Replication-competent oncolytic virus comprising a sequence encoding an antibody or antibody fragment

Abstract

Replication-competent oncolytic virus comprising a sequence encoding an antibody or antibody fragment. The present invention refers to a replication competent oncolytic adenovirus comprising a heterologous nucleic acid inserted into a nonessential region of the adenovirus genome, said heterologous nucleic acid comprising a sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of an SdAb, sdFv, F(ab')2, Fab or scFab, preferably the antibody fragment is an SdAb; wherein the adenovirus is a Delta-24 or Delta-24-RGD.

Description

[0001] Replication-competent oncolytic virus comprising a sequence encoding an antibody or antibody fragment.

[0002] Technical field of the invention

[0003] The invention relates to the field of oncology treatment and, more particularly, to the use of biological therapies.

[0004] Background art.

[0005] Each year, an estimated 400,000 children and adolescents of 0-19 years old develop cancer. Brain and central nervous system (CNS) tumors are the most common cause of cancer- related death in children aged 0-14 years in Europe and the USA, with a globally estimated death in this age range of 100,000. Pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMGs) are high-grade glial tumors with a median overall survival of 9-15 months. These tumors are the most common malignant brain cancer in children and represent the greatest cause of cancer-related deaths under the age of 19 years. Unlike histologically similar lesions in adults, which tend to be restricted to the cerebral hemispheres, pHGG can occur throughout the CNS, with around half occurring in midline locations, particularly the thalamus and the pons. This last location discards surgery as an option and thus confers an inferior prognosis for these tumors. For the DMGs, the standard of care is radiotherapy (RT), being that this treatment is just palliative and extends the median survival by approximately 3 months. In the case of pHGG, chemotherapy is associated with partial or temporary clinical responses but is still not curative for a high percentage of patients. This is also valid for other high-risk pediatric brain tumors, including but not limited to AT / RTs (Atypical teratoid / rhabdoid tumors), medulloblastoma, ependymomas, and other embryonic tumors. Although our understanding of the genomic and epigenomic features of high-risk pediatric brain tumors has seen unprecedented progress in the last decade, this advancement did not result in significant improvements in therapy despite the more than two hundred trials performed. Collectively, these facts emphasize the urgent need for effective treatments for pHGGs and DMGs. Moreover, those who survive in the pHGG group present significant longterm health issues, including neurocognitive dysfunction, endocrine deficits, and secondary cancers. Thus, there is a pressing clinical need to develop novel and radically different treatments to improve these children's survival and quality of life.

[0006] Brief description of the figures

[0007] Figure 1. Intratumoral infusion of oncolytic virus Delta-24-RGD (DNX-2401; in the clinic) followed by radiotherapy in pediatric patients with DIPG. Patients received DNX-2401, and subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. A reduction in tumor size was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months.

[0008] Figure 2. Delta-24-ACT armed virus (A) Schedule (B) prolonged the survival and (C) led to immune memory of mice bearing NP53-derived DIPG tumors in the brain stem. Delta-24- ACT (D) to a proinflammatory phenotype but also to the increase of T-regulatory cells.

[0009] Figure 3. TIM-3 is a therapeutic target for DMGs. (A) Analyses of TIM-3 (HAVCR2) expression in DMG patient samples by scRNAseq and cell lines by WB. (B) In vivo therapeutic effect of a TIM-3 blocking (1 intratumoral (IT) and 2 intraperitoneal (IP) doses). Long-term survivors were subjected to a rechallenge, and brain analyses Ki67. (C) Upregulation of TIM3 in vivo in mouse-bearing orthotopic DMG tumors after RT (upper) or adenoviral infection (lower).

[0010] Figure 4. Combination of Delta-24-RGD and TIM-3 blockade. (A) Schedule of Delta-24- RGD and anti-TIM-3 systemic treatment. (B) In vivo therapeutic effect of combination with systemic anti-TIM-3. (C) Schedule of Delta-24-RGD and anti-TIM-3 Intratumoral treatment. (D) In vivo therapeutic effect of intratumoral combination.

[0011] Figure 5. Construction and testing of TIM-3 single-domain antibody (Nb). (A) mNb5 TIM- 3 binding assay to TIM-3 and other recombinant proteins. (B) TIM-3 binding in cell lines of IgG Ab / TIM-3 mNb5. (C) mNb5 TIM-3 dose-dependent binding assay.

[0012] Figure 6. Functional studies ex-vivo with mNb5 TIM-3 therapeutic single-domain antibody. (A-B) C57 mice (A) CD4 and (B) CD8 T cells expression of activation and cytotoxic markers. (C-D) Pmel mice CD8 T cells expression of activation and cytotoxic markers.

[0013] Figure 7. Therapeutic studies with mNb5 TIM-3 single-domain antibody. (A) Treatment schedule in an XFM orthotopic model. (B) In vivo therapeutic effect of the single-domain antibody compared with the anti-TIM-3 Ab. Figure 8. Representative images of single-domain antibody distribution after intratumoral or systemic (intraperitoneal administration).

[0014] Figure 9. Cloning process of any given single- domain antibody within the genome of our oncolytic virus.

[0015] Figure 10. Therapeutic studies with Delta-24-ACT-mNb5 armed oncolytic virus (Delta- 24-RGD-41BBL-single-domain antibody anti-TIM3 with or without Fc (fragment crystallizable region)).

