Trimeric polypeptides and their use in cancer treatment

Abstract

The present invention relates to a trimeric polypeptide complex comprising three monomeric polypeptides, each monomeric polypeptide comprising an anti-4-1BB specific agonist single chain antibody fragment (scFv), a homotrimerization domain, and a polypeptide region capable of specifically binding to a tumor-associated antigen. The present invention also relates to said trimeric polypeptide complex for use in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to the field of cancer therapeutics, and more specifically to therapeutic agents that are trimeric polypeptide complexes formed by collagen homotrimerization domains. [Background technology]

[0002] Modulation of immune responses with monoclonal antibodies (mAbs) is one of the most promising approaches to cancer immunotherapy. Perhaps the best known is mAb-mediated blockade of the programmed cell death protein 1 (PD-1) inhibitory pathway, which prevents PD-1-mediated immunosuppressive signaling in T cells and restores effector function to anergic tumor-infiltrating T cells. PD-1 / PD-ligand 1 (PD-L1) axis blockade has shown long-lasting, sustained responses in a wide range of cancers, but its efficacy is limited to 10–30% of patients. Another immunotherapeutic approach involves the stimulation of costimulatory receptors, such as 4-1BB, by agonistic mAbs. 4-1BB, also known as CD137, is a member of the TNF receptor (TNFR) superfamily and can be induced on various leukocyte subsets. 4-1BB is a type I single-pass transmembrane receptor with four extracellular cysteine-rich domains (CRDs) and one intracellular signaling domain. On T cells, 4-1BB is expressed after activation via the T cell receptor (TCR). Upon binding to its natural ligand [4-1BB-ligand (4-1BBL), TNFSF9] or agonist mAb, it enhances T cell proliferation and effector function, prevents T cell exhaustion, protects against programmed cell death, and promotes memory cell differentiation, which may support tumor-specific T cell persistence. Anti-4-1BB agonist mAbs have been examined in preclinical cancer models and shown to promote the elimination of a variety of poorly immunogenic tumors. However, extratumoral toxicity remains a major obstacle to the clinical development of full-length anti-human 4-1BB. Anti-hu4-1BB human IgG4 urelumab (BMS-663513) caused dose-dependent hepatotoxicity and was responsible for two deaths. Subsequent studies revealed that lower doses reduced hepatotoxicity but at the cost of reduced efficacy (Segal NH. et al., 2017). The anti-hu4-1BB human IgG2 utomirumab (PF-05082566) has an improved safety profile compared to urelumab but is also less potent as a 4-1BB agonist (Chester C. et al., 2018).

[0003] New strategies are being actively explored to avoid the extratumoral toxicity associated with Fc-FcγR interactions while retaining the antitumor activity associated with 4-1BB costimulation. These approaches aim to restrict 4-1BB costimulation to the tumor microenvironment and draining lymph nodes. Fc-free tumor-specific trimerbodies have recently been reported that target tumor-associated antigens (TAAs) such as EGFR (epidermal growth factor receptor) (Compte M. et al., 2018) or CEA (carcinoembryonic antigen) (Mikkelsen K. et al., 2019) and murine 4-1BB in an agonistic manner. Both trimerbodies are potent costimulatory molecules in vitro, and the EGFR-targeted 4-1BB agonist trimerbodies showed enhanced tumor penetration and potent antitumor activity in immunocompetent mice, while mitigating the systemic cytokine production and T cell-mediated hepatotoxicity associated with IgG-based 4-1BB agonists (Compte M. et al., 2018). More recently, it was disclosed that in liver-specific human EGFR transgenic immune-competent mice, systemic administration of anti-4-1BB agonist IgG caused non-specific immune stimulation and hepatotoxicity, whereas no such immune-related adverse effects were observed in mice treated with Fc-free EGFR-specific 4-1BB agonist trimer bodies (Compte M. et al., 2020).

[0004] Therefore, there is a need in the art for new strategies that allow effective immune stimulation without severe side effects for tumor-targeted therapy. Summary of the Invention

[0005] In a first aspect, the present invention provides a trimeric polypeptide complex comprising three monomeric polypeptides, Each monomeric polypeptide is a) an anti-4-1BB specific agonist single chain antibody fragment (scFv) in which the VH domain is N-terminal to the VL domain; b) a homotrimerization domain selected from the group consisting of type XVIII collagen homotrimerization domain (TIEXVIII), type XV collagen homotrimerization domain (TIEXV) and functionally equivalent variants thereof; and c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Concerning trimeric polypeptide complexes.

[0006] In a second aspect, the present invention provides a trimeric polypeptide complex comprising three monomeric polypeptides, Each monomeric polypeptide is a) anti-4-1BB specific agonist single chain antibody fragments (scFv) whose CDRs comprise the sequences represented by SEQ ID NO:1 or SEQ ID NO:42, SEQ ID NO:2 or SEQ ID NO:43, SEQ ID NO:3 or SEQ ID NO:44, SEQ ID NO:4 or SEQ ID NO:45, SEQ ID NO:5 or SEQ ID NO:46 and SEQ ID NO:6, or functionally equivalent variants thereof; b) a homotrimerization domain selected from the group consisting of type XVIII collagen homotrimerization domain (TIEXVIII), type XV collagen homotrimerization domain (TIEXV) and functionally equivalent variants thereof; and c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Concerning trimeric polypeptide complexes.

[0007] In a third aspect, the present invention relates to a polynucleotide encoding at least one monomeric polypeptide forming part of a trimeric polypeptide according to the invention.

[0008] In a fourth aspect, the present invention relates to a vector comprising a polynucleotide according to the invention.

[0009] In a fifth aspect, the present invention relates to a host cell comprising a vector according to the invention.

[0010] In a sixth aspect, the present invention relates to a combination comprising a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention or a host cell according to the present invention and an immune checkpoint blocker.

[0011] In a seventh aspect, the present invention relates to a pharmaceutical composition comprising a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention or a combination according to the present invention and a pharma- ceutically acceptable excipient.

[0012] In an eighth aspect, the present invention relates to a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention, a combination according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of cancer. [Brief description of the drawings]

[0013] [Figure 1]Design and characterization of humanized Fc-free tumor-targeted 4-1BB agonist trimer body (4-1BBN / CEGFR). (a) Schematic showing the genetic arrangement and domain structure of 4-1BBN / CEGFR as determined by SAXS, and its arrangement in solution. (b) Rigid-body fitting of the model corresponding to 4-1BBN / CEGFR within the SAXS envelope (colored in light grey). Each strand is colored in blue, magenta, and cyan. (c) Experimental (black lines) and calculated (red lines) sensorgrams obtained from BLI showing the interaction between 4-1BBN and 4-1BBN / CEGFR trimer body (1 and 5 nM concentrations) with immobilized hu4-1BB (upper panel), and between anti-huEGFR ATTACK and 4-1BBN / CEGFR trimer body (1 and 5 nM concentrations) with immobilized huEGFR (lower panel). Kinetic rate constants used for the calculated curves are shown in Table V. (d) Simultaneous binding to both immobilized hu4-1BB and huEGFR in solution was demonstrated for 4-1BBLN / CEGFR (green) but not 4-1BBN (black); 5 nM of either trimer body was first bound to immobilized hu4-1BB and then the biosensor was transferred to 10 nM huEGFR. [Diagram 2]4-1BBN / CEGFR trimer body significantly enhances T cell costimulation in vitro in the presence of huEGFR expressing cells and signal 1. Flow cytometric analysis of huFcγRIIb(CD32) expression in CHO and CHOhuFcγRIIb cells (a) and huEGFR expression in 3T3 and 3T3huEGFR cells (b). Cells incubated with PE-conjugated isotype control mAb are shown in grey filled histograms. Fluorescence intensity (horizontal axis) was plotted against relative cell number (vertical axis). Numbers indicate mean fluorescence intensity (MFI). Jurkathu4-1BB cells were co-cultured with CHO or CHOhuFcγRIIb cells (c) and 3T3 or 3T3huEGFR cells (d) in the presence of 10-fold increasing concentrations of 4-1BB IgG, urelumab, 4-1BBN or 4-1BBN / CEGFR antibodies, and luminescence was measured after 6 h at 37°C. Data are presented as fold induction relative to values ​​obtained from unstimulated Jurkathu4-1BB cells. Representative dose-concentration curves are shown and are expressed as mean ± SD (n=3). Significance was determined by unpaired Student's t-test. PBMCs (e) and T cells (f) (1.5×105 / well) isolated from healthy donors were co-cultured with irradiated 3T3 or 3T3huEGFR cells at an E:T ratio of 5:1. Anti-hu4-1BB agonist antibodies (4-1BBIgG or 4-1BBN / CEGFR) and controls were added in 10-fold serial dilutions in the presence or absence of anti-huCD3 mAb (0.05 μg / ml) and IFNγ secretion was analyzed after 72 h (mean ± SD, n = 3). Significance was determined by unpaired Student's t test. (g) Flow cytometric analysis of huEGFR expression (upper panel) or huPD-L1 expression (lower panel) in 3T3huEGFR and MDA-MB-231 cells. Cells incubated with PE-conjugated isotype control mAb are shown in grey-filled histograms.(h) Irradiated EGFR+PD-L1- cells (3T3huEGFR) or EGFR+PD-L1+ cells (MDA-MB-231) (3×104 cells / well) were co-cultured with huPBMCs activated with anti-huCD3 mAb (0.05 μg / ml) in the presence of anti-PD-L1 alone or in combination with 4-1BBN / CEGFR at an E:T ratio of 5:1. Cell-free supernatants were measured for IFNγ by ELISA after 72 hours. Data are shown as mean ± SD (n=3). Significance was calculated by unpaired Student's t-test. One representative experiment out of three independent experiments is shown (a and c). When primary cells were used, at least three different donors were tested. [Diagram 3]4-1BBN / CEGFR trimerbody demonstrated significant tumor growth inhibition in a humanized mouse model. (a) Pharmacokinetic profile of 89Zr-4-1BBN / CEGFR in plasma %ID / ml versus time after iv administration. Data are shown as mean ± SD (n=2–6). (b) Rag2- / -IL2Rγnull mice were inoculated sc with HT29 tumor cells and ip with freshly isolated huPBMCs, and when tumors reached approximately 0.4 cm in diameter, they were randomized into groups (n=7–8 / group) with similar mean tumor size and SD and treated with five ip injections of PBS, CEAN, or 4-1BBN / CEGFR trimerbody (4 mg / kg), or three ip injections of 4-1BB IgG (4 mg / kg). c) Mean tumor volume increase in each group of mice. Data are shown as mean ± SD. Significance was determined by one-way ANOVA adjusted with Bonferroni correction for multiple comparisons. (d) Analysis of huEGFR expression by IHC in NSCLC PDX TP103. (e) NSG mice were inoculated sc with small pre-amplified fragments of TP103 and, when tumors reached approximately 0.5 cm in diameter, randomized into groups (n=6-7 / group) with similar mean tumor size and SD and injected ip with freshly isolated huPBMCs. Mice were treated with PBS or 4-1BBN / CEGFR. (f) Mean tumor volume increase in mice in each group is shown. Data are presented as mean ± SD. Significance was determined by unpaired Student's t-test. For both in vivo assays, mice were weighed weekly to monitor toxicity and euthanized if there were signs of distress and / or 10-15% weight loss. Percentages of CD4+ and CD8+ cells (g) or FoxP3+ cells (h) in tissue sections from PBS- and 4-1BBN / CEGFR-treated mice (mean ± SD, n = 4–5). Significance was calculated by unpaired Student's t-test. (i) Representative IHC staining for CD4 and CD8 is shown. Tumors were harvested at termination from the experiment shown in (c). (j) H&E staining of representative tissue sections of liver from mice treated with PBS, 4-1BB IgG, and 4-1BBN / CEGFR. Scale bars are indicated.(k) Human IFNγ serum levels in mice were examined at week 4 (mean ± SD, n = 4). Significance was calculated by unpaired Student's t test. [Figure 4] Combination of 4-1BBN / CEGFR with a full-length PD-L1 blocking antibody induces tumor regression in a humanized MDA-MB-231 TNBC xenograft model. (a) NSG mice were inoculated sc with MDA-MB-231 tumor cells and injected ip with freshly isolated huPBMCs, and when tumors reached approximately 0.2 cm in diameter, they were randomized into groups (n=5-6 / group) and treated with PBS, or with 4-1BBN / CEGFR or atezolizumab, alone or in combination. (b) The mean tumor volume increase in mice in each group is shown. Data are shown as mean ± SD. Mice were weighed weekly and euthanized when they showed signs of distress and / or had a 10-15% weight loss. Significance was determined by one-way ANOVA adjusted with Bonferroni correction for multiple comparisons. Percentage of cytokeratin (CK)+ cells (c) and CD4 / CD8 ratio of tumor infiltrating lymphocytes (TILs) (d) in tumor sections taken at termination from the experiment shown in (b). Data are shown as mean ± SD (n = 4–6). Significance was calculated by unpaired Student's t test. (e) Representative low-magnification images of H&E and immunohistochemically stained samples for cytokeratin, CD4, and CD8 are shown. Two representative specimens showing partial or complete immune-mediated eradication (IME) of TNBC cells from the combination therapy group are shown. [Diagram 5] Schematic diagram showing the protein structure of anti-hu4-1BB IgG (a) and the gene arrangement (b) and protein structure (c) of the anti-hu4-1BB trimer body. The variable regions from the SAP3.28 antibody are shown in green, and the mouse constant domains in light grey. The scFv-based N-terminal trimer body (4-1BBN) gene construct contains the SAP3.28 scFv genes (VH-linker-VL) connected via a flexible linker (dark grey box) to the human TIEXVIII domain (light blue box). A FLAG-strep tag (light orange box) was added for purification and immunodetection. The arrow indicates the direction of transcription. [Figure 6] Binding assays of 4-1BB IgG and urelumab. Antigen titration ELISA of 4-1BB IgG (a) and urelumab (b) against plastic immobilized hu4-1BB. Data are presented as mean ± SD (n=3). Competitive ELISA of soluble hu4-1BB binding to immobilized hu4-1BBL in the presence of increasing concentrations of 4-1BB IgG or urelumab (c). Percentage of hu4-1BB binding is expressed as (Abs450nm in the presence of competing anti-4-1BB antibody ÷ Abs450nm for soluble hu4-1BB alone) × 100. Data are presented as mean ± SD (n=3). Competitive ELISA of soluble 4-1BB IgG (d) or urelumab (e) against immobilized hu4-1BB in the presence of saturating concentrations of urelumab (d) or 4-1BB IgG (e), respectively. Data shown are means ± SD (n = 3). [Figure 7] Structural characterization of 4-1BBN and 4-1BBN / CEGFR trimer bodies. (a) Reducing SDS-PAGE of purified 4-1BBN and 4-1BBN / CEGFR. SEC-MALS analysis of 4-1BBN (b) and 4-1BBN / CEGFR (c). Black lines correspond to UV absorbance (left axis) and red lines correspond to measured molar mass (right axis). (d) Circular dichroism spectra of 4-1BBN (black line) and 4-1BBN / CEGFR (red line). (e) Thermal denaturation of 4-1BBN (black line) and 4-1BBN / CEGFR (red line) measured by changes in circular dichroism ellipticity at 210 nm and 213 nm, respectively. [Figure 8] SAXS analysis of the configuration of the 4-1BBN trimer body in solution. Rigid-body overlay of the SAXS envelope of 4-1BBN determined from ab initio. A model is constructed (individual chains are colored blue, magenta, and cyan) and fitted to the envelope (colored light gray). [Figure 9]Experimental and theoretical SAXS scattering. Experimental scattering curve (dots) and theoretical scattering calculated from the model (smooth curve) at 6 mg ml-1 concentration. The figure shows the normalized pair-distance distribution function P(r) for 4-1BBN (a) and 4-1BBN / CEGFR (b). Data are vertically offset for clarity. au is arbitrary unit. [Figure 10] Species specificity of 4-1BBN / CEGFR trimer bodies. 4-1BBN / CEGFR bound in a concentration-dependent manner to purified mouse (mo) EGFR, cynomolgus monkey (cy) EGFR and huEGFR immobilized on plastic (a), as well as to cy4-1BB and hu4-1BB, and to a much lesser extent to mo4-1BB (b). Data are presented as the mean ± SD (n=3) of one representative experiment. [Figure 11] Binding of 4-1BBN / CEGFR to cell surface expressed hu4-1BB and huEGFR. 4-1BB IgG was used as a control. The Y-axis indicates cell number and the X-axis indicates fluorescence intensity, expressed on a logarithmic scale. One representative experiment out of three independent experiments is shown. [Figure 12] Effect of 4-1BBN / CEGFR trimer body on EGFR-mediated signaling. (a) Inhibition of A431 cell proliferation. Cells were treated with the indicated doses of 4-1BBN / CEGFR, 4-1BB IgG, cetuximab (positive control) or rituximab (negative control). 72 h after treatment, viable cells were measured in triplicate and plotted against untreated controls. Results are expressed as mean ± SD (n=3). Significance was assessed by unpaired Student's t-test. (b) Inhibition of EGFR phosphorylation. Cells were preincubated with 100 nM of each antibody for 4 h before stimulation with EGF or vehicle for 10 min. The phosphorylation status of EGFR was assessed by Western blotting. [Figure 13]Costimulatory activity of control antibodies. Jurkathu4-1BB reporter cells were co-cultured with 3T3 or 3T3huEGFR cells (a) and CHO or CHOhuFcγRIIb cells (b) in the presence of 10-fold increasing concentrations of mouse IgG1 isotype (moIgG1), human IgG4 isotype (huIgG4) or anti-CEA scFv-based trimeric body (CEAN) and luminescence was measured after 6 h at 37 °C. Data are presented as fold induction relative to values ​​obtained from unstimulated Jurkathu4-1BB cells. One representative experiment out of three independent experiments is shown (mean ± SD, n = 3). Significance was calculated by unpaired Student's t test. [Figure 14] Costimulation studies in human primary cells. Human PBMCs (1.5x105 / well) (a) or isolated T cells (1.5x105 / well) (b) were co-cultured with irradiated 3T3 or 3T3huEGFR cells at an E:T ratio of 5:1. moIG1 isotype or CEAN trimer bodies were added in 10-fold serial dilutions in the presence or absence of anti-huCD3 mAb (0.05 μg / ml) and IFN-γ secretion was analyzed after 72 h. One representative experiment out of three independent experiments is shown. Data are means ± SD (n=3). Significance was calculated by unpaired Student's t-test. [Figure 15] Serum stability of 4-1BBN / CEGFR trimer bodies. ELISA for plastic-immobilized hu4-1BB (a) or huEGFR (b) was performed after incubation in human serum for different times at 37° C. Mean values ​​± SD (n=3) for each time point are shown. [Figure 16] Structural and functional characterization of 4-1BBN / CEGFR after conjugation with p-SCN-Bn-deferoxamine (Df). (a) Reducing SDS-PAGE of unconjugated 4-1BBN / CEGFR and after conjugation with p-SCN-Bn-deferoxamine (Df-1BBN / CEGFR). (b) Functional characterization of 4-1BBN / CEGFR and Df-1BBN / CEGFR by ELISA on plastic-immobilized hu4-1BB and huEGFR. Data are presented as mean ± SD (n=2). [Figure 17] Representative images of CD3+ and FoxP3+ TIL immunostaining in huPBMC-driven humanized NSG mice bearing EGFR+ NSCLC PDXs treated with PBS or 4-1BBN / CEGFR trimer body. Tumors were harvested at termination from the experiment shown in Figure 3C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] We developed a tumor-specific 4-1BB agonist trimerbody that exhibits antitumor activity against a broad range of human tumors and synergizes with immune checkpoint blockers. This approach may provide a way to elicit responses in most cancer patients while avoiding Fc-mediated adverse reactions.

