluation of of compounds of PBMCs 4:1 primary tumor metastasis sizeControl - 20 - i.v., 2000 cells 0 and 72 h 72 h Atezolizumab 0,5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h PDL1-ABD- 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h AFF-PD1(mixed withhuman serumalbumin atlOmg / mL)ATF-Fc4-K5 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h ATF-Fcl-K5 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h ATF-Fc4- 0.5 mg / ml 20 Co-injected i.v.. 2000 cells 0 and 72 h 72 h EndostatinATF-Fcl- 0.5 mg / ml 20 Co-injected i.v.. 2000 cells 0 and 72 h 72 h ULBP2-K5ATF-Fcl-K.5- 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h ULBP2ATF-Fcl / CD16 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h ATF-Fcl / CD3- 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 h Fcl / CD16CD16-Fcl / CD3- 0.5 mg / ml 20 Co-injected i.v., 2000 cells 0 and 72 h 72 hFcl / ATFResultsPrimary tumor evaluation

[0166] The evaluation of anti-tumor efficacy of the constructs in the LXFL 1176 PDX model after 72 hours of exposure is shown in Figure 8. Data are presented as mean ± SEM, a Kruskal-Wallis test was performed (p=0.0065) followed by Mann-Whitney test, **p<0.01. The treatment with ATF-Fc1 / CD16 and ATF-Fc4-Endostatin at 0.5 mg / mL significantly decreased the primary tumor size compared to the control group (**p<0.01) in the presence of PBMC after 3 days post-tumor implantation. The rest of the compounds showed no effect on primary tumor size, probably because the immune cells used in the study were not matched to the tumor cells, leading to insufficient tumor antigenicity and a poor tumor-killing effect of immune cells, primarily by T-cells.Metastasis

[0167] Results are first presented as a comparison of the metastatic dissemination between the different compounds by grouping them according to the fusion construct, i.e., ATF-Fc1 constructs, ATF-Fc4 constructs, Atezolizumab positive control and CD16 constructs.

[0168] Then, all constructs are compiled and presented in a single graph where antitumor efficacy is compared to the negative control.ATF-Fc1 constructs

[0169] The comparison of tumor cell dissemination between the different treatment groups containing ATF-Fc1, namely ATF-FC1-K5, ATF-Fc1-K5-ULBP2 and ATF-Fc1-ULBP2-K5, after 72 hours of exposure is shown in Figure 9. Data are presented as mean ± SEM. Results showed no significant difference in the number of disseminated tumor cells when comparing ATF-Fc1-K5, ATF-Fc1-K5-ULBP2, and ATF-Fc1-ULBP2-K5 treatment groups. It was observed that the construct exhibiting the lowest number of disseminated cells to the CVP was ATF-Fc1 -K5-ULBP2, while placing the ULBP2 domain between ATF-Fc1 and K5 to create construct ATF-Fc1-ULBP2-K5, abolished the anti-metastatic effect showing that the ATF-Fc1-K5-ULBP2 configuration is the most efficacious.CD16 constructs

[0170] The comparison of tumor cell dissemination between the constructs containing CD16, namely ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16 and CD16-Fc1 / CD3-Fc1 / ATF, after 72 hours of exposure is shown in Figure 10. Data are presented as mean ± SEM. Results showed no significant difference in tumor cell dissemination when comparing the different constructs containing the CD16 domain after 3 days of treatment. In addition, the construct conformation of ATF-Fc1 / CD3-Fc1 / CD16 showed the lowest number of disseminated tumor cells to the CVP after 3 days of treatment. This indicates that adding CD3-Fc1 increases the anti-metastatic activity of the construct when this domain is situated between ATF-Fc1 and CD16, and in that order.All constructs

[0171] The evaluation of anti-metastatic efficacy of all constructs on LXFL 1176cancer cells compared to negative control after 72 hours of exposure is shown in Figure 11. Data are presented as mean ± SEM. A one-way ANOVA was performed (p=0.0002) followed by a two-tailed Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. All constructs were at a 0.5 mg / mL dose.

[0172] Results showed that, compared to the control group, treatment with ATF-Fc1 -K5, ATF-Fc1-K5-ULBP2, ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16 and CD16-Fc1 / CD3-Fc1 / ATF significantly reduced the dissemination of tumor cells to the CVP after 3 days of treatment (Figure 11). Previously designed constructs ATF-Fc4-K5, and ATF-Fc4-Endo also significantly reduced the dissemination of tumor cells to the CVP after 3 days of treatment (Figure 11).