[0016] Figure 11. Protocol to generate anti-human CD47 single-domain antibodies (SdAbs). A) Llama immunization process with the extracellular domain of the human CD47 (hCD47) and / or peptide corresponding to the CD47 motif interacting with SIRPa. Llama was immunized 5 times overthe course of 14 weeks. B) Isolation of peripheral blood mononuclear cells (PBMCs) from blood serum at weeks 10 and 14. C) Single heavy-chain variable domains of llama PBMCs were amplified by 2-step PCR and cloned in pADL-20c phagemid vector using Sfil restriction sites. D) Phage display biopanning against hCD47. 5 rounds of panning were conducted to select CD47-binding SdAbs. E) Functional screening performed in the SdAbs to assess their prophagocytic properties. Firstly, SdAbs clones blocking SIRPa binding were identified by biolayer interferometry (BLI). Secondly, phagocytosis assays were carried out to evaluate if SdAbs clones had prophagocytic effects.

[0017] Figure 12. human CD47 (hCD47) single-domain antibodies (SdAbs) library construction.

[0018] A) hCD47 antibodies titers in llama blood serum throughout the immunization process. Antibody titers were determined by ELISA, in which hCD47 protein was coated to the plate. B) Monitoring for the enrichment of CD47-binders after each round of panning. 10-fold dilutions of the eluted phages after each round of panning demonstrated enrichment of phages binding on hCD47.

[0019] Figure 13. Functional screening for human CD47 (hCD47) single-domain antibodies (SdAbs). A) SdAbs clones capability to block SIRPa binding to hCD47 assessed by biolayer interferometry. Among the five clones evaluated, 1A1 and 1B1 clones blocked SIRPa binding.

[0020] B) Phagocytosis assay on human DIPG cells. Conditioned media (CM) of SdAbs transfected cultured were used for this phagocytosis assay. Both SdAbs induced prophagocytic, but 1B1 clone efficacy was higher than 1A1 clone. Figure 14: (A) Analysis of the replicative capacity of the two oncolytic viruses in two different tumor lines. (B) The cytopathic capacity of the two oncolytic viruses was analyzed using an MTS assay in two different tumor lines.

[0021] Figure 15: Western blot analysis of the expression of single-domain antibodies (SdAb), 41BBL, and two viral proteins (E1A and fiber) after infection of tumor cells with 4 different modified viruses. D24RGD-sdAbTIM3, D24RGD-sdAbCD47.1Bl.Fc, D24ACT-sdAbTIM3 (expressing m41BBL), D24ACT-sdAbTIM3-Fc (expressing m41BBL).

[0022] Figure 16: Analysis of the phagocytic capacity of macrophages derived from bone marrow co-cultured with tumor cells infected with the corresponding viruses.

[0023] Figure 17. Analysis of the phagocytic capacity of macrophages derived from bone marrow of nsg mice co-cultured with human tumor cells (DIPG007) treated with medium, IgGl (10 pg / ml), human CD47 antibody (B6H12; 10 pg / ml) Nbl (SdAb-CD47; 1 pg / ml) or Fc-linked Nbl (SdAb-CD47-FC; 5 pg / ml).

[0024] Figure 18. Evaluation of the efficacy of Delta-24-sdAbCD47 in a model of DMG. Therapeutic studies with Delta-24-mSdAbCD47 armed oncolytic virus (Delta-24-RGD-single- domain antibody anti-CD47 with or without Fc).

[0025] Figure 19. Descriptive scheme of the different sequences introduced in the new oncolytic viruses of the invention.

[0026] Figure 20. Evaluation of the neuro-toxicity of the Delta-24-ACT-SdAbTIM-3 and Delta- 24-ACT-SdAbTIM-3-Fc armed oncolytic virus (Delta-24-RGD-41BBL-single-domain antibody anti-TIM3 with or without Fc) injected intratumorally in the brain of healthy mice, measured by body weight and survival.

[0027] Figure 21. Evaluation of the systemic toxicity in numerous organs of the Delta-24-ACT- SdAbTIM-3 armed oncolytic virus (Delta-24-RGD-41BBL-single-domain antibody anti-TIM3 without Fc) injected intratumorally in the brain of healthy mice.

[0028] Figure 22. Evaluation of the systemic toxicity in numerous organs of the Delta-24-ACT- SdAbTIM-3-Fc armed oncolytic virus (Delta-24-RGD-41BBL-single-domain antibody anti-TIM3 with Fc) injected intratumorally in the brain of healthy mice.

[0029] Figure 23. Dot plot graph showing TIM-3 expression in tumor tissue compared to healthy tissue in a variety of organs. TIM-3 is significantly overexpressed in many of them.

[0030] Figure 24. Evaluation of the efficacy of Delta-24-ACT-SdAbTIM3 (Delta-24-RGD-41BBL- single-domain antibody anti-TIM3) in an immunocompetent model of GBM (A; GL261), pediatric osteosarcoma (B; K7M2), lung metastasis (C), breast cancer (D;4T1), and colorectal cancer (D; MC38).

[0031] Brief description of the invention

[0032] An aspect of the invention refers to a replication-competent oncolytic adenovirus comprising a heterologous nucleic acid inserted into a nonessential region of the adenovirus genome, said heterologous nucleic acid comprising a nucleic acid sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv F(ab')2, Fab and scFab, preferably the antibody fragment is a single-domain antibody (SdAb); and wherein the adenovirus is a Delta-24 or Delta-24-RGD virus.