[0015] In addition, we also studied the agonist properties of tumor-specific 4-1BB-agonist trimerbodies. In general, anti-4-1BB-agonist mAbs can be classified as either strong or weak agonists. Strong agonists (e.g., urelumab) can induce signaling activation without FcγR-mediated cross-linking, whereas weak agonists (e.g., utomirumab) require FcγR-mediated cross-linking to significantly induce 4-1BB signaling (Qi et al., Nat Commun., 2019;10:2141). The bivalent (IgG) anti-hu4-1BB antibody derived from the SAP3.28 antibody (International Patent Application WO2017077085) is dependent on the presence of FcγRIIb to induce 4-1BB signaling and can therefore be classified as a weak agonist. Using the hu4-1BB reporting cell line, the inventors observed that the tumor-specific 4-1BB agonist trimerbody according to the present invention exhibits 4-1BB signaling activity without additional cross-linking. This was clearly unexpected, since there was no suggestion in the art that modifying a dimeric antibody specific for 4-1BB into a trimeric antibody as in the present invention would result in an increase in agonist activity significantly above that achieved by the antibody cross-linked by FcγRIIb expressing cells.

[0016] Thus, in a first aspect, the present invention provides a trimeric polypeptide complex (first TPC of the invention) comprising three monomeric polypeptides, Each monomeric polypeptide is a) an anti-4-1BB specific agonist single chain antibody fragment (scFv) in which the VH domain is N-terminal to the VL domain; b) a homotrimerization domain selected from the group consisting of type XVIII collagen homotrimerization domain (TIEXVIII), type XV collagen homotrimerization domain (TIEXV) and functionally equivalent variants thereof; and c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Concerning trimeric polypeptide complexes.

[0017] In another aspect, the present invention provides a trimeric polypeptide complex (second TPC) comprising three monomeric polypeptides, Each monomeric polypeptide is a) anti-4-1BB specific agonist single chain antibody fragments (scFv) whose CDRs comprise the sequences represented by SEQ ID NO:1 or SEQ ID NO:42, SEQ ID NO:2 or SEQ ID NO:43, SEQ ID NO:3 or SEQ ID NO:44, SEQ ID NO:4 or SEQ ID NO:45, SEQ ID NO:5 or SEQ ID NO:46 and SEQ ID NO:6, or functionally equivalent variants thereof; b) Type XVIII collagen homotrimerization domain (TIE XVIII ), collagen XV homotrimerization domain (TIE XV ) and functionally equivalent variants thereof; and c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Concerning trimeric polypeptide complexes.

[0018] As used herein, " Trimeric Polypeptide ComplexThe term "TPC" or "TPC" refers to a complex of three non-covalently bound monomeric polypeptides. Each monomeric polypeptide may be identical or different from the others. In a preferred embodiment, the TPC is a homotrimer, meaning that the three monomers or subunits of the complex are identical. In another preferred embodiment, the TPC is a heterotrimer, meaning that at least one of the three monomers or subunits of the complex is different from the other two. In a more preferred embodiment, the TPC is a homotrimer.

[0019] Anti-4-1BB specific agonist single chain antibody fragment (scFv) As used herein, the term "anti-4-1BB specific agonist single chain antibody fragment (scFv)" refers to a single chain antibody fragment (scFv) capable of specifically binding to and inducing stimulation of 4-1BB.

[0020] "4-1BB", also known as CD137 or TNFRS9, as used herein, refers to activation-induced costimulatory molecule. 4-1BB has only one confirmed ligand [4-1BB-ligand (4-1BBL), TNFSF9], which is expressed on macrophages, activated B cells, and dendritic cells. When 4-1BB binds to its ligand or agonist antibodies, it promotes T cell proliferation, cytokine production, and cytolytic effector function, and protects lymphocytes from programmed cell death. In addition, binding of 4-1BB on NK cells enhances cytokine release (including IFNγ) and antibody-dependent cellular cytotoxicity (ADCC).

[0021] As used herein, the term "single-chain variable fragment" refers to a genetically engineered molecule that contains a variable light chain region and a variable heavy chain region linked by a suitable peptide linker and formed as a genetically fused single-chain molecule. The fragment is a portion of an immunoglobulin molecule that retains the heavy and / or light chain antigen-binding sites, such as heavy chain complementarity determining regions (HCDRs) 1, 2 and 3, light chain complementarity determining regions (LCDRs) 1, 2 and 3, heavy chain variable region (VH), or light chain variable region (VL). Antibody fragments include the well-known Fab, F(ab')2, Fd and Fv fragments, as well as single domain antibodies (dAbs) consisting of one VH domain or one VL domain. The VH and VL domains can be linked via a synthetic linker to form various types of single-chain antibody designs in which the VH / VL domains are paired intramolecularly or intermolecularly when the VH and VL domains are expressed by separate single-chain antibody constructs to form a monovalent antigen-binding site.

[0022] "Specific binding" or "specifically binds" or "binds" refers to a molecule that binds to 4-1BB or an epitope within 4-1BB with higher affinity than other antigens. Typically, an agonist has an equilibrium dissociation constant for binding (K D ) is about 1×10 -8 M or less, for example, about 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 Typically, the K for binding to nonspecific antigens (e.g., BSA, casein) is less than or equal to M D At least 100 times smaller than K D So, "specifically binds." Dcan be measured using standard procedures. However, anti-4-1BB specific agonist single chain antibody fragments may have cross-reactivity to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, such as Macaca fascicularis (cynomolgus monkey, cyno), Pan troglodytes (chimp) or Callithrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds only one antigen or one epitope, a bispecific antibody specifically binds two different antigens or two different epitopes.

[0023] The anti-4-1BB specific agonist single chain antibody fragment forming part of the TPC of the present invention has the ability to induce at least one biological activity of 4-1BB to which the antibody binds, which is induced by the natural ligand of 4-1BB. Exemplary agonist activities include induction of signal transduction through TRAF1 and TRAF2 to activate the NF-κB, AKT, p38MAPK, and ERK pathways, which induces the expression of survival genes encoding survivin, Bcl-2, Bcl-XL, and Bfl-1, and reduces the expression of pro-apoptotic Bim. 4-1BB is involved in T cell proliferation, acquisition of effector functions, survival, and development of T cell memory. A suitable assay for determining whether an anti-4-1BB antibody is an agonist is shown in Example 2 and consists of determining the ability of an antibody targeting EGFR to enhance T cell costimulation.

[0024] Those skilled in the art can know whether a single-chain antibody fragment is 4-1BB specific or not by several assays known in the art. Those skilled in the art can know whether an anti-4-1BB specific single-chain antibody fragment is an agonist or not by several assays known in the art.

[0025] In a preferred embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv) is defined by CDR sequences comprising the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or functionally equivalent variants thereof.

[0026] The term "functionally equivalent variant" is defined below in relation to the homotrimerization domains of the invention as equivalents applicable to functionally equivalent variants of the CDRs disclosed herein.

[0027] In a preferred embodiment, the CDRs have at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% similarity to the sequences of SEQ ID NO:1 or SEQ ID NO:42, SEQ ID NO:2 or SEQ ID NO:43, SEQ ID NO:3 or SEQ ID NO:44, SEQ ID NO:4 or SEQ ID NO:45, SEQ ID NO:5 or SEQ ID NO:46, and / or SEQ ID NO:6. , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0028] "Complementarity determining region (CDR)" is the "antigen binding site" in an antibody. CDR can be defined using various terms: (i) Complementarity determining region (CDR) exists in VH three (HCDR1, HCDR2, HCDR3) and VL three (LCDR1, LCDR2, LCDR3) based on sequence diversity (Wu et al. (1970) J Exp Med 132:211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "Hypervariable Region", "HVR", or "HV" refers to the regions of an antibody variable domain, of which there are three in VH (H1, H2, H3) and three in VL (L1, L2, L3), that are hypervariable in structure as defined by Chothia and Lesk (Chothia et al. (1987) J Mol Biol 196:901-17). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standard numbering and definitions of antigen-binding sites. The correspondence between CDR, HV, and IMGT descriptions is provided in (Lefranc et al. (2003) Dev Comp Immunol 27:55-77). The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include CDRs as defined by any of the Kabat, Chothia or IMGT methods described above, unless otherwise specified herein.

[0029] The anti-4-1BB specific agonist single-chain antibody fragments (scFv) of the present invention are alternatively defined by the framework regions (FR) having the sequences of 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 and SEQ ID NO:14 or functionally equivalent variants thereof.

[0030] In a preferred embodiment, the FR has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112, 113, 114, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 139, 139, 139, 139, 139, 139, 139, 131, 132, 133, 134, 135, 136, 137, 138, 139, 139, 139, 139, 139, 139, 139, 1 , 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0031] As used herein, a "framework region" relates to a portion of a variable domain, either VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without the CDRs.

[0032] In a preferred embodiment, the anti-4-1BB specific agonist scFv is humanized or displays a humanized VL domain and / or a partially humanized VH domain. In a specific embodiment, the anti-4-1BB specific agonist scFv comprises the sequence represented by SEQ ID NO:19.

[0033] "Humanized antibody, single chain antibody fragment, or VL, VH domain" refers to an antibody, single chain antibody fragment, or VL or VH domain in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the framework since the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0034] According to the present invention, antibodies or single chain antibody fragments can be "humanized" to reduce immunogenicity in human individuals. Humanized antibodies improve the safety and efficacy of monoclonal antibody therapy. One common method of humanization is to produce a monoclonal antibody in any suitable animal (e.g., mouse, rat, hamster) and replace its constant region with a human constant region; antibodies engineered in this manner are called "chimeras." Another common method is "CDR grafting," in which a non-human V-FR is replaced with a human V-FR. In CDR grafting, all residues except the CDR regions are of human origin. In certain embodiments, the antibodies described herein are humanized. In certain embodiments, the antibodies described herein are chimeric. In certain embodiments, the antibodies described herein are CDR grafted. Humanization may reduce or have little effect on the overall affinity of an antibody, or may improve affinity for its target after humanization. In certain embodiments, humanization increases affinity for the antibody by 10%. In certain embodiments, humanization increases affinity for the antibody by 25%. In certain embodiments, humanization increases affinity to the antibody by 35%. In certain embodiments, humanization increases affinity to the antibody by 50%. In certain embodiments, humanization increases affinity to the antibody by 60%. In certain embodiments, humanization increases affinity to the antibody by 75%. In certain embodiments, humanization increases affinity to the antibody by 100%. Affinity is suitably measured using surface plasmon resonance (SPR).

[0035] Homotrimerization domain As used herein, the term "homotrimerization domain" refers to the region responsible for non-covalent trimerization between monomers. The homotrimerization domain of the TPC of the present invention is the type XVIII collagen homotrimerization domain (TIE XVIII ), collagen XV homotrimerization domain (TIE XV ) and functionally equivalent variants thereof.

[0036] As disclosed herein, collagen XVIII or collagen XV monomers can be identical or different from each other, as long as the trimerization property is maintained compared to the trimerization property of natural collagen molecule.In certain embodiments, at least one of the monomers is different from the other two.In preferred embodiments, the three monomers are identical to each other, preferably the three monomers of collagen XVIII or collagen XV.

[0037] In one embodiment, the type XVIII collagen homotrimerization domain consists of or comprises SEQ ID NO: 15. In another embodiment, the type XV collagen homotrimerization domain consists of or comprises SEQ ID NO: 16. In another embodiment, the type XVIII collagen homotrimerization domain consists of or comprises SEQ ID NO: 17. In another preferred embodiment, the homotrimerization domain is a humanized type XVIII collagen homotrimerization domain comprising SEQ ID NO: 18.

[0038] As used herein, "functionally equivalent variants thereof" refers to naturally occurring collagen XVIII or collagen XV TIE XVIII and / or TIE XV The term "functionally equivalent variants" is intended to encompass variants in which the amino acid sequence has been modified without adversely affecting to a substantial extent the trimerization properties compared to the native type XVIII or type XV collagen molecule. Such modifications include conservative (or non-conservative) substitution of one or more amino acids for other amino acids, insertion and / or deletion of one or more amino acids, but the trimerization properties of the native type XVIII or type XV collagen protein are substantially maintained, i.e., the variants maintain the ability (disposition) to form trimers with other peptides having the same sequence under physiological conditions.

[0039] Preferably, TIE XVIII and / or TIE XVA variant of is (i) a polypeptide in which one or more amino acid residues have been replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), where such a substituted amino acid may or may not be encoded by the genetic code, (ii) a polypeptide in which one or more modified amino acid residues are present, e.g., residues modified by a substituent group bond, (iii) a polypeptide resulting from alternative processing of the analogous mRNA, and / or (iv) a polypeptide fragment. Fragments include polypeptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The variant may be post-translationally or chemically modified. Such variants should be apparent to one of skill in the art.

[0040] Those skilled in the art will recognize that to determine the corresponding sequence identity of two nucleotide sequences encoding the polypeptides of the present invention, the nucleotide sequence identity value may be appropriately adjusted by taking into account codon degeneracy, conservative amino acid substitutions, and reading frame positioning.

[0041] In the context of the present invention, "conservative amino acid change" and "conservative amino acid substitution" are used synonymously in the present invention. "Conservative amino acid substitution" refers to the interchangeability of residues with similar side chains, and refers to the replacement of one or more amino acids in a native amino acid sequence with another amino acid(s) with similar side chains, resulting in a silent change that does not change the function of the protein. Conservative substitutes for an amino acid in a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains includes serine and threonine; the group of amino acids having amide-containing side chains includes asparagine and glutamine; the group of amino acids having aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains includes lysine, arginine, and histidine; the group of amino acids having aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains includes lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains includes cysteine ​​and methionine. In some embodiments of the invention, preferred conservative amino acid substitutions are valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. XVIII and / or TIE XV It refers to a functionally equivalent variant of the amino acid sequence of one or more amino acids that differs from the other amino acid sequence by one or more conservative amino acid substitutions, which results in a sequence that is obtained as a result of one or more conservative amino acid substitutions. It is well known in the art that one or more amino acids in a polypeptide sequence can be replaced with at least one other amino acid with a similar charge and polarity, such that the substitution results in a silent change in the modified polypeptide that does not change its function compared to that of the unmodified sequence. The present invention further provides that the polypeptide sequence XVIII and / or TIE XV As long as it has the same, similar or equivalent function as TIE XVIII and / or TIE XV A polypeptide sequence refers to any polypeptide sequence which differs by one or more amino acids as a result of either conservative or non-conservative substitutions, and / or as a result of either sequence insertions or deletions, compared to the sequence given by

[0042] "Codon degeneracy" refers to differences in the genetic code that allow for variation in nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide. Those skilled in the art are well aware of the codon bias exhibited by a particular host cell when using nucleotide codons to specify a given amino acid residue. Therefore, for ectopic expression of a gene in a host cell, it is desirable to design or synthesize the gene so that its codon usage is close to the codon usage of the host cell listed in the codon usage table.

[0043] The term "identity", "identical" or "percent identity" in the context of two or more amino acid or nucleotide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid or nucleotide residues when compared and aligned (with gaps introduced, if necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art.