[0173] Figure 11 shows that adding the ULBP2 domain to ATF-Fc1 -K5, forming the ATF-Fc1-K5-ULBP2 construct, increases the anti-metastatic effect on tumor cells, while placing the ULBP2 domain between ATF-Fc1 and K5 affected this anti-metastatic activity.

[0174] Figure 11 also shows that adding the CD3-Fc1 domain to ATF-Fc1 / CD16, forming the ATF-Fc1 / CD3-Fc1 / CD16 construct, increases the anti-metastatic effect on tumor cells, while shifting the order of ATF and CD16 in the construct, creating the CD16-Fc1 / CD3-Fc1 / ATF construct showed similar anti-metastatic activity as the ATF-Fc1 / CD3-Fc1 / CD16 configuration.

[0175] The observed effect on metastasis could be due to the metastatic tumor cells in circulation being readily available to the PBMCs for tumor-killing activity and / or the potent anti-angiogenic activity of the compounds.Conclusions

[0176] ATF-Fc1 / CD16 construct has anti-tumor and anti-metastatic activity on the lung cancer PDX model LXFL 1176 when tested at 0.5 mg / mL in the zebrafish model. Treatment with ATF-Fc1-K5, ATF-Fc1-K5-ULBP2, ATF-Fc1 / CD16, ATF-Fc1 / CD3-Fc1 / CD16 and CD16-Fc1 / CD3-Fc1 / ATF, significantly reduced metastatic dissemination of tumor cells in the ZTX model. Construct configurations ATF-Fc1-K5-ULBP2 and ATF-Fc1 / CD3-Fc1 / CD16 are the best for inhibiting the metastatic dissemination of tumor cells in the ZTX model.Example 7- Constructing ATF-CD16-K5-IL15

[0177] This fusion protein has two heavy chain polypeptides. The first heavy chain sequence has a signal peptide bound to ATF bound to a linker bound to an FC of human IgG1 with mutations creating a hole bound to a linker bound to a linker bound to K5. The second heavy chain sequence has a signal peptide bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to a linker bound to IL15 bound to a linker bound to an FC of human IgG1 with mutations creating a knob bound a linker bound to K5. A diagram of ATF-CD16-K5-IL15 is provided in Figure 12. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 10 and the second heavy chain sequence is set out in SEQ ID No. 11, below. Linkers are shown in italics.

[0178] Heavy chain 1: ATF-Fc-K5 MHSSALLCCLVLLTGVRAMSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFG GQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQL GLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSEPKSADKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPR AG L E KN YC RN P D G D VG G P WC YTAN P RKLYD YC D VP Q C AA

[0179] Heavy Chain 2: CD16-IL15-Fc-K5 MHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFR QAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSGGGGSGGGGSGGGGSNWVNVISDLKK IEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNS LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSEPKSADKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGG GSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRA GLEKNYCRNPDGDVGGPWCYTANPRKLYDYCDVPQCAA

[0180] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >91% as determined by SEC-HPLC at 280 nm. Endotoxin: < 1 Ell / rng. Concentration was 0.35 mg / mL.

[0181] As shown in Figure 13, SDS-PAGE analysis of the construct in β-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain 1 and heavy chain 2) migrate as about 50-70 kDa and 70-100 kDa, respectively. Nonreduced protein migrates about 100-150 kDa.Example 8- Constructing CD16-IL15-ATF-Arresten

[0182] The fusion protein has two heavy chain sequences. The first heavy chain sequence has a signal peptide bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to a linker bound to IL15 bound to a linker bound to a human IgG1 Fc with mutations to create a hole and the second heavy chain sequence has a signal peptide bound to ATF bound to a linker bound to arresten bound to a human IgG1 Fc with a mutation to make a knob (C220A T366W). A diagram of CD16-IL15-ATF-Arresten is provided in Figure 14. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinitychromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 12 and the second heavy chain sequence is set out in SEQ ID No.13, below.

[0183] Heavy chain 1: CD16 vhh-IL15-Fc MHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFR QAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYC AAARRGTNAFLTHDKYGYWGQGTQVTVSSPSGQAGAAASESLFVSNHAYNWVNVIS DLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL ANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEASGGPEEPKSADK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0184] Heavy chain 2: ATF-Arresten-Fc MHSSALLCCLVLLTGVRASNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGG QHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLG LGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSSVDHGFLVTRH SQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINN VCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQI PPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY YANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRTEPKSADKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0185] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >55% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 EU / mg. Concentration was 0.60 mg / mL.