[0033] In an embodiment, the replication-competent oncolytic adenovirus further comprises a sequence encoding a 4-1BB ligand (4-1BBL) operatively linked to a transcriptional control element, preferably wherein the nucleic acid encoding a 4-1BBL encodes a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 95% identical thereto.

[0034] In another embodiment, optionally in combination with any previous or subsequent embodiment of the invention, said nucleic acid comprising a nucleic acid sequence encoding an antibody or antibody fragment further encodes at the N-terminus of the antibody or fragment thereof a signal sequence, preferably said signal sequence is of SEQ ID NO: 17 or a sequence at least 95% identical thereto.

[0035] In another embodiment, optionally in combination with any previous or subsequent embodiment of the invention, the antibody or antibody fragment is an anti-TIM3 antibody, an anti-TIM3 antibody fragment, an anti-TIM3 antibody construct, or an anti-TIM3 single-domain antibody (SdAb).

[0036] In another embodiment, optionally in combination with any previous or subsequent embodiment of the invention, the antibody or antibody fragment is an anti-CD47 antibody or antibody fragment, preferably an anti-CD47 single-domain antibody (SdAb). Preferably, said anti-CD47 antibody or antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9, 11, 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9, 11, 13 or 15 thereto. More preferably, said anti-CD47 antibody or antibody fragment is a single-domain antibody (SdAb). More preferably, the antibody fragment is a single-domain antibody (SdAb), and said singledomain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 13 or 15 thereto, wherein said SdAb comprises or consists of a VH domain comprising all of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of SEQ ID NO: 6; b. HCDR2 consisting of SEQ ID NO: 7; and c. HCDR3 consisting of SEQ ID NO: 8.

[0037] Still more preferably, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 9 or 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9 or 11 thereto, wherein said SdAb comprises or consists of a VH domain comprising all of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of SEQ ID NO: 32; b. HCDR2 consisting of SEQ ID NO: 33; and c. HCDR3 consisting of SEQ ID NO: 34.

[0038] In another embodiment, optionally in combination with any previous or subsequent embodiment of the invention, the heterologous nucleic acid inserted into a nonessential region of the adenovirus genome comprises: a. a transcriptional control element, preferably a promoter; b. a sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element; wherein the 4-1BB (CD137) agonist is preferably a 4-1BB ligand (4-1BBL); and c. a sequence encoding an antibody or antibody fragment as defined in any one of the previous embodiments operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N- terminus; and wherein sequences b) and c) are preferably linked directly or optionally through a linker; and wherein preferably:

[0039] I. the sequence encoding a 4-1BBL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 90% identical hereto; ii. the signal sequence is the sequence set forth in SEQ ID NO: 17 or a sequence at least 95% identical thereto; and iii. the sequence encoding an antibody or antibody fragment is the sequence of an anti-TIM3 antibody or antibody fragment as defined in any one of the previous embodiments or an anti-CD47 antibody or antibody fragment as defined in any one of the previous embodiments.

[0040] In another embodiment, optionally in combination with any previous or subsequent embodiment of the invention, the heterologous nucleic acid inserted into a nonessential region of the adenovirus genome comprises or consists of any one of the sequences set forth in SEQ ID NO: 132 to 143 or SEQ ID NO 22 to 27.

[0041] A further aspect of the invention refers to a pharmaceutical composition comprising a replication competent oncolytic adenovirus according to any of the previous embodiments and pharmaceutically acceptable carrier.

[0042] A still further aspect of the invention refers to a replication competent oncolytic adenovirus according to any of the previous embodiments or a pharmaceutical composition comprising the same, for use as a medicament. Preferably, for use in a method of treatment of cancer in a subject in need thereof, wherein the subject has a cancer selected from a solid tumour, including primary or secondary brain cancer, preferably glioma, head and neck cancer, spinal cord cancer, melanoma, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, chordoma, Ewing's Sarcoma, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, malignant vascular tumors, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, and pancreatic cancer, preferably wherein the patient has a low-grade or high-grade glioma, and wherein the adenovirus is preferably administered intratumorally, intravascularly, or in a neuronal or mesenchymal stem cell carrier, preferably wherein the adenovirus is administered intratumorally.

[0043] Detailed description of the invention

[0044] Definitions

[0045] A replication-competent oncolytic virus shall be understood as a virus, either genetically engineered or naturally occurring, with the unique capability to selectively kill cancer cells while sparing normal tissues.

[0046] Inserted into a nonessential region of the virus genome shall be understood as a viral region that is no necessary for the correct replication of the virus.

[0047] On the other hand, a "promoter" is a control sequence that is a nucleic acid sequence region at which initiation and rate of transcription are controlled. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and / or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cisacting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. The terms "nucleic acid", "polynucleotide", "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein, and relate to any polymeric form of nucleotides of any length and composed of ribonucleotides or deoxyribonucleotides, e.g., deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in either single or double stranded form.

[0048] The term "gene construct" or "nucleic acid construct" as used herein relates to a functional unit required to transfer, and preferably express, a gene or genes of interest in a host cell. This term refers to a nucleic acid molecule, either single- or double-stranded, which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.

[0049] The term "vector" or "cloning vector" as used herein, refers to a construct capable of delivering, and preferably additionally expressing, one or more polynucleotides of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. This term also relates to targeting constructs, which allow for random or site-directed integration of the targeting construct into genomic DNA. Such targeting constructs, preferably, comprise DNA of sufficient length for either homologous recombination or heterologous integration. In a particular embodiment, the vector is an expression vector.