[0044] The percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window. The aligned sequences can be polynucleotide or polypeptide sequences. For optimal alignment of two sequences, a portion of the polynucleotide or amino acid sequence in the comparison window can contain insertions or deletions (i.e., gaps) compared to the reference sequence (not containing insertions or deletions). The percentage of sequence identity is calculated by determining the number of positions where the same nucleotide residue or the same amino acid residue occurs in both sequences being compared to obtain the number of matched positions, then dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence identity between two polypeptide sequences or two polynucleotide sequences can be determined, for example, by using the GAP program in WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc., based on the method of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970), using a set of default parameters for pairwise comparisons (for amino acid sequence comparisons: gap creation penalty = 8, gap extension penalty = 2; for nucleotide sequence comparisons: gap creation penalty = 50, gap extension penalty = 3), or by using the TBLASTN program in the BLAST 2.2.1 software suite, using the BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992) and a set of default parameters for pairwise comparisons (gap creation cost = 11, gap extension cost = 1).

[0045] The percentage of sequence identity between polypeptides and their corresponding functions can be determined using various homology-based search algorithms available for comparing a query sequence to a protein database, including, for example, BLAST, FASTA, and Smith-Waterman. The BLASTX and BLASTP algorithms can be used to provide protein functional information. Many values ​​are examined to assess the reliability of the functional assignment. Useful measurements include "E-value" (also written as "hit_p"), "percent identity", "percent query coverage", and "percent hit coverage". In BLAST, the E-value (i.e., expectation value) represents the number of different alignments with a score equal to or greater than the raw alignment score (S) that would be expected to occur by chance in a database search. Thus, the lower the E-value, the more significant the match. Because the size of the database is a factor in the E-value calculation, the E-values ​​obtained from performing BLAST searches against public databases such as GenBank have generally increased over time for any given query / entry match. Thus, in setting standards for the reliability of polypeptide function prediction, a "high" BLASTX match is considered to have an E-value of the top BLASTX hit less than 1E-30; a moderate BLASTX is considered to have an E-value between 1E-30 and 1E-8; and a low BLASTX is considered to have an E-value higher than 1E-8. Percent identity refers to the percentage of perfectly matched amino acid residues along the length of the sequence portion aligned by the BLAST algorithm. In setting standards for the reliability of polypeptide function prediction, a "high" BLAST match is considered to have a percent identity of at least 70% to the top BLAST hit; a moderate percent identity value is considered to be between 35% and 70%; and a low percent identity is considered to be less than 35%. Of particular interest in protein function assignment is the use of a combination of E-values, percent identity, query coverage, and hit coverage.Query coverage refers to the percentage of the query sequence that is displayed in the BLAST alignment, and hit coverage refers to the percentage of the database entries that are displayed in the BLAST alignment. For the purpose of defining the polypeptides that are functionally encompassed by the present invention, the function of the polypeptide is inferred from the function of the protein homologs such as SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, and the polypeptides of the present invention are those that are either (1) hit_p<1e-30 or identity %>35% and query coverage>50% and hit coverage>50%, or (2) hit_p<1e-8 and query coverage>70% and hit coverage>70%.

[0046] TIE XVIII Functionally equivalent variants of also include sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:15, the sequence of SEQ ID NO:17 or the sequence of SEQ ID NO:18.

[0047] TIE XV Functionally equivalent variants of also include sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:16.

[0048] The ability of functionally equivalent variants to form trimers can be determined by conventional methods known to those skilled in the art. For example, as a simple example, the ability of functionally equivalent variants to form trimers can be determined using standard chromatography techniques. Thus, the variant to be evaluated is placed under suitable trimerization conditions, and the complex is subjected to a standard chromatographic assay under non-denaturing conditions so that the final complex (trimer) formed is not altered. When the variant is properly trimerized, the molecular size of the complex is three times heavier than that of one molecule of the variant. The molecular size of the complex can be determined using standard methods such as analytical centrifugation, mass spectrometry, size exclusion chromatography, and sedimentation velocity.

[0049] TIE XVIII and / or TIE XV The TIE may be derived from any subject, preferably a mammal, such as a mouse, rat, monkey, human, etc. In a preferred embodiment, the TIE XVIII In another preferred embodiment, the TIE XV In another preferred embodiment, the TIE XVIII is derived from mouse type XVIII collagen. XV is derived from mouse type XV collagen. In a more preferred embodiment, the TIE XVIII is a small homotrimerization domain of mouse type XVIII collagen.

[0050] TIE XVIII and / or TIE XV can be used to generate, among other trimeric polypeptide complexes (TPCs), functionally active mono- and bispecific trivalent N-terminal TPCs, trivalent C-terminal TPCs, mono- and bispecific trivalent N / C-terminal TPCs; and mono- and bispecific hexavalent single-chain N / C-terminal TPCs. Additionally, single domains (V HH) antibodies or growth factors (e.g., VEGF). Thus, monospecific or multispecific (e.g., bi-, tri-, tetra-specific, etc.) multivalent (e.g., trivalent, tetravalent, pentavalent, or hexavalent) recombinant molecules with different combinations of specificity and valency can be readily generated. In certain embodiments, the TIE XVIII and / or TIE XV is used to generate monospecific TCPs. In a preferred embodiment, the TIE XVIII and / or TIE XV are used to generate monospecific or bispecific TCPs.

[0051] Polypeptide regions capable of specifically binding to tumor-associated antigens The TPC according to the invention may be monospecific, i.e. it comprises a polypeptide region capable of specifically binding to a tumor-associated antigen, but it may also comprise one or more polypeptide regions capable of specifically binding to tumor-associated antigens present on the surface of tumor cells. This results in a bispecific antibody comprising a region that binds to 4-1BB and exerts an agonistic effect, and a polypeptide region that binds to a tumor-associated antigen. It will be understood that the number of monomers in the TPC that comprise a region capable of specifically binding to a tumor-associated antigen may be one, two or three. In a preferred embodiment, one of the monomeric polypeptides comprises a polypeptide region capable of specifically binding to a tumor-associated antigen. In another preferred embodiment, two of the monomeric polypeptides comprise a polypeptide region capable of specifically binding to a tumor-associated antigen. In another preferred embodiment, three monomeric polypeptides comprise a polypeptide region capable of specifically binding to a tumor-associated antigen.

[0052] The term "specific binding" has been defined in detail above for the agonist of the anti-4-1BB specific agonist single chain antibody fragment and applies equally to the region capable of specifically binding to the tumor associated antigen. Typically, the antibody has a binding capacity of approximately 10 -7 Less than M, e.g., about 10 -8 Less than M, 10 -9 Less than M or 10-10 The term "K D " or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present invention have an affinity for approximately 10, as determined, for example, using surface plasmon resonance (SPR) technology on a BIACORE instrument. -7 Less than M, e.g., about 10 -8 Less than M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) binds to the antigen.

[0053] As used herein, the term "tumor-associated antigen" or "TAA" refers to any antigen that can match a patient's cancer status or type with an appropriate immunotherapy product or regimen. TAA may be expressed by the cancer cells themselves or may be associated with non-cancerous components of the tumor, such as neovasculature or other stroma associated with the tumor. Among the tumor antigens that are expressed by tumor cells and can serve as targets for immune effector mechanisms are proteins, generally glycoproteins, peptides, carbohydrates, and glycolipids. Non-limiting examples of tumor-associated antigens include AFP (alpha (α)-fetoprotein), AIM-2 (interferon-inducible protein 2 absent in melanoma), ART-4 (adenocarcinoma antigen 4 recognized by T cells), BAGE (B antigen), BCMA, CAMEL (CTL-recognized antigen on melanoma), C16a, CD19, CD20, CD22, CD30, CD3, CD40, CD33, CD123, VEGF, IL-6, MUC-1, endoglucanase, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL- Lin, DLL, B7-H3, CEA (carcinoembryonic antigen), DAM (melanoma differentiation antigen), Ep-CAM (epithelial cell adhesion molecule), ErB3, FAP, gpA33, Her2, IGF-1R, CD-5, FAP, MAGE (melanoma antigen), MART-1 / Melan-A (melanoma antigen-1 / melanoma antigen A recognized by T cells), MC1R (melanocortin 1 receptor), MET, MUC-1, NY-ESO-1 (New York esophageous 1), OA1 (ocular albinism type 1 protein), P-cachelin, PD-L1, PSMA (prostate specific membrane antigen), SART-1, -2, -3 (squamous antigen rejection tumor 1, 2, 3), survivin-2B (surviving intron 2), TRP (tyrosinase-related protein). Antigens may be expressed on the surface of tumor cells or secreted. In a preferred embodiment, antigens are cell surface antigens. Those skilled in the art can determine the presence of serum antibodies of patients against potential tumor antigens, for example, by using SEREX (serological identification of antigens by recombinant expression cloning), in which serum is reacted with a cDNA library derived from tumor cells to identify target antigens.

[0054] In one embodiment, the TAA is EGFR.

[0055] In another embodiment, the TAA is CEA.

[0056] In one embodiment, the region capable of specifically binding to a TAA does not have agonistic potential for said TAA.

[0057] In one embodiment, the region capable of specifically binding to a TAA is an antibody, more preferably a "single chain antibody, nanobody or "non-immunoglobulin agent", the term being defined above in the context of anti-4-1BB specific agonist single chain antibody fragments and is equally applicable to regions capable of specifically binding to tumor-associated antigens.

[0058] In a preferred embodiment, the polypeptide region capable of specifically binding to a TAA is located N-terminal or C-terminal to the homotrimerization domain. In a preferred embodiment, the TAA is located C-terminal to the homotrimerization domain.

[0059] In a preferred embodiment, when the molecule capable of specifically binding to the anti-4-1BB specific agonistic single-chain antibody fragment is located N-terminal to the homotrimerization domain, the molecule capable of specifically binding to the tumor-associated antigen is located C-terminal to the homotrimerization domain. In another preferred embodiment, when the polypeptide region capable of specifically binding to the tumor-associated antigen is located C-terminal to the homotrimerization domain, the anti-4-1BB specific agonistic single-chain antibody fragment is located N-terminal to the homotrimerization domain.

[0060] In a preferred embodiment, the tumor-associated antigen is epidermal growth factor receptor (EGFR). As used herein, the term "epidermal growth factor receptor" or "EGFR" is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. It refers to a tyrosine kinase that regulates signal transduction pathways and cell growth and survival, and shows affinity for EGF molecules. The ErbB family of receptors consists of four closely related subtypes: ErbB1 (epidermal growth factor receptor [EGFR]), ErbB2 (HER2 / neu), ErbB3 (HER3), and ErbB4 (HER4), as well as their variants (e.g., deletion mutant EGFR, such as Humphrey et al. (Proc. Natl. Acad. Sci. USA, 1990, 87: 4207-4211)). Non-limiting examples of molecules capable of binding to EGFR include natural ligands epidermal growth factor (EGF), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin (EPR), transforming growth factor-α (TGF-α), and epigene (EPG). In one embodiment, the molecule capable of specifically binding to EGFR does not have agonistic potential. In a preferred embodiment, the EGFR is human.

[0061] In a preferred embodiment, the polypeptide region capable of specifically binding to EGFR is an antibody.

[0062] "Antibody" is used in a broad sense to include immunoglobulin molecules, including monoclonal antibodies, including mouse, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, trimeric or multimeric antibodies, single chain antibodies, single domain antibodies, antibody mimetics, and any other modified structure of an immunoglobulin molecule that contains an antigen-binding site of the required specificity. A "full-length antibody molecule" is composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of CH1, hinge, CH2 and CH3 domains). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with framework regions (FR). Each VH and VL is composed of three CDR and four FR segments arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the amino acid sequence of the heavy chain constant region. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant region.

[0063] In a preferred embodiment, the anti-EGFR antibody is an scFv, a nanobody or an antibody mimetic.

[0064] As used herein, the term "single chain antibody" refers to a genetically engineered molecule comprising a variable light chain region and a variable heavy chain region joined by a suitable peptide linker, formed as a genetically fused single chain molecule.

[0065] As used herein, the term "nanobody" refers to a single domain antibody (sdAb), which is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is capable of selectively binding to a specific antigen.

[0066] As used herein, the term "antibody mimetic" refers to any compound that can specifically bind to an antigen in a manner similar to an antibody, but is not necessarily structurally related to an antibody. "Mimetic" compounds include compounds in which the chemical structure of a compound required for functional activity is replaced with another chemical structure that mimics the conformation of the compound. Examples of mimetics include peptidic compounds in which the peptide backbone is replaced with one or more benzodiazepine molecules (see, for example, James, GL et al. (1993) Science 260:1937-1942), or oligomers that mimic peptide secondary structures using amide bond isosteres and / or modifications of the natural peptide backbone, including chain extensions or heteroatom incorporation; examples of which include azapeptides, oligocarbamates, oligoureas, β-peptides, γ-peptides, oligo(phenyleneethynylene), vinylic sulfonopeptides, poly-N-substituted glycines (peptoids), and the like. Methods for preparing peptidomimetic compounds are well known in the art and are detailed, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0067] In another preferred embodiment, the anti-EGFR antibody is a nanobody.

[0068] In another preferred embodiment, the EGFR antibody is an anti-EGFR (huEGFR) single domain antibody (VHH). In a more preferred embodiment, the anti-EGFR (huEGFR) single domain antibody (VHH) nucleotide sequence comprises the sequence represented by SEQ ID NO: 20. In another preferred embodiment, the anti-EGFR (EGFR) single domain antibody (VHH) CDR sequence is CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 26), CDR3 (SEQ ID NO: 27). In another preferred embodiment, the anti-EGFR (huEGFR) single domain antibody (VHH) FR sequence is FR1 (SEQ ID NO: 21), FR2 (SEQ ID NO: 22), FR3 (SEQ ID NO: 23), FR4 (SEQ ID NO: 24).

[0069] In another preferred embodiment, the EGFR antibody is a humanized anti-EGFR (huEGFR) single domain antibody (VHH). In a more preferred embodiment, the humanized anti-EGFR (huEGFR) single domain antibody (VHH) comprises the sequence represented by SEQ ID NO: 28. In another preferred embodiment, the anti-EGFR (huEGFR) single domain antibody (VHH) CDR sequence is CDR1 (SEQ ID NO: 33), CDR2 (SEQ ID NO: 34), CDR3 (SEQ ID NO: 35). In another preferred embodiment, the anti-EGFR (huEGFR) single domain antibody (VHH) FR sequence is FR1 (SEQ ID NO: 29), FR2 (SEQ ID NO: 30), FR3 (SEQ ID NO: 31), FR4 (SEQ ID NO: 32).

[0070] In another preferred embodiment, the tumor-associated antigen is carcinoembryonic antigen (CEA). As used herein, the term "carcinoembryonic antigen" or "CEA", also known as CEACAM1, BGP1, BGPI, CD66a, BGP, refers to carcinoembryonic antigen-related cell adhesion molecule 1. The human gene encoding said protein is shown in the Ensembl database under the accession number ENSG00000079385.

[0071] A linker region between the homotrimerization domain, the anti-4-1BB specific agonistic single-chain antibody fragment and the polypeptide region capable of specifically binding to a tumor-associated antigen. The different elements of the monomeric polypeptides forming the TPC according to the invention may be directly linked to each other or may be connected via amino acid spacers or linkers.

[0072] In one embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv), the homotrimerization domain, and / or the polypeptide region capable of specifically binding to a tumor-associated antigen are directly connected. In another embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv) and the homotrimerization domain are directly connected. In another preferred embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv) and the polypeptide region capable of specifically binding to a tumor-associated antigen are directly connected. In another preferred embodiment, the homotrimerization domain and the polypeptide region capable of specifically binding to a tumor-associated antigen are directly connected. In another preferred embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv), the homotrimerization domain, and the polypeptide region capable of specifically binding to a tumor-associated antigen are directly connected.

[0073] In another embodiment, the anti-4-1BB specific agonist single antibody fragment (scFv), the homotrimerization domain, and / or the polypeptide region capable of specifically binding to a tumor-associated antigen are connected by an amino acid linker or spacer. In another embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv) and the homotrimerization domain are connected by an amino acid linker or spacer. In another preferred embodiment, the anti-4-1BB specific agonist single chain antibody fragment (scFv) and the polypeptide region capable of specifically binding to a tumor-associated antigen are connected by an amino acid linker or spacer. In another preferred embodiment, the homotrimerization domain and the polypeptide region capable of specifically binding to a tumor-associated antigen are connected by an amino acid linker or spacer.

[0074] In another embodiment, the agonist of the anti-4-1BB specific agonist single chain antibody fragment (scFv) is connected via an amino acid linker to the homotrimerization domain, which is connected by an amino acid spacer to the polypeptide region capable of specifically binding to the tumor-associated antigen.

[0075] As disclosed herein, a spacer is an insert that connects or links peptides of appropriate length and characteristics. In general, the spacer functions as a hinge region between the domains, allowing the individual domains to move independently of each other while maintaining their three-dimensional form. In this sense, a preferred spacer would be a hinge region that is characterized by structural ductility or flexibility that allows this movement. The length of the spacer can vary, and typically the number of amino acids in the spacer is 100 amino acids or less, preferably 50 amino acids or less, more preferably 40 amino acids or less, even more preferably 30 amino acids or less, and even more preferably 20 amino acids or less.

[0076] Alternatively, a suitable spacer can be based on the sequence of 10 amino acid residues of the upper hinge region of mouse IgG3, which has been used to generate coiled-coil dimerized antibodies (Pack P. and Pluckthun, A., 1992, Biochemistry 31:1579-1584) and can be useful as a spacer peptide according to the present invention. It can also be the corresponding sequence of the upper hinge region of human IgG3 or other human Ig subclasses (IgG1, IgG2, IgG4, IgM and IgA). The sequences of human Ig are not expected to be immunogenic in humans. Additional spacers that can be used in the present invention include peptides of the amino acid sequence GAP, AAA.