[0186] As shown in Figure 15, SDS-PAGE analysis of the construct in β-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for30 minutes with Coomassie Blue. As a result the reduced proteins migrate at about 70-100 kDa. Non-reduced protein migrates about 100-150 kDa.Example 9 -Constructing ATF-CD16-CD16-K5

[0187] The fusion protein has two heavy chain sequences and one light chain sequence. The first heavy chain sequence has a signal peptide (underlined) bound to a CD16 single variable domain on a heavy chain (VHH) antibody (italics) bound to a human IgG1 Fc with mutations to create a knob (bold) bound to a linker bound to K5 (bold underlined). The second heavy chain sequence has a signal peptide bound to ATF bound to a human IgG1 Fc with a mutation to make a hole bound to a linker bound to K5. This second heavy chain sequence is shown as heavy chain 1: ATF-Fc-K5 in Example 7, above. The light chain sequence has a signal peptide (underlined) bound to a CD16 VHH antibody (italics) bound to a human kappa constant light (CL) chain (bold). The first heavy chain sequence is set out in SEQ ID No. 14 below, the second heavy chain sequence is set out in SEQ ID No. 10 above and the light chain sequence is set out in SEQ ID No. 2 below.Heavy Chain 1: CD16-FC1-K5 MHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFRQAPGK EREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFL THDKYGYI / I / GQGTQVTV'SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCM FGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPW CYTAN PRKLYDYCDVPQCAALight Chain: CD16-CLMHSSALLCCLVLLTGVRAQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFL THDKYGYWGQGTQVTVSSRTVAAPSVFI FPPSDEQLKSGTASWCLLN N FYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

[0188] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >89% as determined by SEC-HPLC at 280 nm. Endotoxin: <1 Ell / rng. Concentration was 0.62 mg / mL. The formulation buffer was PBS.

[0189] As shown in Figure 17, SDS-PAGE analysis of the construct in β-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result the reduced proteins (heavy chain and light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 100-150 kDa.

Claims

WHAT IS CLAIMED IS:

1. A compound comprising:- One or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide and- One or more binding peptides from a T cell or NK cell activator, operatively linked to a second immunoglobulin G (IgG) antibody peptide.

2. A compound comprising:- One or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, operatively linked to a first immunoglobulin G (IgG) antibody peptide,- One or more binding peptides from a T cell activator, operatively linked to a second immunoglobulin G (IgG) antibody peptide and- One or more binding peptides from an NK cell activator, operatively linked to a third immunoglobulin (IgG) antibody peptide.

3. The compound of claim 1 wherein the urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to the one or more binding peptides from a T-cell or NK cell activator, which is operatively linked to the one or more IgG antibody peptides.

4. The compound of the preceding claims, wherein the first and / or the second IgG is a fragment of an Fc fusion peptide.

5. The compound of the preceding claims, wherein the first and / or the second IgG is a fragment of a human Fc fusion peptide.

6. The compound of the preceding claims, wherein the Fc fusion peptide is an IgG protein.

7. The compound the preceding claims, wherein the Fc fusion protein is selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide.

8. The compound of claims 1 or 2, wherein the first and / or the second IgG is a constant heavy chain peptide of human lgG1.

9. The compound of claims 1 or 2, wherein the first or the second IgG is a human kappa constant light (CL) chain.

10. The compound of the preceding claims, further comprising a linker peptide between the binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist and the first IgG antibody peptide.

11. The compound of the preceding claims, further comprising a linker peptide between the binding peptide from a T cell or NK cell activator and the second IgG antibody peptide.

12. The compound of the preceding claims, further comprising a kringle domain 5 from a plasminogen operatively linked to the C terminal of the one or more IgG antibody peptides.

13. A compound comprising:- A first heavy chain polypeptide and a second heavy chain polypeptide in which one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist, is operatively linked to the first and / or the second heavy chain polypeptide and - one or more binding peptides from a T cell or NK cell activator is operatively linked to the first and / or the second heavy chain polypeptide.

14. A compound comprising:- A first light chain polypeptide, a second light chain polypeptide and a first heavy chain polypeptide and a second heavy chain polypeptide in which, - one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to one or more of the light or heavy chain polypeptides and- one or more binding peptides from a T cell or NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides.

15. A compound comprising:- A first light chain polypeptide, a second light chain polypeptide, a first heavy chain polypeptide and a second heavy chain polypeptide in which- one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist is operatively linked to the one or more of the light or heavy chain polypeptides, - one or more binding peptides from a T cell activator is operatively linked to the one or more of the light or heavy chain polypeptides and - one or more binding peptides from an NK cell activator is operatively linked to one or more of the light or heavy chain polypeptides.

16. A compound comprising an antibody capable of binding uPAR, T cells and NK cells and having activity of a uPAR antagonist and a T-cell and NK cell activator.