[0050] The term "expression cassette", as used herein relates to a gene construct operatively linked to an expression control sequence. The gene construct can be obtained through the use of techniques widely known in the prior art.

[0051] The term "cancer" or "tumor" or "tumor disease" as used herein, refers to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighboring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether or not they can spread by invasion and metastasis, tumors are classified as being either benign or malignant: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumors are tumors that are capable of spreading by invasion and metastasis. As used herein, the term cancer includes, but is not limited to, the following types of cancer including primary or secondary (metastatic) brain tumors head and neck cancer, spinal cord cancer, melanoma, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, chordoma, Ewing's Sarcoma, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, malignant vascular tumors, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer and the like.

[0052] As used herein, "secondary brain cancer" or "metastatic brain cancer" or "metastatic brain tumor" or "secondary brain tumor" occurs when cancer cells spread to the brain from a primary cancer elsewhere in the body, forming a tumor or tumors in the brain. Such brain cancers are about ten times more common than a cancer that starts or originates in the brain, which is known as "primary brain cancer." Cancer cells can break away from the primary tumor site and travel through blood and lymphatic vessels. Metastases most often appear in the brain at the junction of two types of brain tissue, referred to as gray matter and white matter. This junction is rich with blood vessels of very narrow diameter, and metastatic cells often lodge there.

[0053] The terms "subject" "individual" or "patient" are used interchangeably herein and refer to an individual, such as a human, a nonhuman primate (e.g., chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. In a particular embodiment of the invention, the subject is a mammal, preferably a human, more preferably a pediatric or young adult.

[0054] The term "mammal" includes, but is not restricted to, domestic and farm mammals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. The terms "pediatric", "pediatric patient" and "pediatric individual or subject" are used interchangeably herein to refer to human individuals from the age of greater than 0 year to 14 years or less. The term young adult ("aya") are used to individuals from the age of 15 to 39 or less.

[0055] "Administering" or "administration of" a medicament to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self-administration, and / or indirect administration, which may be the act of prescribing a drug. E.g., a physician who instructs a patient to self-administer a medicament or provides a patient with a prescription for a drug is administering the drug to the patient.

[0056] The term "antibody" refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular target. The term includes whole antibodies and antibody fragments or any antigen binding portion or single chains thereof and combinations thereof; for instance, the term "antibody" in particular includes bivalent antibodies and bivalent bispecific antibodies, more particularly, the term antibody further includes a "single-domain antibody" or "SdAb" which refers to a protein comprising or consisting of the soluble single antigen-binding V-domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et al., 2017. Front Immunol. 8:1603). A typical type of antibody comprises at least two heavy chains ("HC") and two light chains ("LC") interconnected by disulfide bonds. Each "heavy chain" comprises a "heavy chain variable domain" (abbreviated herein as "VH") and a "heavy chain constant domain" (abbreviated herein as "CH"). The heavy chain constant domain typically comprises three constants' domains, CHI, CH2, and CH3. Each "light chain" comprises a "light chain variable domain" (abbreviated herein as "VL") and a "light chain constant domain" ("CL"). The light chain constant domain (CL) can be of the kappa type or of the lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FW" or "FR"). Each VH and VL is composed of three complementarity-determining regions (CDRs) and four framework regions (FRs), arranged from amino-terminus (N) to carboxy-terminus (C) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The present disclosure inter alia presents VH and VL sequences as well as the subsequences corresponding to CDR1, CDR2, and CDR3.

[0057] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927- 948 ("Chothia" numbering scheme).

[0058] Accordingly, a person skilled in the art would understand that the sequences of FR1, FR2, FR3 and FR4 are equally disclosed. For a particular VH, FR1 is the subsequence between the N- terminus of the VH and the N-terminus of HCDR1, FR2 is the subsequence between the C- terminus of HCDR1 and the N-terminus of HCDR2, FR3 is the subsequence between the C- terminus of HCDR2 and the N-terminus of HCDR3, and FR4 is the subsequence between the C -terminus of HCDR3 and the C-terminus of the VH. Similarly, for a particular VL, FR1 is the subsequence between the N-terminus of the VL and the N-terminus of LCDR1, FR2 is the subsequence between the C-terminus of LCDR1 and the N-terminus of LCDR2. FR3 is the subsequence between the C-terminus of LCDR2 and the N-terminus of LCDR3, and FR4 is the subsequence between the C-terminus of LCDR3 and the C-terminus of the VL.

[0059] The expression "functionally equivalent variant", when referred to a CDR refers to a peptide sequence which, when present in an antibody or antibody fragment in combination with the other CDRs of the antibody or antibody fragment, results in an antibody or antibody fragment which substantially preserves its ability of specifically binding to the antigen.

[0060] As used herein, the terms "specific binding", "selective binding", "selectively binds", and "specifically binds", refer to an antibody or antibody fragment binding to an epitope on a predetermined antigen with high affinity.

[0061] As used herein, the term "high affinity" for an antibody or antibody fragment refers to an antibody or antibody fragment having a Ko of 10-7IVI or less, preferably IO-8IVI or less, more preferably IO-9M or less and even more preferably 1010M or less for a target antigen. Typically, the antibody or antibody fragment (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10-7M, such as approximately less than IO-8M, IO-9M or 1010M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BIACORE instrument using the predetermined antigen, e.g., TIM-3 or CD47, as the analyte and the antibody or antibody fragment as the ligand, or Scatchard analysis of binding of the antibody or antibody fragment to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. Accordingly, an antibody or antibody fragment that "specifically binds to TIM-3 or to CD47" refers to an antibody or antibody fragment that binds to soluble or cell bound TIM-3 or CD47 with a KD of 10-7M or less, such as approximately less than IO-8M, IO-9M or 1010M or even lower.