[0077] In a particular embodiment, the spacer is a peptide with structural flexibility (i.e., a flexible linking peptide or "flexible linker") and comprises two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. In another particular embodiment, the spacer is a peptide comprising repeats of amino acid residues, in particular Gly and Ser, or any other suitable repeats of amino acid residues. Virtually any flexible linker can be used as a spacer according to the present invention.

[0078] In a preferred embodiment, the spacer is a flexible linker. In a more preferred embodiment, the flexible linker is between 1 and 18 residues. In an even more preferred embodiment, the flexible linker is 5 residues, 15 residues, 17 residues, or 18 residues, preferably 15 residues.

[0079] In more preferred embodiments, the flexible linker is at least 1 residue, at least 2 residues, at least 3 residues, at least 4 residues, at least 5 residues, at least 6 residues, at least 7 residues, at least 8 residues, at least 9 residues, at least 10 residues, at least 11 residues, at least 12 residues, at least 13 residues, at least 14 residues, at least 15 residues, at least 16 residues, at least 17 residues, or at least 18 residues. In even more preferred embodiments, the flexible linker is 15 residues in length.

[0080] In a preferred embodiment, the homotrimerization domain is directly linked to either the anti-4-1BB specific agonist single chain antibody fragment or to a region capable of specifically binding to a tumor-associated antigen. In another preferred embodiment, the homotrimerization domain is directly linked to either the anti-4-1BB specific agonist single chain antibody fragment and to a region capable of specifically binding to a tumor-associated antigen. In a preferred embodiment, the homotrimerization domain is directly linked to either the anti-4-1BB specific agonist single chain antibody fragment or to a region capable of specifically binding to a tumor-associated antigen via a flexible linker. In another preferred embodiment, the homotrimerization domain is directly linked to either the anti-4-1BB specific agonist single chain antibody fragment and to a region capable of specifically binding to a tumor-associated antigen via a flexible linker.

[0081] In a more preferred embodiment, the flexible linker is at least 1 residue, at least 2 residues, at least 3 residues, at least 4 residues, at least 5 residues, at least 6 residues, at least 7 residues, at least 8 residues, at least 9 residues, at least 10 residues, at least 11 residues, at least 12 residues, at least 13 residues, at least 14 residues, at least 15 residues, at least 16 residues, at least 17 residues, or at least 18 residues. In a more preferred embodiment, the flexible linker is 17 and / or 18 residues long. In an even more preferred embodiment, the anti-4-1BB specific agonist single-chain antibody fragment is linked to the homotrimerization domain via a linker 18 residues long and / or the region capable of specifically binding to a tumor-associated antigen is linked to the homotrimerization domain via a linker 16 residues long.

[0082] In a preferred embodiment, the 18 residue long linker is SEQ ID NO: 47. In another preferred embodiment, the 16 residue long linker is SEQ ID NO: 36.

[0083] In a preferred embodiment, at least one monomer of the TPC further comprises a tag suitable for detection and / or purification of the trimeric polypeptide.Non-limiting examples of tags include affinity purification tags such as tag peptides; illustrative non-limiting examples of said tags include polyhistidine [poly(His)] sequences, peptide sequences recognizable by antibodies that can be used to purify the resulting fusion protein by immunoaffinity chromatography, such as epitopes derived from hemagglutinin of fever viruses, c-myc tags, Strep tags, etc.In another preferred embodiment, multiple monomers of the TPC further comprise tags suitable for detection and / or purification of the trimeric polypeptide.

[0084] In a particular embodiment, each of the three monomeric polypeptides comprises one affinity purification tag, which, if different from each other (e.g. affinity purification tags "a", "b" and "c", where tag "a" is recognized by binding to substance A, tag "b" is recognized by binding to substance B and tag "c" is recognized by binding to substance C), are subjected to a three-step affinity purification procedure designed to selectively recover only those TPCs of the invention that exhibit affinity for the corresponding substances (A, B and C). The affinity purification tags can be fused directly in tandem or can be fused to the monomeric polypeptides via a cleavable linker, i.e. a peptide segment that comprises an amino acid sequence that can be specifically cleaved by enzymatic or chemical means (i.e. a recognition / cleavage site). In certain embodiments, the cleavable linker comprises an amino acid sequence cleavable by a protease, such as enterokinase, Arg C endoprotease, Glu C endoprotease, Lys C endoprotease, factor Xa, or in another particular embodiment, the cleavable linker comprises an amino acid sequence cleavable by a chemical reagent, such as, for example, cyanogen bromide, which cleaves methionine residues, or any other suitable chemical reagent. Cleavable linkers are useful when subsequent removal of the affinity purification tag is desired.

[0085] In a preferred embodiment, the three monomer polypeptides contain the same affinity purification tag. The tag can be located at any position of the monomer, in particular the C-terminal or N-terminal to the homotrimerization domain. In a more preferred embodiment, the tag is at the N-terminus of the anti-4-1BB specific agonist single chain antibody fragment. In a more preferred embodiment, the tag is a His6-myc tag or a strep-Flag tag. In a more preferred embodiment, the tag is a flap tag of SEQ ID NO: 37 and / or a StrepII tag of SEQ ID NO: 38.

[0086] In another preferred embodiment, the monomer further comprises a moiety that increases the circulating half-life of the trimeric polypeptide. According to the present invention, "half-life" is the period of time required for the concentration or amount of a compound in the body to decrease to half of a given concentration or amount. The given concentration or amount does not have to be the maximum observed during the observation period, nor the concentration or amount present at the start of administration, since the half-life is completely independent of the concentration or amount selected as the "starting point".

[0087] Non-limiting strategies for increasing suboptimal half-life profiles for therapeutic dosing are known to those of skill in the art and include genetic fusion of a pharmacologically active peptide or protein with a naturally occurring long half-life protein or protein domain (e.g., Fc fusion, transferrin fusion, or albumin fusion); genetic fusion of a pharmacologically active peptide or protein with an inactive polypeptide, such as XTEN (also known as recombinant PEG or "rPEG"), homoamino acid polymer (HAP; HAPylated), proline-alanine-serine polymer (PAS; PASylated), or elastin-like peptide (ELP; ELPylated); increasing the hydrodynamic radius by chemical conjugation of a pharmacologically active peptide or protein with a repeating chemical moiety, such as PEG (PEGylated) or hyaluronic acid; significantly increasing the negative charge fusing a pharmacologically active peptide or protein by polysialylation; or genetic fusion of a pharmacologically active peptide or protein with human GG These include fusing the molecule of interest with negatively charged, highly sialylated peptides (e.g., the carboxy-terminal peptide [CTP; of the chorionic gonadotropin (CG) β chain]), which are known to extend the half-life of native proteins such as the β subunit; non-covalent binding to normally long half-life proteins such as HSA, human IgG, or transferrin by attaching a peptide or protein-binding domain to a biologically active protein; and chemical conjugation of peptides or small molecules to long half-life proteins such as human IgG, Fc portions, or HSA.

[0088] In preferred embodiments, the half-life can be increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to a trimeric polypeptide that does not include any moiety that increases the TPC circulating half-life.

[0089] The moiety responsible for increasing the circulating half-life of the TPC may be present in one of the monomers of the TPC, two of the monomers of the TPC, or three of the TPC monomers. Further, the moiety responsible for increasing the circulating half-life of the TPC may be present at the N-terminus of the monomer, the C-terminus of the monomer, N-terminus to the homotrimerization domain, or C-terminus to the homotrimerization domain.

[0090] In another preferred embodiment, the moiety that increases the circulatory half-life of the trimeric polypeptide is an albumin fragment or an albumin binding moiety.

[0091] The term "binding moiety" refers to a domain that specifically binds to an antigen or epitope, independent of a different epitope or antigen-binding domain. A binding moiety may be a domain antibody (dAb) or a domain that is a derivative of a non-immunoglobulin protein scaffold, such as a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, adnectin, affibody, avimer, GroEl, transferrin, GroES and fibronectin, which binds to a ligand other than the natural ligand. In a preferred embodiment, the moiety binds to serum albumin.

[0092] All the above terms and embodiments are equally applicable to the present disclosure.

[0093] Polynucleotides, Vectors and Host Cells In another embodiment, the present invention relates to a polynucleotide encoding at least one monomeric polypeptide forming part of a trimeric polypeptide of the present invention.

[0094] As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer having deoxyribonucleotide or ribonucleotide bases. In certain embodiments, the polynucleotide has ribonucleotide bases. In preferred embodiments, the polynucleotide has deoxyribonucleotide bases. In more preferred embodiments, the polynucleotide encodes at least one, at least two, or at least three of the monomeric polypeptides that form part of the trimeric polypeptide according to the present invention.

[0095] In a preferred embodiment, the polynucleotide further comprises a sequence encoding a signal sequence, located 5' to the sequence encoding the polypeptide and in the same open reading frame as said sequence. As used herein, the term "signal sequence" or "signal peptide" refers to a peptide of relatively short length, generally 5-30 amino acid residues, that directs a protein synthesized in a cell to the secretory pathway. Signal peptides usually contain a series of hydrophobic amino acids that adopt a secondary alpha-helical structure. In addition, many peptides contain a series of positively charged amino acids that may contribute to the protein adopting a topology suitable for its transport. Signal peptides tend to have a motif at their carboxyl terminus that is recognized by peptidases, which are capable of hydrolyzing the signal peptide to generate a free signal peptide and a mature protein. The signal peptide may be cleaved once the protein of interest has reached the appropriate location. Any signal peptide may be used in the present invention. In a preferred embodiment, the signal sequence is the signal sequence of oncostatin M.

[0096] In another aspect, the present invention relates to a vector comprising a polynucleotide according to the present invention.

[0097] As used herein, the term "vector" or "expression vector" refers to a replicating DNA construct used to express at least one polynucleotide in a cell, preferably a eukaryotic cell. The choice of expression vector depends on the choice of host. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids derived from E. coli, including pCR1, pBR322, pCR3.1, pCMV3, pMB9 and their derivatives, and broader host range plasmids, such as M13 and filamentous single-stranded DNA phages. These vectors may contain additional separate cassettes for expressing selectable markers used to initially select clones that have incorporated foreign DNA during the transformation protocol. Expression vectors preferably contain an origin of replication. Expression vectors may also contain one or more multiple cloning sites.

[0098] The expression vector may also include a prokaryotic origin of replication, which is necessary for the vector to amplify in bacteria. In addition, the expression vector may also include a selection gene for bacteria, such as a gene encoding a protein that confers resistance to antibiotics, such as ampicillin, kanamycin, chloramphenicol, etc. The expression vector may also include one or more multiple cloning sites. A multiple cloning site is a polynucleotide sequence that includes one or more unique restriction sites. Non-limiting examples of restriction sites include EcoRI, SacI, KpnI, SmaI, XmaI, BamHI, XbaI, HincII, PstI, SphI, HindIII, AvaI, or any combination thereof.

[0099] The polynucleotide(s) expressed in the vector of the present invention, and the RNA or DNA constructs necessary for the preparation of the expression vector of the present invention can be obtained by conventional molecular biology methods described in general laboratory manuals, for example, "Molecular cloning: a laboratory manual" (eds. Joseph Sambrook, David W. Russell, 2001, 3rd ed., Cold Spring Harbor, New York) or "Current protocols in molecular biology" (eds. FMA Usubel, R. Brent, RE Kingston, DD Moore, JA Smith, JG Seidman and K. Struhl, vol. 2, Greene Publishing Associates and Wiley Interscience, New York, NY, updated September 2006).

[0100] In another aspect, the present invention relates to a host cell comprising the vector according to the invention.

[0101] The term "host cell" is used to refer not only to a particular subject cell, but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in the progeny, either due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein. The host cell may be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., yeast, insect or plant cell), which may be prepared by conventional genetic engineering techniques, including inserting the nucleic acid of the present invention into a suitable expression vector, transforming a suitable host cell with the vector, and culturing the host cell under conditions that allow expression of the polypeptide portion of the monomeric polypeptide that constitutes the TPC of the present invention. The nucleic acid of the present invention may be placed under the control of a suitable promoter, which may be an inducible promoter or a constitutive promoter. Depending on the expression system, the polypeptide may be recovered from the extracellular phase, the periplasm, or the cytoplasm of the host cell.

[0102] Suitable vector systems and host cells are well known in the art, as evidenced by the extensive literature and resources available to those skilled in the art. Since the present invention also relates to the construction of vectors and the use of the nucleic acids of the present invention in host cells, the following provides general considerations related to such uses, as well as specific considerations in carrying out this aspect of the invention.

[0103] Generally, prokaryotes are preferred for the initial cloning of the nucleic acid of the present invention and for the construction of the vector of the present invention. For example, in addition to the specific strains mentioned in the more specific disclosure below, strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli X1776 (ATCC No. 31537) may be mentioned as examples. Of course, these examples are intended to be illustrative rather than limiting.

[0104] Prokaryotes can also be used for expression, as efficient purification and protein refolding strategies are available. The aforementioned strains, as well as bacilli such as E. coli W3110 (F-, λ-, prototrophic strain, ATCC No. 273325), Bacillus subtilis, or other enterobacteria such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species can be used.

[0105] Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector usually has a replication site and a marking sequence that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed with pBR322, a plasmid derived from E. coli species. The pBR322 plasmid contains genes for ampicillin resistance and tetracycline resistance, thus providing an easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that can be used by the microorganism for expression.

[0106] The promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems, and the tryptophan (trp) promoter system. Although these are the most commonly used, other microbial promoters have been discovered and utilized, and details of their nucleotide sequences have been published, allowing those skilled in the art to ligate them functionally with plasmid vectors. Certain genes from prokaryotes may be efficiently expressed in E. coli from their own promoter sequences, without the need to add another promoter by artificial means.

[0107] In addition to prokaryotes, eukaryotic microbes such as yeast cultures may be used. Among eukaryotic microbes, Saccharomyces cerevisiase, or common baker's yeast, is the most commonly used, although many other strains are commonly available. For expression in Saccharomyces, for example, the plasmid YRp7 is commonly used. This plasmid already contains the trp1 gene and provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, for example ATCC number 44076 or PEP4-1. The presence of the trp1 lesion as a feature of the yeast host cell genome provides an effective environment for detecting transformants by growth in the absence of tryptophan.

[0108] Suitable promoting sequences in yeast vectors include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, termination sequences associated with these genes are also ligated 3' to the sequence to be expressed in the expression vector to provide polyadenylation and termination of the mRNA.

[0109] Other promoters which have the added advantage of transcription being controlled by growth conditions are the promoter regions for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsible for maltose and lactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

[0110] In addition to microorganisms, cultures of cells derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is viable, whether from vertebrate or invertebrate culture. However, interest is greatest in vertebrate cells, and propagation of vertebrates in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines, as well as W138, BHK, COS-7, human embryonic kidney (HEK) 293, and MDCK cell lines. In addition, the baculovirus-insect cell expression system is widely used to produce recombinant proteins and antibodies.

[0111] Expression vectors for such cells usually contain (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, together with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences.

[0112] For use in mammalian cells, control functions in expression vectors are often provided by viral material; for example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus (CMV), and most frequently simian virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful, since both are easily obtained from the virus as fragments that also contain the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided that the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication is included. In addition, it is possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell system.

[0113] The origin of replication may be provided by construction of the vector with a foreign origin, which may be derived, for example, from SV40 or other viruses (e.g., polyoma, adeno, etc.), or may be provided by the host cell's chromosomal replication machinery. If the vector is integrated into the host cell's chromosome, the latter is often sufficient.

[0114] In producing monomeric polypeptides constituting the TPC of the invention, it may be necessary to further process the polypeptide, for example by introducing non-proteinaceous functions into the polypeptide, by subjecting the material to appropriate refolding conditions (e.g. by using the generally applicable strategies suggested in WO 94 / 18227), or by cleaving off undesired peptide portions of the monomer (e.g. expression-enhancing peptide fragments that are undesired in the final product).

[0115] In view of the above discussion, a method for recombinantly producing said TPC of the invention or said monomeric polypeptides constituting said TPC of the invention is also part of the present invention, as is a vector capable of carrying and / or replicating the nucleic acid of the invention in a host cell or cell line. According to the present invention, the expression vector can be, for example, a virus, a plasmid, a cosmid, a minichromosome or a phage.

[0116] Another aspect of the invention is a transformed cell (i.e., a host cell of the invention) useful in the above-described methods, carrying a nucleic acid of the invention and capable of replicating, the host cell may be a microorganism such as a bacterium, yeast, or protozoa, or may be a cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. The cell may also be transfected.

[0117] Yet another aspect of the invention relates to stable cell lines producing the monomeric polypeptides constituting the TPC of the invention or polypeptide parts thereof, preferably said cell lines harboring and expressing the nucleic acid of the invention. Of particular interest are cells derived from the mammalian cell lines HEK and CHO.

[0118] All of the above terms and embodiments are equally applicable to this aspect of the invention.

[0119] Therapeutic combinations In another aspect, the present invention relates to a combination comprising a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, or a host cell according to the present invention and an immune checkpoint blocker.

[0120] By "combination" is meant various combinations of the trimeric polypeptide of the invention, the polynucleotide according to the invention, the vector according to the invention or the host cell according to the invention with immune checkpoint blockers in compositions, in combination mixtures composed of separate formulations of single active compounds such as "tank mixes", and in combined use of single active ingredients when applied sequentially, i.e. one by one over a reasonably short period of time such as a few hours or days, or applied by simultaneous administration.