17. The compound of claims 13-15, wherein the heavy chain polypeptides comprise a constant heavy chain peptide of human lgG1.

18. The compound of claims 13-16, wherein the first heavy chain polypeptide comprises a human lgG1 modified with a T366S L368A Y407V hole and the the second heavy chain polypeptide comprises a human lgG1 modified with a T366W knob.

19. The compound of claims 13-16, wherein the heavy chain polypeptides comprise a fragment of an Fc fusion peptide.

20. The compound of claim 19, wherein the Fc fusion peptide is an IgG protein.

21. The compound of claim 19, wherein the Fc fusion peptide is selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide.

22. The compound of claims 13-21, wherein the light chain polypeptide comprises a human kappa constant light (CL) chain.

23. The compound of claims 13-22, further comprising a linker peptide between the one or more binding peptides from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist and the one or more heavy or light chains polypeptides.

24. The compound of claims 13-23, further comprising a linker peptide between the one or more binding peptides from a T-cell or NK-cell activator and the one or more heavy or light chains polypeptides.

25. The compound of claims 13-24, further comprising a kringle domain 5 from a plasminogen operatively linked to the C terminal of one or more of the heavy or light chain polypeptides.

26. The compound of the preceding claims, wherein the binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist having activity of a uPAR antagonist comprises an amino terminal fragment (ATF) of uPAR having uPAR binding activity.

27. The compound of the preceding claims, wherein the binding peptide from a T cell activator comprises an anti-CD3 single variable domain on a heavy chain (VHH) antibody.

28. The compound of the preceding claims, wherein the binding peptide from a NK cell activator comprises an anti-CD16 VHH antibody.

29. The compound of the preceding claims, wherein the binding peptide from a NK cell activator comprises ULBP2.

30. The compound of the preceding claims, wherein the binding peptide from a T-cell or NK cell activator comprises IL15.

31. The compound of claim 13, wherein the first heavy chain polypeptide is coded by a first heavy chain sequence and the second heavy chain polypeptide is coded by a second heavy chain sequence selected from a group of first heavy chain sequence and second heavy chain sequence combinations consisting of SEQ ID No. 12 and SEQ ID No. 13 or SEQ ID No. 10 and SEQ ID No. 11.

32. The compound of claim 14, wherein the first heavy chain polypeptide and the second heavy chain polypeptide are coded by a heavy chain sequence and the first light chain polypeptide and the second light chain polypeptide are coded by a light chain sequence selected from a group of heavy chain sequence and light chain sequence combinations consisting of SEQ ID No. 1 and SEQ ID No. 2 or SEQ ID No. 7 and SEQ ID No. 8.

33. The compound of claim 15, wherein the first heavy chain polypeptide is coded by a first heavy chain sequence, the second heavy chain polypeptide is coded by a second heavy chain sequence and the first light chain polypeptide and the second light chain polypeptide are coded by a light chain sequence selected from a groupof first heavy chain sequence, second heavy chain sequence and light chain sequence combinations consisting of- SEQ ID No. 3, SEQ ID No. 4 and SEQ ID. No. 2; or- SEQ ID No. 5, SEQ ID No. 4 and SEQ ID No. 6.

34. A compound comprising a first heavy chain encoded by SEQ ID No. 14, a second heavy chain encoded SEQ ID No. 10, and a light chain encoded by SEQ ID No. 2.

35. A compound comprising a sequence consisting of SEQ ID No. 9.

36. A compound comprising a compound at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the compound of claims 31-35.

37. A compound selected from the group consisting of ATF-Fc1 / CD16, ATF-Fc1 / CD3- Fc1 / CD16, CD16-Fc1 / CD3-Fc1 / ATF and ATF-Fc1-K5-ULBP2.

38. A compound consisting of ATF-Fc1-ULBP2-K5.

39. A compound consisting of ATF-CD16-K5-IL15.

40. A compound selected from the group consisting of CD16-IL15-ATF-Arresten and ATF-CD16-CD16-K5.

41. A compound comprising a compound substantially homologous to the compound of the preceding claims and having the activity of the compound of the preceding claims.

42. One or more nucleotides encoding the compound of the preceding claims.

43. A vector containing the compound of the preceding claims.

44. The compound of the preceding claims, wherein the compound exhibits anticancer activity.

45. A method of treating cancer comprising administration of a composition comprising the compound of the preceding claims.

46. A use of the compound of the preceding claims for treatment of cancer.

47. A method for inhibiting metastasis in a subject with cancer, the method comprising administering an effective amount of the compound of the preceding claims to the subject.

48. The method or use of the preceding claims, further comprising one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

49. The method or use of claims 45-48, wherein the cancer is a solid cancer.

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