[0062] The functionality of the antibody or antibody fragment carrying the functionally equivalent variant of the CDRs can also be determined by measuring their "half maximum effective concentration" (EC50), which is the half-maximal effect concentration (concentration for 50% of maximal effect, EC50), which refers to the concentration that elicits 50% of the maximal effect. Accordingly, the antibodies or antibody fragments disclosed herein preferably have an EC50 of at least 30 nM, at least 29 nM, at least 28 nM, at least 27 nM, at least 26 nM, at least

[0063] 25 nM, at least 24 nM, at least 23 nM, at least 22 nM, at least 21 nM, at least 20 nM, at least

[0064] 19 nM, at least 18 nM, at least 17 nM, at least 16 nM, at least 15 nM, at least 14 nM, at least

[0065] 13 nM, at least 12 nM, at least 11 nM, at least 10 nM. It will be understood that the functionally equivalent variants of the antibodies or antibody fragments disclosed herein will maintain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% of the EC50 of the antibody or antibody fragment to which the variant refers to.

[0066] A functionally equivalent variant of a CDR sequence may be a polypeptide sequence derivative of said sequence comprising the addition, deletion or substitution of one or more amino acids. The substitution of one amino acid by other in the functionally equivalent variant is a conservative substitution.

[0067] As used herein, the term "conservative substitution" refers to the replacement of an amino acid by another amino acid having similar chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

[0068] Alanine (A), Serine (S), Threonine (T);

[0069] Aspartic acid (D), Glutamic acid (E);

[0070] Asparagine (N), Glutamine (Q);

[0071] Arginine (R), Lysine (K);

[0072] Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0073] The expression "functionally equivalent variant", when referred to a FR refers to a peptide sequence which, when present in an antibody or antibody fragment in combination with the other FRs of the antibody or antibody fragment, results in an antibody or antibody fragment which substantially preserves its ability of specifically binding to the antigen. The term "specific binding", as well as suitable values for the parameters used to measure binding (KD and EC50) have been defined above in the context of the functionally equivalent variant of the CDRs.

[0074] A functionally equivalent variant of a FR sequence may be a polypeptide sequence derivative of said sequence comprising the addition, deletion or substitution of one or more amino acids. The substitution of one amino acid by other in the functionally equivalent variant is a conservative substitution. The term "conservative substitution" has been defined in the context of the functionally equivalent variant of the CDRs.

[0075] The expression "functionally equivalent variant", when referred to a certain VH, VL or sdAb refers to a polypeptide resulting from the modification, deletion or insertion or one or more amino acids and which substantially preserves the activity of its reference polypeptide wherein, if the variant results from the modification of one or more residues, then the modification can be a conservative amino acid substitution or not.

[0076] The term "specific binding", as well as values for the parameters used to measure binding (KD and EC50) and "conservative substitution" have been defined above in the context of the functionally equivalent variant of the CDRs.

[0077] The term "neutralizing" or "neutralize", as used herein, refers to an antibody or antibody fragment that is able to block the TIM3-mediated activity or that is able to block the SIRPa binding to CD47. This refers to an antibody or antibody fragment that is capable of neutralizing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the TIM3-mediated activity when determined using any standard assay for determining the TIM3-mediated activity, or of the binding of SIRPa to CD47 when determined using any standard assay for determining the binding of SIRPa to CD47. The TIM3- mediated activity is determined as described in the examples of the application and illustrated in Figure 3B, Figure 4B-D, Figure 6, Figure 7B, Figure 10 or Figure 23, i.e., by determining the capacity of re-estimulated T cells and the anti-tumor response in the probability of survival in DIPG mouse models. The binding of SIRPa to CD47 is determined as described in the application examples and illustrated in Figures 16 and 17, i.e., by measuring the phagocytic capacity of macrophages derived from bone marrow in tumor cell lines.

[0078] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may also be included in a pharmaceutical composition described herein, provided that they do not adversely affect the desired characteristics of the pharmaceutical composition.

[0079] The term "sequence identity" refers to a percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. In the present case, the sequence identity is obtained using the global alignment tool "EMBOSS Needle" using the default settings (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(Wl):W580-4). The global alignment tool is available at: https: / / www.ebi.ac.uk / Tools / psa / .

[0080] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein comprising one variable and one constant domain of the light chain of an antibody attached to one variable and one constant domain of the heavy chain of an antibody, wherein the heavy and light chains are linked together through a short peptide.

[0081] The term "antibody construct" as used herein, refers to constructs based on an antibody or antibody fragment that are typically generated by genetic engineering techniques. As used herein, the antibody construct refers to a construct that comprises at least an antigen-binding fragment of an antibody that retain the ability to specifically bind to an antigen (e.g., TIM3). Examples of antibody constructs include scFv, (scFv , scFv-Fc, minibody, diabody or bispecific antibody.

[0082] The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker. The term also includes a disulfide stabilized variable fragment (dsFv). Methods of stabilizing scFvs with disulfide bonds are disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10):1239-45.

[0083] The terms "treatment" and "therapy", as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intent to cure and / or alleviate a disease and / or symptoms with the goal of remediating the health problem. The terms "treatment" and "therapy" include preventive and curative methods, since both are directed to the maintenance and / or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.