[0121] The combination of the trimeric polypeptide of the invention, the polynucleotide of the invention, the vector of the invention, or the host cell of the invention with an immune checkpoint blocker may be formulated for simultaneous, separate, or sequential administration thereof. This means that the combination of the two compounds: - As a combination where the two compounds are part of the same pharmaceutical formulation and are always administered together - As a combination of two units each containing one of the substances offering the possibility of simultaneous, sequential or separate administration This means that it can be administered.

[0122] In a particular embodiment, the trimeric polypeptide of the invention, the polynucleotide according to the invention, the vector according to the invention, or the host cell according to the invention is administered independently (i.e. in two units) but simultaneously with the immune checkpoint blocker.

[0123] In another specific embodiment, the trimeric polypeptide of the invention, the polynucleotide according to the invention, the vector according to the invention, or the host cell according to the invention is administered first, and then the immune checkpoint blocker is administered separately or sequentially.

[0124] In yet another specific embodiment, the immune checkpoint blocker is administered first, and then the trimeric polypeptide of the invention, the polynucleotide according to the invention, the vector according to the invention or the host cell according to the invention is administered separately or sequentially as defined.

[0125] Furthermore, the compounds may be administered in the same or different dosage forms or by the same or different routes of administration (e.g., one compound may be administered topically and the other compound may be administered orally). Preferably, both compounds are administered orally.

[0126] "Immune checkpoint blockers" or "immune checkpoint inhibitors" as used herein refer to a group of molecules that inhibit proteins called checkpoints, which are a type of signal to control antigen recognition of T cell receptors (TCRs) during the immune response. Immune checkpoints are inhibitory regulators of the immune system that are crucial to control the duration and extent of immune responses to maintain self-tolerance, prevent autoimmunity, and minimize secondary tissue damage. These immune checkpoints are often overexpressed on tumor cells or on non-transformed cells within the tumor microenvironment, compromising the ability of the immune system to mount an effective anti-tumor response. These molecules can effectively function as "brakes" that downregulate or inhibit adaptive immune responses. Thus, these molecules are useful agents to prevent cancer cells from evading the patient's immune system. One of the major mechanisms of anti-tumor immune destruction is known as "T cell exhaustion," which results from chronic exposure to antigens causing the upregulation of inhibitory receptors. These inhibitory receptors function as immune checkpoints to prevent uncontrolled immune responses.

[0127] PD-1 and its co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin and mucin domain-3 (Tim-3), and lymphocyte activation gene-3 (Lag-3; CD223), are often referred to as checkpoint regulators. They function as molecular "gatekeepers" that allow extracellular signals to determine whether or not cell cycle progression and other intracellular signaling processes should proceed.

[0128] The inhibition of immune checkpoint can be performed by inhibition at DNA, RNA or protein level.In some embodiments, inhibitory nucleic acid (e.g., dsRNA, siRNA or shRNA) can be used to inhibit the expression of inhibitory molecule.In other embodiments, the inhibitor of immune checkpoint is a polypeptide that binds to inhibitory molecule, such as a soluble ligand, or / antibody or its antigen-binding fragment.

[0129] In one embodiment, the checkpoint inhibitor is a biological therapeutic or a small molecule. In a preferred embodiment, the immune checkpoint blocker is an antibody. In another embodiment, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.

[0130] The term "antibody" has been defined previously as being equally applicable to this aspect of the invention.

[0131] In further embodiments, the immune checkpoint inhibitor inhibits a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In further embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Immune checkpoint blockers include antibodies, or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-Ll monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PDl antibody), CT-011 (anti-PDl antibody), BY55 monoclonal antibody, AMP224 (anti-PDLl antibody), BMS-936559 (anti-PDLl antibody), MPLDL3280A (anti-PDLl antibody), MSB0010718C (anti-PDLl antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Ligands of checkpoint proteins include, but are not limited to, PD-Ll, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0132] In certain embodiments, the immune checkpoint blocker is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (OPDIVO®), ipilimumab (YERVOY®), and pembrolizumab (KEYTRUDA®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, OPDIVO®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, KEYTRUDA®, Merck); ipilimumab (anti-CTLA-4 antibody, YERVOY®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, Imfinzi®, AstraZeneca); avelumab (anti-PD-L1 antibody), and atezolizumab (anti-PD-L1 antibody, Tecentriq®, Genentech).

[0133] In some embodiments, the immune checkpoint blocker is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirlumab, IPH2101, pembrolizumab (Keytruda®), and tremelimumab.

[0134] In some embodiments, the immune checkpoint blocker is the anti-PD-1 antibody REGN2810 (Regeneron); an antibody that binds to PD-1, pidilizumab (CureTech), also known as CT-011; avelumab (BAVENCIO®, Pfizer / Merck KGaA), a fully human IgG1 anti-PD-L1 antibody, also known as MSB0010718C.

[0135] In some embodiments, the immune checkpoint blocker is an inhibitor of T cell immunoglobulin mucin-containing protein-3 (TIM-3).TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367 and MBG453.

[0136] In some embodiments, the immune checkpoint blocker is an inhibitor of T cell immune receptor with Ig and ITIM domains (i.e., TIGIT), an immune receptor on certain T cells and NK cells. TIGIT inhibitors that can be used in the present invention include anti-TIGIT monoclonal antibodies BMS-986207 (Bristol-Myers Squibb) (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibodies (NCT03119428).

[0137] In some embodiments, the immune checkpoint blocker is an inhibitor of lymphocyte activation gene-3 (LAG-3). The LAG-3 inhibitors that can be used in the present invention include BMS-986016 and REGN3767 and IMP321.

[0138] The immune checkpoint blocker that can be used in the present invention includes OX40 agonist.OX40 agonist that is currently in clinical trial includes PF-04518600 / PF-8600 (Pfizer);GSK3174998 (Merck);MEDI0562 (Medimmune / AstraZeneca);MEDI6469;and BMS-986178 (Bristol-Myers Squibb).

[0139] The immune checkpoint blocker that can be used in the present invention includes CD137 (also called 4-1BB) agonist.Non-exemplary and non-limiting examples of CD137 agonist include utomilumab (PF-05082566, Pfizer) and urelumab (BMS-663513, Bristol-Myers Squibb).

[0140] Immune checkpoint blockers that can be used in the present invention include CD27 agonists. CD27 agonists include varlilumab (CDX-1127, Celldex Therapeutics).

[0141] The immune checkpoint blocker that can be used in the present invention includes glucocorticoid-induced tumor necrosis factor receptor (GITR) agonist.GITR agonist that is currently in clinical trials includes TRX518 (Leap Therapeutics); GWN323; INCAGN01876 (Incyte / Agenus), MK-4166 (Merck), and MEDI1873 (Medimmune / AstraZeneca).

[0142] The immune checkpoint blocker that can be used in the present invention includes inducible T cell costimulator (ICOS, also known as CD278) agonist.Exemplary and non-limiting examples of ICOS agonist include MEDI-570 (Medimmune); GSK3359609 (Merck); and JTX-2011 (Jounce Therapeutics).

[0143] The immune checkpoint blocker that can be used in the present invention includes killer IgG-like receptor (KIR) inhibitor.The exemplary non-limiting examples of KIR include lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb); IPH2101 (1-7F9, Innate Pharma); and IPH4102 (Innate Pharma).

[0144] Immune checkpoint blockers that can be used in the present invention include CD47 inhibitors that inhibit the interaction between CD47 and signal regulatory protein alpha (SIRPa). Illustrative, non-limiting examples of CD47 / SIRPa inhibitors include ALX-148 (Alexo Therapeutics), TTI-621 (SIRPa-Fc, Trillium Therapeutics; CC-90002 (Celgene); and Hu5F9-G4 (Forty Seven, Inc.).

[0145] Immune checkpoint blockers that can be used in the present invention include CD73 inhibitors. Illustrative and non-limiting examples of CD73 inhibitors include MEDI9447 (Medimmune; and BMS-986179).

[0146] Immune checkpoint blockers that may be used in the present invention include agonists of the stimulator of interferon genes protein (STING, also known as transmembrane protein 173, or TMEM173). Illustrative, non-limiting examples of agonists of STING include MK-1454 (Merck); and ADU-S100.

[0147] Immune checkpoint blockers that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors include pexidartinib (PLX3397, Plexxikon; and IMC-CS4 (LY3022855, Lilly).

[0148] The immune checkpoint blocker that can be used in the present invention includes NKG2A receptor inhibitor.An exemplary and non-limiting example of NKG2A receptor inhibitor includes monalizumab (IPH2201, Innate Pharma).

[0149] In a preferred embodiment, the immune checkpoint blocker is a PD-L1 inhibitor.

[0150] As used herein, the term "PD-L1," also known as CD274, PDCD1L1, B7-H, B7-H1, PDCD1LG1, PDL1, B7H1, refers to programmed death-ligand 1. The human gene encoding said protein is represented in the Ensembl database under the accession number ENSG00000120217.

[0151] Small molecules that bind to PD-L1 are disclosed in WO2015034820. These compounds consist of a tricyclic aromatic structure containing an ortho-substituted biphenyl moiety. The biological activity of these compounds was established by homogeneous time-resolved fluorescence (HTRF) binding assays. Representative examples of such compounds are BMS-8 and BMS-202.

[0152] In some embodiments, the immune checkpoint blocker is an anti-PD-LI binding antagonist selected from YW243.55.S70, MPOL3280A, MEOI-4736, MSB-0010718C, or MOX-1105. MOX-1105, also known as BMS-936559, is an anti-PD-LI antibody described in WO2007 / 005874. Antibody YW243.55.S70 is an anti-PD-LI antibody described in WO2010 / 077634.

[0153] In a preferred embodiment, the PD-L1 inhibitor is a PD-L1 antibody.

[0154] In a more preferred embodiment, the PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0155] In some embodiments, the immune checkpoint blocker is MDPL3280A (Genentech 1 Roche), a human Fe-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Patent Publication No. 20120039906. Other anti-PD-L1 binding agents useful as immune checkpoint blockers for the combinations of the invention include YW243.55.S70 (see WO2010 / 077634), MDX-1105 (also known as 8MS-936559), and the anti-PD-L1 binding agents disclosed in WO2007 / 005874.

[0156] In another preferred embodiment, the immune checkpoint blocker is a PD-1 inhibitor. In some embodiments, the anti-PDL1 antibody is MSB0010718C. MSB0010718C (also called A09-246-2; Merck Serono) is a monoclonal antibody that binds to PD-L1.

[0157] In some embodiments, the immune checkpoint blocker is an antibody against PD-1, which binds to the programmed cell death 1 receptor (PD-1) and blocks the receptor from binding to the inhibitory ligand PDL-1, abolishing the tumor's ability to suppress the host's anti-tumor immune response.

[0158] In some embodiments, the immune checkpoint blocker is an anti-PD-1 antibody selected from MDX-1106, Merck3475, or CT-011.In some embodiments, the immunomodulatory agent is a PD-1 inhibitor, such as AMP-224.

[0159] In some embodiments, the immune checkpoint blocker is pidilizumab (CT-011; Cure Tech), a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611.

[0160] In some embodiments, the immune checkpoint blocker is nivolumab (CAS Registry Number: 946414-94-4). Alternative names for nivolumab include MOX-1106, MOX-1106-04, ON0-4538, or BMS-936558. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US8,008,449, EP2161336, and WO2006 / 121168.

[0161] In some embodiments, the immune checkpoint blocker is the anti-PD-1 antibody pembrolizumab. Pembrolizumab (also called lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUOA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, US8,354,509, WO2009 / 114335, and WO2013 / 079174.

[0162] Other anti-PD1 antibodies useful as immune checkpoint blockers in the combinations disclosed herein include AMP514 (Amplimmune), and the anti-PD1 antibodies disclosed in US8,609,089, US2010028330, and / or US20120114649.

[0163] In another preferred embodiment of the combination of the present invention, the PD-1 inhibitor is pembrolizumab or nivolumab.

[0164] Pharmaceutical Compositions In another aspect, the present invention relates to a pharmaceutical composition comprising a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention, or a combination according to the present invention and a pharma- ceutically acceptable excipient.

[0165] The TPC according to the present invention, the polynucleotide according to the present invention, the vector according to the present invention, the host cell according to the present invention, or the combination according to the present invention can be part of a pharmaceutical composition that includes a vehicle suitable for its administration to a subject, in which case the TPC, the polynucleotide, the vector, the host cell, or the combination will be administered to a subject in a pharmaceutical dosage form suitable for the purpose and will include at least one pharma- ceutically acceptable vehicle. Thus, in certain embodiments, the TPC, the polynucleotide, the vector, the host cell, or the combination will be part of a pharmaceutical composition that includes at least one vehicle, preferably a pharma- ceutically acceptable vehicle, in addition to the TPC, the polynucleotide, the vector, the host cell, or the combination as an active ingredient. The term "vehicle" generally includes any diluent or excipient that is administered together with the active ingredient. Preferably, the vehicle is a pharma- ceutically acceptable vehicle for its administration to a subject, i.e., a vehicle (e.g., excipient) approved by a regulatory agency, such as the European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA), or included in a generally recognized pharmacopoeia (e.g., the European Pharmacopoeia, the United States Pharmacopoeia, etc.) for use in animals, and more particularly in humans.

[0166] TPC, polynucleotide, vector, host cell or combination thereof can be dissolved in any suitable medium for administration.Non-limiting examples of the medium in which active ingredient can be dissolved, suspended or form emulsion with active ingredient include water, ethanol, water-ethanol mixture or water-propylene glycol mixture, oil including petroleum-derived oil, animal oil, vegetable oil or synthetic oil, such as peanut oil, soybean oil, mineral oil, sesame oil, organic solvent, such as acetone, methyl alcohol, ethyl alcohol, ethylene glycol, propylene glycol, glycerin, diethyl ester, chloroform, benzene, toluene, xylene, ethylbenzene, pentane, hexane, cyclohexane, tetrahydrofuran, carbon tetrachloride, chloroform, methylene chloride, trichloroethylene, perchloroethylene, dimethyl sulfoxide (DMSO), etc.

[0167] Also included are the solid form preparations of pharmaceutical compositions that are intended to be converted into liquid form preparations for oral or parenteral administration immediately before use.This type of liquid form includes solutions, suspensions and emulsions.General descriptions of the various pharmaceutical dosage forms of active ingredients, the vehicles used and their preparation methods are described, for example, in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzan 5, SA de Ediciones, 1993 and Remington's Pharmaceutical Sciences (edited by AR Gennaro), 20th edition, Williams & Wilkins PA, USA (2000).

[0168] In a non-limiting manner, the administration route for the TPC, polynucleotide, vector, host cell, or combination includes, inter alia, non-invasive pharmacological administration routes, such as oral, gastrointestinal, intranasal, or sublingual routes, and invasive administration routes, such as parenteral routes. In a particular embodiment, the TPC is administered in pharmaceutical form by parenteral route (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc.). "Administration by parenteral route" is understood as an administration route consisting of administering the compound of interest by injection, thus requiring the use of a syringe and needle. There are different types of parenteral punctures, depending on the tissue that the needle reaches: intramuscular (the compound is injected into muscle tissue), intravenous (the compound is injected into a vein), subcutaneous (the compound is injected under the skin), and intradermal (the compound is injected between layers of the skin). The intrathecal route is used to administer drugs that do not cross the blood-brain barrier well into the central nervous system, whereby the drug is administered to the space surrounding the spinal cord (the intrathecal space). In preferred embodiments, administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intrathecal.

[0169] medical use In another aspect, the present invention relates to a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention, a combination according to the present invention, or a pharmaceutical composition for use in the treatment of cancer.

[0170] Alternatively, the present invention relates to a method for treating cancer, comprising administering to a subject in need thereof a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention, a combination, or a pharmaceutical composition according to the present invention.

[0171] Alternatively, the present invention relates to a trimeric polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a host cell according to the present invention, a combination, or a pharmaceutical composition according to the present invention for the preparation of a medicament for the treatment of cancer.

[0172] As used herein, the term "treatment" refers to any type of therapy that aims to terminate, improve, or reduce susceptibility to cancer. Thus, "treatment", "treat", and equivalent terms refer to obtaining a desired pharmacological or physiological effect, and are directed to any treatment of cancer in mammals, including humans. The effect may be preventive, in that it provides a complete or partial prevention of the disorder and / or the adverse effects caused by it. In other words, "treatment" includes (1) preventing the disease, e.g., arresting its onset, (2) interrupting or terminating the disorder or at least its associated symptoms, so that the patient no longer suffers from the disease or its symptoms, e.g., regressing the disease or its symptoms by restoring or repairing lost, missing, or defective functions, or stimulating an inefficient process, or (3) alleviating, mitigating, or improving the disease or its associated symptoms, where alleviation is used in a broad sense to refer to at least reducing the magnitude of a parameter or symptom, such as inflammation, pain, difficulty breathing, or inability to move independently.

[0173] As disclosed herein, the terms "cancer" and "tumor" refer to a physiological condition in a mammal characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, gastrointestinal tract, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid gland. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and intraepithelial carcinoma, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforma, hairy cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myelodisplastic syndrome, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic sarcoma and other sarcomas, ovarian tumors, pheochromocytoma, polycythermia vera, and leukemia. vera, primary brain tumors, small cell lung tumors, squamous cell carcinoma of both ulcerative and papillary types, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor or carcinoma, veticulum cell sarcoma, and Wilm's tumor. Cancer examples also include astrocytoma, gastrointestinal stromal tumor (GIST), glioma or glioblastoma, renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), and pancreatic neuroendocrine carcinoma.