[0084] The term "immune cell stimulatory agonist" shall be understood as a substance that produce a positive response by binding to a receptor on the immune cell included by not limited to T cells.

[0085] The term "about" when referred to a given amount or quantity indicates that a number can vary between ± 20%, preferably ± 10%, its indicated value.

[0086] Description of embodiments

[0087] Methods and compositions of the present invention include the construction and verification of oncolytic viruses (e.g. adenoviruses) comprising heterologous nucleic acid encoding an antibody or antibody fragment, in particular a single-domain antibody (SdAb), preferably in combination with an immune cell stimulatory receptor agonist, that exhibits enhanced and even synergistic anti-tumor effects compared to the unmodified oncolytic virus (i.e. genetically similar or identical oncolytic virus not containing heterologous nucleic acid encoding an antibody or antibody fragment, in particular a single-domain antibody (SdAb), preferably in combination with an immune cell stimulatory receptor agonist).

[0088] As already indicated, a single-domain antibody (SdAb) is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain). In particular, in the present invention, the single-domain antibodies are preferably engineered from heavy-chain antibodies found in camelids; these are called VHH fragments. Nevertheless, nanobodies derived from light chains have also been shown to bind specifically to target epitopes and are also included within the scope of the present invention.

[0089] Therefore, a first aspect of the invention refers to a replication-competent oncolytic virus comprising a heterologous nucleic acid inserted into a nonessential region of the virus genome, said heterologous nucleic acid comprising a sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, preferably the antibody fragment is a single-domain antibody (SdAb).

[0090] According to the present invention, replication-competent oncolytic viruses include any naturally occurring (e.g., from a "field source") or modified replication-competent oncolytic virus. The oncolytic virus, in addition to expressing one or more antibodies or antibody fragments, in particular SdAbs, preferably in combination with one or more immune cell stimulatory receptor agonists, may, for example, be modified to increase selectivity of the virus for cancer cells.

[0091] Replication-competent oncolytic viruses according to the invention include, but are not limited to, oncolytic viruses that are a member in the family of myoviridae, siphoviridae, podpviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adenoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hepadnaviridae, retroviridae, cyctoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, leviviridae, picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae, and barnaviridae.

[0092] Particular examples of replication-competent oncolytic viruses for use in the practice of the invention include adenovirus, retrovirus, reovirus, rhabdovirus, Newcastle Disease virus (NDV), polyomavirus, vaccinia virus, herpes simplex virus, picornavirus, coxsackie virus, and parvovirus. Adenoviruses are particularly preferred replication-competent oncolytic viruses according to the present invention.

[0093] Adenoviruses (Ad) are large (~36 kb) DNA viruses that infect humans but display a broad host range. Physically, adenovirus is icosahedral, containing a double-stranded, linear DNA genome. Approximately 50 human adenovirus serotypes are divided into six families based on molecular, immunological, and functional criteria. By adulthood, virtually every human has been infected with the more common adenovirus serotypes, the major effect being cold-like symptoms.

[0094] Adenoviral infection of host cells results in adenoviral DNA being maintained episomally, which reduces the potential genotoxicity associated with integrating vectors. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually most epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild diseases, such as respiratory / digestive tract diseases in humans.

[0095] Members of any of the 57 human adenovirus serotypes (HAdV-l to 57) may incorporate heterologous nucleic acid encoding an antibody or antibody fragment, in particular a singledomain antibody (SdAb), preferably in combination with an immune cell stimulatory receptor agonist, according to the invention. Human Ad5 is genetically and biochemically well characterized (GenBank M73260; AC 000008 (gi 1561605291 ref | AC_000008.11 Human adenovirus 5, complete genome)). Thus, in a preferred embodiment, the oncolytic adenovirus is a replication-competent Ad5 serotype or a hybrid serotype comprising an Ad5 component. The adenovirus may be a wild-type strain but is preferably genetically modified to enhance tumor selectivity, for example, by attenuating the ability ofthe virus to replicate within normal quiescent cells without affecting the ability of the virus to replicate in tumor cells. Non-limiting examples of replication-competent oncolytic adenoviruses encompassed by the present invention include primarily Delta-24 and Delta-24-RGD.

[0096] In one particularly preferred embodiment, the replication-competent oncolytic adenovirus is

[0097] Delta-24 or Delta-24-RGD. Delta-24 is described in U.S. Patent Application Publication Nos. 20030138405 and 20060147420, each of which are incorporated herein by reference. The Delta-24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24-base-pair deletion within the CR2 portion of the E1A gene that encompasses the area responsible for binding Rb protein (nucleotides 923-946) corresponding to amino acids 122- 129 in the encoded E1A protein (Fueyo J et al., Oncogene, 19:2-12 (2000)). Delta-24-RGD further comprises an insertion of the RGD-4C sequence (which binds strongly to v|33 and v|35 integrins) into the HI loop of the fiber knob protein (Pasqualini R. et al., Nat Biotechnol, 15:542-546 (1997)). The E1A deletion increases the selectivity of the virus for cancer cells; the RGD-4C sequence increases the infectivity of the virus in tumors that express integrins.

[0098] Therefore, a preferred embodiment of the present invention refers to a replication- competent oncolytic adenovirus comprising a heterologous nucleic acid inserted into a nonessential region of the adenovirus genome, said heterologous nucleic acid comprising a nucleic acid sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, preferably the antibody fragment is a single-domain antibody (SdAb); wherein the replication-competent oncolytic adenovirus is Delta-24 or Delta-24-RGD.