[0174] The trimeric polypeptides, polynucleotides, vectors, host cells, combinations, or pharmaceutical compositions according to the present invention are useful for the treatment of any cancer or tumor, including, but not limited to, breast tumor, heart tumor, lung tumor, small intestine tumor, colon tumor, spleen tumor, kidney tumor, bladder tumor, head tumor, neck tumor, ovarian tumor, prostate tumor, brain tumor, pancreatic tumor, skin tumor, bone tumor, bone marrow tumor, hematoma, thymus tumor, uterine tumor, testicular tumor, and liver tumor.

[0175] In various embodiments, the cancer of the patient to be treated is metastatic cancer or is a refractory and / or recurrent cancer that is refractory to first, second, or third line therapy. In another embodiment, the treatment is a first, second, or third line therapy. As used herein, the phrase "first line" or "second line" or "third line" refers to the order of treatment that the patient undergoes. The first line treatment regimen is the first treatment, and the second or third line treatment is performed after the first or second line therapy, respectively. Thus, the first line treatment is the first treatment for a disease or condition. For cancer patients, the first line treatment can be surgery, chemotherapy, radiation therapy, or a combination of these. The first line treatment is also referred to as primary therapy or primary treatment by those skilled in the art. Typically, the next chemotherapy regimen is administered to the patient because the patient did not show a good clinical response to the first line therapy, showed only a subclinical response, or the first line treatment was discontinued. In this context, "chemotherapy" is used in its broadest sense to encompass not only classical cytotoxic chemotherapy but also molecular targeted therapy and immunotherapy.

[0176] In a preferred embodiment, the cancer is EGFR positive.

[0177] In another preferred embodiment, the cancer is also positive for an immune checkpoint, whose inhibitor is included in the combination of the present invention used to treat said cancer. Illustrative, non-limiting examples of immune checkpoints have been described above and are equally applicable to this aspect of the present invention. More particularly, the cancer is positive for PD-L1.

[0178] The term "positive", as used herein to refer to EGFR, indicates that the "amount" or "level" of EGFR in a tumor or cancer is higher than that observed in a non-positive tumor or normal cell. Expression levels can be measured by methods known to those skilled in the art and disclosed herein. The term "level of expression" or "expression level" generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetically encoded information and / or epigenetic information) is converted into structures that exist and function within a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or modifications of polynucleotides and / or polypeptides (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0179] "Increased expression," "increased expression level," "increased level," "elevated expression," "elevated expression level," or "elevated level" are used interchangeably to refer to increased expression or increased level of a biomarker in an individual compared to a control, such as, for example, one or more individuals without a disease or disorder (e.g., cancer), an internal control (e.g., housekeeping biomarker), or the median expression level of the biomarker in samples from a group / population of patients.

[0180] In a more preferred embodiment, the cancer is colorectal cancer, breast cancer, pancreatic cancer, thyroid cancer, prostate cancer, ovarian cancer, head and neck cancer or lung cancer. In another more preferred embodiment, the breast cancer is triple negative breast cancer and the lung cancer is small cell lung cancer.

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[0182] The present invention is illustrated by the following examples which should be considered as merely illustrative and not as limiting the scope of the invention. EXAMPLES

[0183] material and method mouse Mouse NOD.Cg-Prkdc SCID IL2rg tm1Wjl / SzJ (NSG) female mice were provided by Charles River, Hsd: athymic Nude-Foxn1nu female mice were provided by Envigo RMS SPAIN SL, and 129S4-Rag2tm1.1Flv Il2rgtm1.1Flv / J (Rag2- / -IL2Rγ-deficient) female mice were kept in the animal facility of CIMA. Animals were kept under specific pathogen-free conditions with a 12-h light / 12-h dark daily cycle and had free access to sterile water and food. All animal experimental procedures were in accordance with the European Union Directive 86 / 609 / EEC and Recommendation 2007 / 526 / EC implemented in Spanish law under RD1201 / 2005. The animal experimental protocols were approved by the respective ethical committees of animal experiments at the participating institutions (IDIPHISA, imas12, CIEMAT and CIMA) and were carried out in strict adherence to the guidelines set out in the "International Guiding Principles for Biomedical Research Using Animals" established by the Council for International Organizations of Medical Sciences (CIOMS). The experimental study protocols were further approved by local authorities (PROEX094 / 15, 108 / 15, 076 / 19 and 166 / 19).

[0184] Antibodies and cell lines The commercially available antibodies used in the experiments are listed in Table I.

[0185] [Table 1-1]

[0186] [Table 1-2]

[0187] [Table 1-3]

[0188] The recombinantly produced antibodies are listed in Table II.

[0189] [Table 2]

[0190] HEK293 (CRL-1573), MDA-MB-231 (HTB-26), A431 (CRL-1555), NIH / 3T3 (CRL-1658) and CHO-K1 (CCL-61) cells were obtained from the American Type Culture Collection and cultured at 37°C in humidified 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Lonza) supplemented with 2 mM L-glutamine, 10% (vol / vol) heat-inactivated fetal calf serum (FCS) (Merck Life Science), and antibiotics (100 units / mL penicillin, 100 mg / mL streptomycin) (all from Life Technologies), referred to as DMEM complete medium (DCM). NIH / 3T3 cells expressing huEGFR (3T3 huEGFR ) (Estrada C. et al., 1992) was kindly provided by Dr. A. Villalobo (IIBm, Madrid, Spain). The hu4-1BB expressing HEK293 cell line (HEK239 hu4-1BB ) were generated by transfection with the expression vector pCMV3-Flag-TNFRSF9 (SinoBiological) and selected in DCM with 500 μg / mL G418 (Life Technologies). CHO-K1 cells expressing human FcγRIIb (CD32) were obtained from Promega (#JA2251). Cell lines were routinely screened for the absence of mycoplasma contamination by PCR using the Mycoplasma Plus™ primer set (Biotools B&M Labs).

[0191] Construction of expression vectors To generate the SAP3.28scFv-based N-terminal trimer body, a DNA fragment encoding the FLAG-strepII-SAP3.28HL(VH-linker-VL)scFv was synthesized by Geneart AG and subcloned as HindIII / NotI into the expression vector pCR3.1-MFE23N (Cuesta AM. et al., 2009) to obtain pCR3.1-FLAG-strepII-SAP3.28HL-N-myc / His. The C-terminal myc / His tag sequence was removed by PCR from the plasmid using Fw-CMV and Stop-XbaI-Rev primers (Table III).

[0192] [Table 3]

[0193] The Flag-strepII-SAP3.28HL scFv gene was cloned into the homotrimerization vector (TIE) derived from human collagen XVIII. XVIII ) domain and an anti-human EGFR single domain antibody (VHH; EGa1) (Schmitz KR et al., 2013), resulting in the bispecific trimer body expression vector pCR3.1-FLAG-strepII-SAP3.28HL-N18 / C18EGa1. All sequences were verified using primers FwCMV and RvBGH (Table III).

[0194] Recombinant antibody expression and purification HEK293 cells were transfected with the appropriate expression vectors by calcium phosphate precipitation and selected in DCM containing 500 μg / mL G418 to generate the stable cell lines 293-SAP3.28N and 293-SAP3.28. N / CEGa1 was generated. Conditioned medium was collected and purified using the Strep-Tactin® purification system (IBA Lifesciences) with the AKTA Prime plus system (Cytiva). Purified antibodies were dialyzed overnight at 4°C against PBS + 150 mM NaCl, pH 7.0, analyzed by SDS-PAGE under reducing conditions, and stored at 4°C. Purified antibodies were tested for endotoxin levels by Pierce's Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit according to the manufacturer's specifications (Thermo Fisher Scientific). Endotoxin levels of purified antibody stocks were less than 0.25EU / ml by LAL test.

[0195] Western blotting Protein samples were separated on 10-20% Tris-glycine gels under reducing conditions, transferred onto nitrocellulose membranes (Thermo Fisher Scientific), and probed with anti-FLAG or anti-Strep-tagII mAb, followed by incubation with DyLight800-conjugated goat anti-mouse (GAM) IgG. Visualization and quantitative analysis of protein bands were performed using an Odyssey® infrared imaging system (LI-COR Biosciences).

[0196] ELISA Mouse (mo-), cynomolgus monkey (cy-) and hu4-1BB-human IgG1 Fc chimeras (R&D Systems, #937-4B, #9324-4B, #838-4B) (5 μg / ml), or mo-, cy- and huEGFR-human IgG1 Fc chimeras (R&D Systems, #1280-ER, #10366-ER, #344-ER) (5 μg / ml) were immobilized on Maxisorp ELISA plates (NUNC Brand Products) overnight at 4°C. After washing and blocking with 200 μl PBS+5% BSA (Merck Life Science), 100 μl of purified protein solution was added and incubated for 1 h at room temperature. Wells were washed three times with PBS+0.05% Tween-20 and 100 μl of anti-FLAG or anti-Strep-tagII mAb was added and incubated for 1 h at room temperature. Plates were washed as above and 100 μl of HRP-conjugated GAM IgG (GAM-HRP) was added to each well. Plates were then washed and developed using OPD (Merck Life Science). Recombinant His-tagged hu4-1BB (Sino Biological, #10014-H08H) (3 μg / mL) was immobilized overnight at 4 °C, wells were washed and blocked, and anti-hu4-1BB antibodies were added in triplicate at 10-fold serial dilutions to obtain equilibrium binding curves. After 1 h of incubation, wells were washed and incubated with HRP-conjugated goat anti-human IgG (GAH-HRP) (1:1000) or GAM-HRP (1:1000), respectively. Plates were developed using TMB (Merck Life Science) and absorbance was measured at 450–570 nm. For competitive ELISA, wells were coated with hu4-1BBL human IgG1 Fc chimera (Abcam, #ab217567) (5 μg / mL) overnight at 4°C. Plates were washed and blocked as described above. Recombinant His-tagged hu4-1BB (1 μg / mL) was preincubated with increasing concentrations (0-10 μg / mL) of urelumab or 4-1BB IgG (SAP3.28 IgG) for 30 min, after which the mixture was transferred in triplicate to hu4-1BBL-coated wells and incubated for 1 h.Wells were washed and incubated with Tetra-His mAb (αHis4) and then with GAM-HRP (1:1000) for 1 hour. Plates were washed and developed with TMB. To further evaluate the epitope binding of both anti-hu4-1BB mAbs, His-tagged hu4-1BB was immobilized (3 μg / mL) on Maxisorp ELISA plates overnight at 4°C. After washing and blocking as above, urelumab or 4-1BB Ig was added in triplicate for 1 hour at a pre-determined concentration of 10 μg / mL to achieve saturation. After washing the wells, the other antibody not used in the previous step was added to the wells at 1 μg / mL. After 1 hour, plates were washed and 100 μl of GAM-HRP or GAH-HRP (1:000) was added to each well. Plates were then washed and developed with TMB.

[0197] mass spectrometry 2 μl of protein samples were desalted using a ZipTip® C4 microcolumn (Merck Millipore) and eluted onto a GroundSteel massive 384 target (Bruker Daltonics) with 0.5 μl of SA (sinapic acid, 10 mg / ml in [70:30] acetonitrile:trifluoroacetic acid 0.1%) matrix. An Autoflex III MALDI-TOF / TOF spectrometer (Bruker Daltonics) was used in linear mode with the following settings: 5000-40000 Th window, linear positive mode, ion source 1: 20 kV, ion source 2: 18.5 kV, lens: 9 kV, 120 ns pulsed ion extraction, high gated ion suppression up to 1000 Mr. Mass calibration was performed externally using Protein 1 standard calibration mixture (Bruker Daltonics). Data acquisition, peak peaking and subsequent spectral analysis were performed using Flex Control 3.0 and Flex Analysis 3.0 software (Bruker Daltonics).

[0198] Size Exclusion Chromatography-Multi-Angle Laser Light Scattering (SEC-MALS) Static light scattering measurements were performed at 25°C using a Superdex 200 Increase 10 / 300 GL column (Cytiva) connected in-line to a DAWN-HELEOS light scattering detector and an Optilab rEX refractive index detector (Wyatt Technology). The excluded volume of the column was 8.6 mL, and no absorbance was observed in any of the injections (no aggregated protein was observed). The column was equilibrated with running buffer (PBS + 150 mM NaCl, 0.1 μm filtered) and the SEC-MALS system was calibrated with a sample of 1 g / L BSA in the same buffer. A 100 μL sample of each of the two trimer bodies at 1.1 g / L in running buffer was then injected onto the column at a flow rate of 0.5 mL / min. Data acquisition and analysis were performed using ASTRA software (Wyatt Technology). The reported molar masses correspond to the center of the chromatographic peak. Based on multiple measurements on 1 g / L BSA samples under identical or similar conditions, we estimate the experimental error in molar mass to be approximately 5%.

[0199] Circular dichroism Circular dichroism measurements were performed using a Jasco J-810 spectropolarimeter (JASCO). Spectra were recorded at 25 °C for protein samples at 0.1 g / L in PBS + 150 mM NaCl using a quartz cuvette with a 0.2 cm path length. Thermal denaturation curves from 5 to 95 °C were recorded for the same protein sample and cuvette by increasing the temperature at a rate of 1 °C / min and observing the denaturation at 210 nm (4-1BB). N ) or 213nm (4-1BB N / C The changes in ellipticity of EGFR were recorded.

[0200] Small-angle X-ray scattering (SAXS) SAXS experiments were carried out at beamline B21 at the Diamond Light Source (Didcot). Proteins were concentrated and prepared at 4°C prior to data collection. 4-1BBN and 4-1BB at concentrations of 3mg / ml and 6mg / ml were used. N / C40 μl of the EGFR sample was delivered to a Shodex Kw-403 column at 4 °C via an in-line Agilent 1200 HPLC system using a running buffer composed of 50 mM Tris (pH 7.5) + 150 mM NaCl. The continuously eluting sample was exposed for 300 s during 10 s acquisition blocks using an X-ray wavelength of 1 Å and a distance of 3.9 m from the sample to the detector (Pilatus 2M). The data covered a momentum transfer range of 0.032 < q < 3.695 Å−1. The frame recorded immediately before elution of the sample was subtracted from the protein scattering profile. The data, buffer subtraction, scaling, integration, and checking for the possibility of radiation damage to the sample were analyzed using the Scatter software package (www.bioisis.net). The data set corresponding to 3 mg / mL of 4-1BBN could not be analyzed further due to aggregation. The Rg value was calculated using the Guinier approximation assuming a very small angle q < 1.3 / Rg. The maximum particle distribution Dmax and the distance distribution were calculated from the scattering pattern by GNOM, and the shape estimation was performed using DAMMIF / DAMMIN. All of these programs were included in the ATSAS package (Petoukhov MV. et al., 2012). An interactive PDB-based model was created for the two antibodies based on the template obtained using the program RaptorX. The real-space scattering profile of this model was calculated using the program FoXS.

[0201] Dynamics studies using biolayer interferometry Interactions between appropriate antibodies and immobilized antigens were investigated using biolayer interferometry on an Octet RED96 system (Fortebio). Purified antigens, hu4-1BB and huEGFR (R&D), were immobilized on AR2G biosensors using standard amine-reactive chemistry. Briefly, biosensors were activated with EDC and s-NHS, then incubated for 30 min in a 5 μg / mL antigen solution in 10 mM acetate buffer at pH 5.0, followed by quenching with ethanolamine. All binding studies were performed using kinetic buffer (PBS + 0.1% BSA + 0.05% Tween 20). 4-1BB N and 4-1BB N / C To examine the interaction of EGFR with hu4-1BB, 1 nM and 5 nM of the antibody were incubated with hu4-1BB immobilized biosensors for 2 h, and then dissociation was measured in kinetic buffer for 2 h. N / C The same antibody concentrations and time lengths were used for the interaction of EGFR and ATTACK antibodies with the huEGFR immobilized biosensor. Immobilized hu4-1BB in solution and 4-1BB for huEGFR N and 4-1BB N / C Tandem binding of EGFR was examined by binding 5 nM of either antibody or kinetic buffer alone to duplicate hu4-1BB immobilized biosensors for 1 hour followed by dissociation for 1 hour. One of the duplicate biosensors was then introduced to 10 nM huEGFR in kinetic buffer while the other was maintained in kinetic buffer. After 1 hour of secondary binding, secondary dissociation was measured for 1 hour.

[0202] In vitro 4-1BB-dependent NF-κB activation assay 4-1BB-dependent activation assays of activated nuclear factor kappa B (NF-kappa B) were performed using thaw-and-use (T&U) GloResponse™ NFkappaB-luc2 / 4-1BB Jurkat cells (Promega, #JA2351) according to the manufacturer's instructions. To perform the general assay, 1x105 Jurkat cells / well were seeded in Assay Buffer (RPMI + 1% FCS) in white-walled 96-well plates (Merck Life Science). Anti-4-1BB agonists and control antibodies were added in 10-fold serial dilutions. Human EGFR-negative cells (3T3) or EGFR-positive cells (3T3 huEGFR ), and CHO-K1 cells (CHO) or CHO-K1 cells expressing human FcγRIIb (CHO huFcγRIIb )(2×10 4 cells / well) and Jurkat 4-1BB Cells were stimulated for 6 hours at 37°C. Bio-Glo™ Luciferase Assay Reagent (Promega) was added and luciferase activity was assessed using a Tecan Infinite F200 plate-reading luminometer (Tecan Trading AG). Experiments were performed in triplicate and data were reported as x-fold induction relative to values ​​obtained from unstimulated cells (mean ± SD). Data were analyzed and plotted using GraphPad Prism fitting software 6.01.

[0203] Inhibition of cell proliferation and signaling through EGFR A431 cells were seeded in DCM in 96-well plates. After 24 hours, the medium was diluted with equimolar concentrations (0.19–50 nM) of cetuximab, rituximab, and 4-1BB. N / C The medium was then replaced with DMEM + 1% FCS containing EGFR or 4-1BB IgG and incubated for 72 hours. Viability was assessed using a CellTiter-Glo luminescent assay (Promega, #G7570). Experiments were performed in triplicate. For EGFR signaling studies, A431 cells were starved overnight in DMEM 1% FCS, then treated with 0.1 μM cetuximab, rituximab, 4-1BB, or 5% erythrocyte sedimentation (CES) or 5% CO. For EGFR signaling studies, A431 cells were starved overnight in DMEM 1% FCS, then treated with 0.1 μM cetuximab, rituximab, 4-1BB, or 5% CO. N / CThe cells were incubated in serum-free DMEM for 4 hours in the presence of EGFR or 4-1BB IgG, followed by incubation with 25 ng / mL human EGF (MiltenyiBiotec GmbH, #130-097-749) for 10 minutes. Samples were separated on a 12% Tris-glycine gel under reducing conditions, transferred to a nitrocellulose membrane, blocked, and incubated with rabbit anti-human fluorophore-EGFR (Tyr1068) mAb, followed by incubation with IRDye800-donkey anti-rabbit antibody. Concurrently, anti-β-actin mouse mAb was added as a loading control, followed by IRDye700-donkey anti-mouse IgG. Visualization and quantitative analysis of protein bands were performed using the Odyssey system.

[0204] Isolation of human PBMCs and T cells Cells from healthy donors were isolated from small leukocyte reduction chambers (LRS chambers) collected after the apheresis procedure (Neron S. et al., 2007). The LRS chamber contents were drained into a sterile tube, rinsed with up to 50 ml of PBS, and huPBMCs were isolated by density gradient centrifugation (2000 rpm, 20 min at room temperature) using Ficoll-Paque (Cytiva Life Sciences). ACK lysis buffer (Life Technologies) was added to remove residual red blood cells (RBCs), and huPBMCs were washed, counted, and resuspended to the desired final concentration. Human T cells were purified using the Pan T Cell Isolation Kit (Human) (Milteny Biotech, #130-096-535) according to the manufacturer's instructions. Cells were washed, counted, and resuspended to the desired final concentration.

[0205] Human PBMC and T cell activation assays Human PBMCs or isolated T cells (1.5 × 10 5 Cells / well) were seeded in triplicate in flat-bottom 96-well plates in RPMI supplemented with 10% FCS and 50 μM β-mercaptoethanol (Life Technologies) and 45 Gy irradiated target cells (3T3 or 3T3 hEGFR) at an effector / target ratio of 5:1. Anti-hu4-1BB agonist antibodies and controls were added in 10-fold serial dilutions in the presence of 0.05 μg / ml of anti-huCD3 (OKT3) mAb. After 72 hours, cell-free supernatants were analyzed for cytokine secretion by ELISA. Irradiated EGFR+PD-L1 cells (3T3 huEGFR ) or EGFR+PD-L1+ cells (MDA-MB-231) (3 × 10 4 cells / well) were treated with anti-PD-L1 (atezolizumab) alone (10 μg / ml) or 4-1BB N / C huPBMCs (1.5 × 10 5 Cell-free supernatants were measured for IFNγ by ELISA (Diaclone, #851560005) after 72 hours.

[0206] Flow cytometry Cells expressing hu4-1BB (2.5 × 10 5 Cells (1000 x 1000 cells / well) were incubated with purified antibodies (3 μg / ml) for 1 h on ice, washed, incubated with anti-FLAG mAb for 30 min on ice, and detected with PE-conjugated F(ab')2 GAM IgG antibody. Purified anti-hu4-1BB IgG1 mAb (clone SAP3.28), kindly provided by M. Glennie (University of Southampton, UK), was used as a control. To examine the expression of human FcγRIIb, cells were incubated with anti-huCD32 mAb and PE-GAM-F(ab')2. Cell surface expression of huEGFR and huPD-L1 was measured in 3T3, 3T3 hEGFRand MDA-MB-231 cells were analyzed after incubation with PE-conjugated anti-huEGFR and APC-conjugated anti-huPD-L1 mAbs. Samples were analyzed on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmbH). A minimum of 20,000 events were acquired per sample, and data were evaluated using FCS Express V3 software (De Novo Software, Glendale, CA, USA).

[0207] Serum stability Refined 4-1BB N and 4-1BB N / C EGFR was incubated in 60% human serum at 37° C. for 4 days. Samples were frozen at −80° C. at various time points until the end of the experiment and analyzed for binding activity to hu4-1BB and huEGFR by ELISA (as described above). To calculate the time corresponding to the percentage decay of binding activity, the binding activity of the samples at 0 hours was taken as 100%.

[0208] 4-1BB N / C EGFR conjugation and 89 Zr label 4-1BB N / C EGFR (1 mg) was conjugated with p-SCN-Bn-deferoxamine (Macrocyclics) using a method previously reported (Vosjan MJ. et al., 2010). Zirconium-89 ( 89 Zr)(T 1 / 2 = 78.4 hours, β + = 22.6%; commercially available at approximately 2.7 GBq / ml in 1 M oxalic acid) was obtained from Cyclotron VU. N / CRadiolabeling of EGFR was achieved using conventional methods previously described and purified on a PD-10 column (Life Technologies) (Vosjan MJ. et al., 2010). Radiolabeling yield (RCY) was determined as the % radioactivity recovered in each collected fraction out of the total radioactivity used, correcting the measurements for decay over time. Determination of 89Zr radioactivity was performed using a dose calibrator VDC-405 (Veenstra Instruments) using a recently reported conversion factor (or dial setting factor) (Garcia-Torano E. et al., 2019). Radiochemical purity (RQP) was defined as the % of radioactivity of the radionuclide present in the desired radiopharmaceutical form out of the total radioactivity and was analyzed by instant thin layer chromatography (ITLC). Experiments were performed in triplicate.

[0209] Pharmacokinetic studies Six-week-old female athymic nude mice were administered 242.35 kBq (14 μg) of IgG in PBS. 89 Zr labeled 4-1BB N / C EGFR was injected iv (tail vein). For pharmacokinetic studies, blood samples were collected in heparinized tubes and centrifuged at 3000 rpm for 10 min to obtain plasma. Plasma radioactivity concentrations were calculated as percent injected dose per mL (%ID / mL) and plotted against time after injection. Plasma concentrations of radioactivity versus time were analyzed by compartmental methods using nonlinear regression (Brown AM. et al., 2001). Initial estimates of pharmacokinetic parameters were calculated using the curve stripping method with the add-in program PKSolver (Zhang Y. et al., 2010). The best-fitting compartment model was determined using the Akaike Information Criterion (AIC). Blood clearance (Cl 血液 ) is calculated as plasma clearance (Cl 血漿 ) was estimated from Cl 血液 =Cl 血漿 / BP, where BP is the blood-to-plasma ratio.

[0210] Humanized colon cancer cell line-derived xenograft (CDLX) model HT29 cells (1 x 106 ) were placed in 6-week-old Rag2 mice. - / - IL2Rγ-deficient female mice were transplanted sc into the dorsal cavity, followed by fresh huPBMCs (1 × 10 7 Tumor growth was monitored by caliper measurements 3 times a week, and when tumors reached approximately 0.4 cm in diameter, mice were randomly assigned to treatment (n=7-8 / group). Measurements were performed in random order by an investigator blinded to treatment assignment. CEA was administered 5 times every 3 days. N Or 4-1BB N / C Mice were treated with ip injections of EGFR trimerbody (4 mg / kg) or ip injections of 4-1BB IgG (4 mg / kg) three times weekly. MDA-MB-231 cells (2 × 10 6 1 × 10 cells were resuspended in PBS and mixed with Matrigel (30%). The cells were transplanted sc into the dorsal right flank of 6-week-old NSG female mice, followed by freshly isolated huPBMCs (1 × 10 7 Tumor growth was monitored by caliper measurement three times a week. Tumor-bearing mice (0.2 cm diameter) were randomly divided into four groups (n=5–6 / group) and investigators were blinded to treatment allocation. Five doses of 4-1BB were administered every 3 days. N / C Mice were treated with ip injections of EGFR trimerbody (4 mg / kg) or ip injections of PD-L1 IgG (4 mg / kg) three times weekly, either alone or in combination. Mice were weighed twice weekly to monitor toxicity. Mice were euthanized upon signs of distress and / or 10-15% weight loss.

[0211] Humanized Patient-Derived Xenograft (PDX) Models In this study, pre-amplified lung PDX TP103 was selected according to histology, genetic background (EGFR and TP53 mutations), and huEGFR cell surface expression (Quintanal-Villalonga A. et al., 2019). 3Tumors were cut into 100x fragments and implanted sc through a small incision into the dorsal cavity of anesthetized 6-week-old NSG female mice. Tumor growth was monitored by caliper measurements every 3-4 days, and when tumors reached approximately 0.5 cm in diameter, mice were randomized into groups (n=6-7 / group) with similar mean tumor size and SD and implanted with freshly isolated huPBMCs (1x10) from healthy donors. 7 The mice were injected i.p. with 4-1BB (cells / mouse) every 3 days. N / C Mice were treated with five ip injections of EGFR (4 mg / kg). Mice were weighed weekly to monitor toxicity. Mice were euthanized if they experienced more than 10-15% weight loss, if tumor size reached 1.0 cm in diameter at any dimension, if tumors ulcerated, or if the mice showed signs of distress.

[0212] Immunohistochemistry Immunohistochemical staining was performed on 4 μm thick sections of formalin-fixed, paraffin-embedded samples. Slides were incubated with mouse mAbs listed in Table I using a Bond™ automated system (Leica Microsystems) according to the manufacturer's instructions. Nuclei were counterstained with Harris hematoxylin.

[0213] statistical analysis Statistical analysis was performed using GraphPad Prism software version 6.0. In vitro experiments were usually performed in triplicate and values ​​are presented as mean ± SD from one of at least three separate experiments. Significant differences (P values) were determined applying a two-tailed unpaired Student's t-test assuming normal distribution. P values ​​were shown in the corresponding figures for each experiment. EC50 was calculated using a nonlinear regression curve (log agonist vs. normalized response - variable response). Mean tumor volumes were presented for each group as mean ± SD using scatter plots. To assess differences between treatment groups, P values ​​were determined by one-way analysis of variance (ANOVA) adjusted with Bonferroni correction for multiple comparison testing.

[0214] Example 1.4-1 Generation and characterization of BB agonist humanized trimerbodies Anti-hu4-1BB trimer bodies were generated using scFv-encoding genes derived from the anti-hu4-1BB agonist SAP3.28 mAb (Figure 5a) (WO / 2017 / 077085), which binds to hu4-1BB CRD-1. SAP3.28 IgG (hereafter referred to as 4-1BB IgG) is a chimeric molecule that presents a humanized VL domain, a partially humanized VH domain that preserves the mouse FR3 region to retain antigen binding, and the Fc region of mouse IgG1 (WO / 2017 / 077085). Similar to urelumab (Chin S. M. et al., 2018), which recognizes the N-terminus of CRD-1, 4-1BB IgG does not block the hu4-1BB receptor / hu4-1BBL interaction (Figures 6a-c). Furthermore, we showed that the epitopes of 4-1BB IgG and urelumab do not overlap (Figures 6d and e). We designed an SAP3.28 scFv-based anti-hu4-1BB N-terminal trimer body (4-1BBN) by fusing SAP3.28 scFv to the homotrimerization (TIEXVIII) domain derived from human collagen XVIII using a flexible linker (Figure 5b and c), and synthesized an anti-EGFR EGa1 V HH The antibody (Schmitz KR. et al., op. cit. 2013) was fused to the C-terminus of 4-1BBN to produce 4-1BB N / C Bispecific trimeric bodies were engineered by generating a construct called EGFR (Figure 1a). Both trimeric bodies were purified from conditioned medium from stably transfected HEK293 cells by Strep-Tactin affinity chromatography with protein yields of >95% purity (3.5 mg / L and 4.5 mg / L, respectively) (Figure 7a). Mass spectrometry (using MALDI-TOF, not shown) confirmed the absence of signal sequences in the purified antibodies. 4-1BB N and 4-1BB N / CSEC-MALS experiments for both EGFRs yielded major peaks with molar masses of 111 kDa and 160 kDa, respectively (Figure 7b and c), which are consistent with trimeric molecules. Smaller volume minor peaks with molar masses of 217 kDa and 340 kDa indicate the presence of dimers of trimers, as previously observed for other trimeric bodies (Compte M. et al., 2018). Circular dichroism measurements show a pronounced b-sheet structure and cooperative thermal denaturation (Tm = approx. 60 °C; Figure 7d and e). Small angle X-ray scattering (SAXS) was used to investigate the three-dimensional structure of both trimeric bodies. 4-1BB N The trimer bodies showed a flat distribution with a clear TIEXVIII core in the center and the scFvs partially elongated in the same plane like the spokes of a wheel (Fig. 8 and 9; Table IV).

[0215] [Table 4] TIFF2024510947000007.tif35169

[0216] [Table 5]

[0217] 4-1BB N / C The EGFR trimer body maintains the same planar structure as 4-1BBN, and in addition, small EGFR VHH domains are interspersed among the 4-1BB scFvs, resembling a six-bladed shuriken (Fig. 1b; Fig. 9; Table IV). Biolayer interferometry (BLI) was used to determine the binding of 4-1BBN and 4-1BB to hu4-1BB. N / C 4-1BB binds to EGFR and huEGFR N / C The association and dissociation rates of EGFR and anti-EGFR ATTACK antibody (Harwood SL. et al., 2017) were measured (Figure 1c).

[0218] The bispecific ATTACK antibody evolved from a tandem trimer body format (Alvarez-Cienfuegos A. et al., 2016) that combines three EGFR-binding VHH antibodies with one CD3-binding scFv (Harwood SL et al., supra, 2017). All interactions were of high affinity (low picomolar KD values), indicating the functional trivalency of the trimer body for the antigen displayed on the biosensor surface (Table V). The kinetics of huEGFR binding by these trivalent antibodies are consistent with previous studies (Compte M. et al., supra; Harwood SL et al., supra). In supplementary experiments, 4-1BB binds EGFR-binding VHH antibodies with a CD3-binding scFv. N and 4-1BB N / C EGFR was first loaded onto hu4-1BB immobilized on the biosensor surface, and then the biosensor was transferred into a buffer solution containing huEGFR. N / C EGFR is 4-1BB N Unlike , it was able to bind soluble huEGFR while still bound to immobilized hu4-1BB, further confirming its bivalency and ability to bind both antigens simultaneously (Figure 1d). N / C EGFR bound to mouse (mo-), cynomolgus monkey (cy-) and huEGFR (Figure 10a), and also to cy4-1BB and hu4-1BB, but with much lower binding to mo4-1BB (Figure 10b). The ability to detect hu4-1BB and huEGFR in a cellular context was analyzed by flow cytometry. N / C The EGFR trimerbody bound to wild-type HEK293 (EGFR+) cells, HEK293 cells transfected to express hu4-1BB on the cell surface (HEK293hu4-1BB), and mouse 3T3 cells expressing huEGFR (3T3huEGFR), but not to wild-type 3T3 cells (Figure 11). In contrast, 4-1BB IgG bound only to HEK293hu4-1BB cells (Figure 11). N / C To further evaluate the multivalent binding of EGFR, the inventors studied its ability to inhibit proliferation and EGFR phosphorylation in A431 cells (Schmitz KR. et al., supra).N / C Both the EGFR and EGF competitive inhibitor cetuximab (Li S. et al., 2005), but not anti-human CD20 rituximab or the parental 4-1BB IgG, inhibited A431 proliferation in a dose-dependent manner (P=0.003 and P=0.0005 for high doses of both antibodies, respectively, compared with equimolar doses of control antibody) (Figure 12a) and also inhibited EGFR phosphorylation (Figure 12b).