[0099] Oncolytic adenoviruses injected into a tumor induce cell death and release of new adenovirus progeny that, by infecting the neighbor cells, generates a treatment wave that, if not halted, may lead to the total destruction of the tumor. Significant antitumor effects of Delta-24-RGD have been shown in cell culture systems and in malignant glioma xenograft models. Aspects of the current invention are directed at enhancing this anti-tumor efficacy by inducing a more efficacious antitumor immune response, while maintaining their oncolytic capacity.

[0100] An important element of the present invention is the fact that said heterologous nucleic acid comprises a sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, preferably the antibody fragment is a single-domain antibody (SdAb). In this sense and in particular, in another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the nucleic acid encoding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-TIM3 or anti-CD47 antibody or antibody fragment, optionally linked or fused to a Fc fragment.

[0101] In another preferred embodiment, said antibody or antibody fragment is an anti-TIM3 antibody fragment selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [SGSISSIIAMA] (SEQ ID NO:

[0102] 1), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 capable of specifically binding to an epitope of the human TIM3 protein; b. HCDR2 comprises, consists, or consists essentially of [IHTGGSTH] (SEQ ID NO:

[0103] 2), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 capable of specifically binding to an epitope of the human TIM3 protein; and c. HCDR3 comprises, consists, or consists essentially of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 capable of specifically binding to an epitope of the human TIM3 protein.

[0104] In another particular embodiment, the antibody or antibody fragment is an anti-TIM3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a SdAb, scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a heavy chain domain (VH) comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. a HCDR1 consisting of [SGSISSIIAMA] (SEQ ID NO: 1); b. a HCDR2 consisting of [IHTGGSTH] (SEQ ID NO: 2); and c. a HCDR3 consisting of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3).

[0105] In another more particular embodiment of the replication competent oncolytic adenovirus according to this first aspect of the invention, the antibody or antibody fragment is an anti- Tl M3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a SdAb, scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises a heavy chain domain (VH) comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. a HCDR1 consisting of [SGSISSI I AMA] (SEQ ID NO: 1); b. a HCDR2 consisting of [IHTGGSTH] (SEQ ID NO: 2); and c. a HCDR3 consisting of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3).

[0106] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-TIM3 antibody fragment, optionally linked or fused to a Fc fragment, wherein the antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having an amino acid sequence set forth in anyone of SEQ ID NO: 4 or 29 or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4 or 29 thereto, or said single-domain antibody (SdAb) is encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 5 or SEQ ID NO: 30.

[0107] In some embodiments, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-TI M3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain, optionally linked or fused to a Fc fragment, comprises a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide sequence set forth in sequences SEQ ID NO: 4 or SEQ ID NO: 29; or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 5 or SEQ ID NO: 30; wherein said VH domain comprises at least one or more, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides, wherein a) HCDR1 comprises, consists, or consists essentially of [SGSISSI I AMA] (SEQ ID NO: 1), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 capable of specifically binding to an epitope of the human TIM3 protein; b) HCDR2 comprises, consists, or consists essentially of [IHTGGSTH] (SEQ ID NO: 2), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 capable of specifically binding to an epitope of the human TIM3 protein; and c) HCDR3 comprises, consists, or consists essentially of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 capable of specifically binding to an epitope of the human TIM3 protein.

[0108] In another particular embodiment according to the first aspect of the invention, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-TIM3 antibody fragment wherein the antibody fragment is a single-domain domain antibody having the a amino acid sequence set forth in anyone of SEQ ID NO: 4 or 29 or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4 or 29 thereto, or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 5 or SEQ ID NO: 30; wherein said single-domain antibody comprises or consists of a heavy chain domain (VH) comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. a HCDR1 consisting of [SGSISSI I AM A] (SEQ ID NO: 1); b. a HCDR2 consisting of [IHTGGSTH] (SEQ ID NO: 2); and c. a HCDR3 consisting of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3).

[0109] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-TIM3 antibody, an anti-TIM3 antibody fragment, an anti-TIM3 antibody construct, or an anti-TIM3 single-domain antibody (SdAb).

[0110] In a preferred embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the anti-TIM3 antibody or antibody fragment is an anti-TIM3 single-domain antibody (SdAb).

[0111] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the anti-TIM3 SdAb comprises:

[0112] (a) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 50, 51 and 52, or functionally equivalent variants thereof;

[0113] (b) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 55, 56 and 57, or functionally equivalent variants thereof;

[0114] (c) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 60, 61 and 62, or functionally equivalent variants thereof;

[0115] (d) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 65, 66 and 67, or functionally equivalent variants thereof;

[0116] (e) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 70, 71 and 72, or functionally equivalent variants thereof;

[0117] (f) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 75, 76 and 77, or functionally equivalent variants thereof;

[0118] (g) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 80, 81 and 82, or functionally equivalent variants thereof;

[0119] (h) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 85, 86 and 87, or functionally equivalent variants thereof;

[0120] (i) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 90, 91 and 92, or functionally equivalent variants thereof; (j) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 95, 96 and 97, or functionally equivalent variants thereof;

[0121] (k) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 100, 101 and 102, or functionally equivalent variants thereof;

[0122] (l) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 105, 106 and 107, or functionally equivalent variants thereof;

[0123] (m)the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 110, 111 and 112, or functionally equivalent variants thereof;

[0124] (n) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 115, 116 and 117, or functionally equivalent variants thereof;

[0125] (o) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 120, 121 and 122, or functionally equivalent variants thereof;

[0126] (p) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 125, 126 and 127, or functionally equivalent variants thereof; or

[0127] (q) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 1, 2 and 3, or functionally equivalent variants thereof.