[0219] Example 2. Fc-free EGFR-targeted 4-1BB agonist humanized trimerbody significantly enhances T cell costimulation in the presence of EGFR-expressing cells The agonist activity of the three SAP3.28-derived antibodies and urelumab was assessed using NF-κB-luc2 / 4-1BB Jurkat cells, which constitutively express hu4-1BB on the cell surface and a luciferase reporter driven by an NF-κB response element (JurkatNF-κB). NF-κB The reporter cells were huFcγRIIb (CHO huFcγRIIb ) or huEGFR(3T3 huEGFR ) and non-transfected CHO or 3T3 cells as negative controls; cell surface expression of huFcγRIIb and huEGFR was demonstrated by flow cytometry (Figures 2a and b). Bivalent (4-1BB IgG or urelumab) or trivalent (4-1BB N Or 4-1BB N / CTitrations of anti-hu4-1BB antibodies against EGFR were added to co-cultured cells. In the absence of Fc- or EGFR-mediated antibody cross-linking on the target cell surface (i.e., when co-cultured with non-transfected CHO or 3T3 cells), 4-1BB IgG showed little to no induction on untreated Jurkat NF-κB cells at all concentrations tested, while both anti-hu4-1BB trimerbodies showed approximately 10-fold induction, and urelumab showed approximately 20-fold induction (Figure 2c and d). In the presence of FcγRIIb-mediated cross-linking (i.e., when CHOhuFcγRIIb was used as the target cell), 4-1BB IgG induced dose-dependent activation of NF-κB with a 26-fold induction (P=0.0008), and urelumab's induction increased further to 40-fold (P=0.003) (Figure 2c). Neither trimer body showed increased induction via FcγRIIb (Fig. 2c). Trimer body-mediated 4-1BB signaling was significantly enhanced when target cells expressed huEGFR (P=0.0008), with a 40-fold increase in NF-κB luciferase reporter activity (Fig. 2d). Induction by 4-1BB IgG, urelumab, and 4-1BBN was not affected by huEGFR expression (Fig. 2d). Negative control antibodies moIgG1, huIgG4, and CEAN, a trimer body that recognizes CEA, showed no activation (Figs. 13a and b). Next, we used huPBMCs or T cells from healthy donors to investigate the activation of irradiated 3T3 or 3T3 huEGFR The effect of anti-hu4-1BB antibody on IFNγ secretion when co-cultured with cells was examined in both the presence and absence of a suboptimal dose of anti-huCD3 mAb. N / C EGFR trimerbodies showed a dose-dependent activating effect on IFNγ secretion only when huPBMCs or T cells were cocultured with EGFR+ cells; no induction was observed in EGFR− cells (Figures 2e and f). Under these conditions, the effects of 4-1BB IgG and CEAN were minimal and independent of EGFR expression (Figure 2e; Figure S14). These data support the conclusion that 4-1BB N / CWe show that EGFR induces potent EGFR-dependent T cell costimulation and IFNγ secretion, which requires an initial signaling through the TCR / CD3 complex (signal 1). N / C In the presence of the EGFR and PD-L1 blocking antibody atezolizumab, irradiated EGFR+PD-L1-(3T3 huEGFR ) cells or EGFR+PD-L1+ (MDA-MB-231) cells (Figure 2g). When combined with a suboptimal dose of anti-huCD3 mAb, 4-1BB N / C EGFR trimer body significantly enhanced IFNγ secretion (P=0.0007 3T3 huEGFR cells; P=0.0002 MDA-MB-231 cells) (Fig. 2h). 4-1BB N / C When huPBMCs were cocultured with MDA-MB-231 cells in the presence of EGFR, the addition of atezolizumab significantly increased IFNγ levels ( P = 0.02) ( Fig. 2 h ).

[0220] Example 3. 89Zr-labeled 4-1BB N / C Pharmacokinetics of EGFR trimer body 4-1BB N / C The EGFR trimerbody retained nearly 100% of its initial binding activity after 4 days at 37° C. in human serum (FIGS. 15a and b). N / C Chelation of EGFR trimer bodies with p-SCN-Bn-deferoxamine (Df) did not change the migration pattern on SDS-PAGE or reduce the binding activity (Fig. 16a and b). 89 Zr]Zr-Df-4-1BB N / C The RCY (radiolabeling yield) and RQP (radiochemical purity) of EGFR were 40% and 95%, respectively. The AIC values ​​were 89 Zr]Zr-Df-4-1BB N / C For EGFR 1 and 2 compartments, the values ​​were 10.97 and -22.66, respectively, so 4-1BB N / C The disposition of EGFR trimer bodies was better explained by a two-compartment model (Table VI).

[0221] [Table 6]

[0222] After intravenous administration 89 Zr]Zr-Df-4-1BB N / C EGFR elimination was biphasic, with a half-life of 7.3 h in the rapid distribution phase and 66.8 h in the slow distribution phase (Figure 3a). The volume of distribution at steady state was 66.5 mL (2.63 L / Kg) and plasma clearance was 0.97 mL / h (37.6 mL / Kg / h). With a blood-to-plasma ratio of 0.62, the obtained blood clearance value was very low (0.062 L / Kg / h) compared to cardiac output (21.7 L / Kg / h in mice), which is generally desirable for developing drugs for use in low-dose regimens (Toutain PL. et al., 2004).

[0223] Example 4. Antitumor activity of Fc-free EGFR-targeted 4-1BB agonist humanized trimerbody We demonstrate that 4-1BB is a potent immunizing agent in a huPBMC-driven humanized immunoavatar mouse model. N / C The antitumor activity of the EGFR trimerbody was tested. - / - IL2Rγ null Mice were injected intraperitoneally (ip) with huPBMCs and then subcutaneously (sc) inoculated with human HT-29 CRC cells (Fig. 3b). Although the engrafted human T cells are activated and generate pathogenic xenoreactivity, a process called xenograft-versus-host disease (xGVHD) (Nervi B. et al., 2007), engrafted human T cells can be modulated by therapeutic agents and are a valuable model for testing immunomodulatory strategies (Carroll RG. et al., 2008; Mutis T. et al., 2006; Sanmamed MF. et al., 2015). Mice with tumors reaching approximately 0.4 cm in diameter were treated with trimer body (CEA) for 5 doses at 3 / 4 day intervals, as shown in Fig. 3b. N Or 4-1BB N / CThe subjects were treated with either ip injections of EGFR or equimolar doses of 4-1BB IgG three times a week. The doses and treatment schedules were designed similar to those performed with anti-mo4-1BB agonists in an immunocompetent model of CRC (Compte M. et al., supra). N / C The EGFR-treated group showed significantly slower tumor growth compared to the untreated group (P = 0.01) and the CEAN-treated group (P = 0.004) (Figure 3c). N / C The EGFR trimerbody demonstrated antitumor activity in vivo that was comparable to that of 4-1BB IgG (FIG. 3c).

[0224] We next sought to determine whether antitumor effects also occurred in a huPBMC-driven humanized NSG mouse model (TP103, Fig. 3d and e) bearing EGFR+ NSCLC PDXs. As shown in Fig. 3f, 4-1BB N / C EGFR-treated mice had suppressed tumor growth compared to the control group. The improved tumor growth control was accompanied by a significant change in the TIL infiltration pattern. Both groups showed a diffuse infiltration of CD3+ T lymphocytes surrounding and involved tumor cell nests (Figure 17). PBS-treated mice had more CD4+ T cells, with a CD4 / CD8 ratio of 2.8 (Figure 3g-i). 4-1BB N / C In the EGFR treatment group, a significant increase in the number of CD8+ T cells (P = 0.04) was observed, accompanied by a decrease in the number of Foxp3+ cells (P = 0.01) (Figures 3g-i; Figure S17).

[0225] The present inventors have demonstrated that 4-1BB IgG or 4-1BB N / CWe compared toxicity profiles in huPBMC-driven humanized NSG mice that were administered EGFR trimerbody (6 mg / kg) once a week for 3 weeks and euthanized 1 week later. Liver histological studies revealed that 4-1BB IgG treatment exacerbated xGVHD. Details of liver infiltration in representative mice from each treatment group are shown in Figure 3j, with the group treated with an IgG-based 4-1BB agonist showing extensive perivascular mononuclear cell infiltration. We next examined human IFNγ concentrations in serum samples taken at the time of sacrifice. 4-1BB IgG treatment significantly increased the risk of developing xGVHD compared with 4-1BB. N / C It significantly increased IFNγ levels compared with EGFR treatment ( P = 0.001), but levels were similar to those in PBS-treated mice ( Fig. 3 k ).

[0226] Example 5.4-1BB N / C Combination of EGFR and atezolizumab induces tumor regression 4-1BB N / C The therapeutic potential of the combination of EGFR and PD-L1 blocker atezolizumab was investigated in huPBMC-driven humanized NSG mice bearing human EGFR+PD-L1+MDA-MB-231 (Fig. 2g) TNBC xenografts (Fig. 4a). Atezolizumab monotherapy was able to inhibit tumor growth by approximately 60%, whereas 4-1BB N / C EGFR monotherapy suppressed tumor growth by approximately 90% (Figure 4b). N / C The combination of EGFR resulted in further suppression of tumor growth (Figure 4b). In the PBS-treated group, large nests of neoplastic pleomorphic cells were observed that strongly expressed cytokeratin (CK) and were accompanied by dense lymphocytic infiltration (Figures 4c and d). Importantly, 4-1BB N / C The percentage of CK+ cells was significantly lower in the EGFR monotherapy group (P = 0.04) and the combination therapy group (P = 0.0002) than in the atezolizumab monotherapy group (Figure 4c). In the combination therapy, the percentage of CK+ cells was at most 30% in 5 of 6 mice, and TNBC cells were completely eradicated in 1 mouse (Figure 4e). This reduction in tumor burden was due to the combination therapy with 4-1BB. N / CThis was associated with a significant increase in the percentage of CD8 + T cells in the EGFR-treated group ( P = 0.03 and P = 0.04) ( Fig. 4 d and e ).

[0227] Example 6. 4-1BB including 1D8 anti-4-1BB ScFv as disclosed in Compte et al. (Nature Communications, 2018, doi:10.1038 / s41467-018-07195-w) N / C 4-1BB containing SAP3.28 anti-4-1BB ScFv against EGFR anti-4-1BB trimer body N / C Improving the pharmacokinetic properties of EGFR trimerbodies 1D8 N / C Pharmacokinetic studies of EGa1 were performed using 1D8 N / C The study was carried out in female CD-1 mice (n=24) and immunocompetent Balb / C female mice (n=24) with a single intravenous (iv) injection of EGa1 trimer body at 1 mg / kg. Blood samples were taken from three mice per group at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, and 48 h. Serum was collected after centrifugation and stored at -20°C. Antibody concentrations in serum were analyzed by ELISA against plastic-immobilized m4-1BB (3 μg / ml). After washing and blocking, serum from different time points was added and incubated for 1 h at room temperature. Wells were washed and horseradish peroxidase (HRP)-conjugated anti-FLAG mAb (1 μg / ml) (M2; catalogue no. ab49763, Abcam) (1:1000 dilution) was added. After washing, plates were developed with o-phenylenediamine dihydrochloride (OPD). Pharmacokinetic parameters were calculated using Prism software (GraphPad Software).

[0228] In CD-1 mice, 1D8 N / C The circulating half-life of EGa1 was 16.1 hours (Table 2). N / C The serum half-life of EGa1 was not affected by mouse genetic background, and a similar pharmacokinetic profile was observed in BALB / c mice (Table 3).

[0229] [Table 7]

[0230] [Table 8]

[0231] SAP3.28 N / C Pharmacokinetic studies of EGa1 were performed using a 242.35 kBq (14 μg) (0.56 mg / kg) dose in PBS. 89 Zr labeled SAP3.28 N / C The study was carried out in 6-week-old female athymic nude mice injected iv (tail vein) with EGa1 trimer body. For pharmacokinetic studies, blood samples were collected in heparinized tubes and centrifuged at 3000 rpm for 10 min to obtain plasma. Radioactivity concentration in plasma was calculated as percent injected dose per mL (%ID / mL) and plotted against time after injection. Plasma concentration of radioactivity versus time was analyzed by compartmental methods using nonlinear regression. Initial estimates of pharmacokinetic parameters were calculated using the curve stripping method with the add-in program PKSolver. The best-fitting compartment model was determined using Akaike's Information Criterion (AIC). Blood clearance (Cl blood) was estimated from plasma clearance (Cl plasma) using the following relationship: Cl blood = Cl plasma / BP, where BP is the blood-to-plasma ratio.

[0232] After intravenous administration 89 Zr]Zr-Df-SAP3.28 N / C The elimination of EGa1 was biphasic, with a half-life of 7.3 h in the rapid distribution phase and 66.8 h in the slow distribution phase (Table 4). The volume of distribution at steady state was 66.5 mL (2.63 L / kg), and the plasma clearance was 0.97 mL / h (37.6 mL / kg / h). The blood-to-plasma ratio was 0.62, so the obtained blood clearance value was very low (0.062 L / kg / h) compared with the cardiac output (21.7 L / kg / h in mice).

[0233] [Table 9]

[0234] As shown by the comparative data above, the 4-1BB antibody according to the present invention is characterized by the presence of the SAP3.28 anti-4-1BB ScFv. N / C The half-life of the EGFR trimer body was measured using 4-1BB containing 1D8 anti-4-1BB ScFv as disclosed in Compte et al. (Nature Communications, 2018, doi:10.1038 / s41467-018-07195-w). N / C The half-life of the antibody according to the invention is much longer than that of the EGFR anti-4-1BB trimerbody. The difference in half-life of these two antibodies is about 7-fold in the rapid distribution phase and about 4-fold in the slow distribution phase. This difference is so significant that it cannot be attributed solely to methodological differences in the pharmacokinetic assays used to characterize both antibodies. The longer half-life of the antibody according to the invention is clearly unexpected and is an advantage over the antibodies of the prior art, since a high half-life value is a desirable property for a drug that is to be administered in a low-dose regimen.

Claims

1. A trimeric polypeptide complex comprising three monomeric polypeptides, each monomeric polypeptide a) V Ｈ Domain is V Ｌ an anti-4-1BB specific agonist single chain antibody fragment (scFv) N-terminal to the domain; b) Type XVIII collagen homotrimerization domain (TIE) ＸＶＩＩＩ ), type XV collagen homotrimerization domain (TIE ＸＶ a homotrimerization domain selected from the group consisting of: c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Trimeric polypeptide complexes.

2. A trimeric polypeptide complex comprising three monomeric polypeptides, each monomeric polypeptide a) anti-4-1BB specific agonistic single chain antibody fragments (scFv) whose CDRs comprise the sequences set forth in SEQ ID NO:1 or SEQ ID NO:42, SEQ ID NO:2 or SEQ ID NO:43, SEQ ID NO:3 or SEQ ID NO:44, SEQ ID NO:4 or SEQ ID NO:45, SEQ ID NO:5 or SEQ ID NO:46, and SEQ ID NO:6, or functionally equivalent variants thereof; b) Type XVIII collagen homotrimerization domain (TIE) ＸＶＩＩＩ ), type XV collagen homotrimerization domain (TIE ＸＶ a homotrimerization domain selected from the group consisting of: c) a polypeptide region capable of specifically binding to a tumor-associated antigen Including, Trimeric polypeptide complexes.

3. The trimeric polypeptide complex according to claim 1 or 2, wherein the anti-4-1BB specific agonist scFv is humanized or presents a humanized VL domain and / or a partially humanized VH domain.

4. The trimeric polypeptide complex of claim 3, wherein the anti-4-1BB agonist scFv comprises the sequence represented by SEQ ID NO:

19.

5. 5. The trimeric polypeptide according to claim 1, wherein the region capable of specifically binding to the tumor-associated antigen is located C-terminal to the homotrimerization domain.

6. 6. The trimeric polypeptide complex of claim 1, wherein the tumor-associated antigen is EGFR.

7. The trimeric polypeptide complex of claim 6 , wherein the polypeptide region capable of specifically binding to EGFR is an antibody.

8. 8. The trimeric polypeptide complex of claim 7, wherein the anti-EGFR antibody is a humanized anti-EGFR (huEGFR) single domain antibody (VHH).

9. The trimeric polypeptide complex of claim 8, wherein the humanized anti-EGFR (huEGFR) single domain antibody (VHH) comprises the sequence represented by SEQ ID NO:

28.

10. 10. The trimeric polypeptide complex according to any one of claims 1 to 9, wherein the anti-4-1BB agonist single-chain antibody fragment (scFv), the homotrimerization domain and / or the region capable of specifically binding to a tumor-associated antigen are linked directly or via a spacer.

11. The trimeric polypeptide of claim 10, wherein the spacer is a flexible linker having 1 to 18 residues.

12. 12. The trimeric polypeptide of claim 1, wherein at least one of the monomers further comprises a tag suitable for detection and / or purification of the trimeric polypeptide.

13. 3. The trimeric polypeptide of claim 1 or 2, wherein the monomeric polypeptide comprises SEQ ID NO: or a sequence represented by SEQ ID NO:

14. A polynucleotide encoding at least one monomeric polypeptide forming part of a trimeric polypeptide according to any one of claims 1 to 13.

15. 15. The polynucleotide of claim 14, comprising a sequence number or a sequence represented by a sequence number.

16. A vector comprising the polynucleotide of any one of claims 14 or 15.

17. A host cell comprising the vector of claim 16.

18. A combination comprising a trimeric polypeptide described in any one of claims 1 to 13, a polynucleotide described in any one of claims 14 or 15, a vector described in claim 16, or a host cell described in claim 17, and an immune checkpoint blocker.

19. The combination according to claim 18, wherein the immune checkpoint blocker is a PD-L1 inhibitor.

20. The combination of claim 19, wherein the PD-L1 inhibitor is a PD-L1 antibody.

21. 21. The combination of claim 20, wherein the PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

22. The combination of claim 18, wherein the immune checkpoint blocker is a PD-1 inhibitor.

23. 23. The combination of claim 22, wherein the PD-1 inhibitor is pembrolizumab or nivolumab.

24. A pharmaceutical composition comprising the trimeric polypeptide of any one of claims 1 to 13, the polynucleotide of any one of claims 14 or 15, the vector of claim 16, the host cell of claim 17, or the combination of any one of claims 18 to 23, and a pharmaceutically acceptable excipient.

25. 25. The trimeric polypeptide of any one of claims 1 to 13, the polynucleotide of any one of claims 14 or 15, the vector of claim 16, the host cell of claim 17, the combination of any one of claims 18 to 23, or the pharmaceutical composition of claim 24 for use in the treatment of cancer.

26. 26. The trimeric polypeptide, polynucleotide, vector, host cell, combination, or pharmaceutical composition of claim 25, wherein the cancer is EGFR positive.

27. 27. The trimeric polypeptide, polynucleotide, vector, host cell, combination, or pharmaceutical composition of claim 26, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, thyroid cancer, prostate cancer, ovarian cancer, head and neck cancer, and lung cancer.

28. 28. The trimeric polypeptide, polynucleotide, vector, host cell, combination, or pharmaceutical composition of claim 27, wherein the breast cancer is triple-negative breast cancer and the lung cancer is small cell lung cancer.

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