[0128] Functionally equivalent variants of a CDR sequence according to the invention include CDR sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 50 to 52, 55 to 57, 60 to 62, 65 to 67, 70 to 72, 75 to 77, 80 to 82, 85 to 87, 90 to 92, 95 to 97, 100 to 102, 105 to 107, 110 to 112, 115 to 117, 120 to 122, 125 to 127, or 1 to 3. It is also contemplated that functionally equivalent variants of a CDR sequence comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 50 to 52, 55 to 57, 60 to 62, 65 to 67, 70 to 72, 75 to 77, 80 to 82, 85 to 87, 90 to 92, 95 to 97, 100 to 102, 105 to 107, 110 to 112, 115 to 117, 120 to 122, 125 to 127 or 1 to 3. Likewise, it is also contemplated that variants comprise deletions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequences of any one of the sequences set forth in SEQ ID NO: 50 to 52, 55 to 57, 60 to 62, 65 to 67, 70 to 72, 75 to 77, 80 to 82, 85 to 87, 90 to 92, 95 to 97, 100 to 102, 105 to 107, 110 to 112, 115 to 117, 120 to 122, 125 to 127 or 1 to 3.

[0129] Functionally equivalent variants of a CDR sequence according to the invention will preferably maintain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the capacity of the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 50 to 52, 55 to 57, 60 to 62, 65 to 67, 70 to 72, 75 to 77, 80 to 82, 85 to 87, 90 to 92, 95 to 97, 100 to 102, 105 to 107, 110 to 112, 115 to 117, 120 to 122, 125 to 127 or 1 to 3 to bind to its cognate antigen when being part of the antibody or antibody fragment of the invention. This capacity to bind to its cognate antigen may be determined as a value of affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment to its cognate antigen. The term "specific binding", as well as suitable values for the parameters used to measure binding (KD and EC50) have been defined above in the context of the functionally equivalent variant of the CDRs. In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the anti-TIM3 SdAb comprises:

[0130] (a) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 50, 51 and 52, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 203, 204, 205 and 206, or functionally equivalent variants thereof;

[0131] (b) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 55, 56 and 57, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 207, 208, 209 and 206, or functionally equivalent variants thereof;

[0132] (c) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 60, 61 and 62, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 210, 211, 212 and 206, or functionally equivalent variants thereof;

[0133] (d) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 65, 66 and 67, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 213, 214, 215 and 206, or functionally equivalent variants thereof;

[0134] (e) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 70, 71 and 72, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 216, 217, 218 and 206, or functionally equivalent variants thereof;

[0135] (f) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 75, 76 and 77, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 219, 220, 221 and 206, or functionally equivalent variants thereof;

[0136] (g) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 80, 81 and 82, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 222, 220, 223 and 206, or functionally equivalent variants thereof;

[0137] (h) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 85, 86 and 87, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 224, 225, 226 and 206, or functionally equivalent variants thereof;

[0138] (i) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 90, 91 and 92, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 219, 225, 227 and 228, or functionally equivalent variants thereof;

[0139] (j) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 95, 96 and 97, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 229, 230, 212 and 228, or functionally equivalent variants thereof;

[0140] (k) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 100, 101 and 102, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 231, 225, 232 and 206, or functionally equivalent variants thereof;

[0141] (l) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 105, 106 and 107, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 233, 234, 235 and 206, or functionally equivalent variants thereof; (m)the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 110, 111 and 112, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 236, 225, 237 and 238, or functionally equivalent variants thereof;

[0142] (n) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 115, 116 and 117, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 239, 225, 240 and 206, or functionally equivalent variants thereof;

[0143] (o) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 120, 121 and 122, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 241, 225, 242 and 238, or functionally equivalent variants thereof;

[0144] (p) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 125, 126 and 127, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 224, 225, 243 and 238, or functionally equivalent variants thereof; or

[0145] (q) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 1, 2 and 3, or functionally equivalent variants thereof, and the FR1, FR2, FR3 and FR4 regions comprising, respectively, the sequences set forth in SEQ ID NO: 299, 300, 301 and 302, or functionally equivalent variants thereof.

[0146] Functionally equivalent variants of a FR sequence according to the invention include FR sequences having at least approximately 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 203 to 243 or 299 to 302. It is also contemplated that functionally equivalent variants of a FR sequence comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 203 to 243 or 299 to 302. Likewise, it is also contemplated that variants comprise deletions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 203 to 243 or 299 to 302.

[0147] Functionally equivalent variants of a FR sequence according to the invention will preferably maintain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the capacity of the corresponding amino acid sequence of any one of the sequences set forth in SEQ ID NO: 203 to 243 or 299 to 302 to bind to its cognate antigen when being part of an antibody or antibody fragment of the invention. This capacity to bind to its cognate antigen may be determined as a value of affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment to its cognate antigen.

[0148] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the anti-TIM3 SdAb comprises:

[0149] (a) the sequence set forth in SEQ ID NO: 49, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 50, 51 and 52, or functionally equivalent variants thereof;

[0150] (b) the sequence set forth in SEQ ID NO: 54, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 55, 56 and 57, or functionally equivalent variants thereof;

[0151] (c) the sequence set forth in SEQ ID NO: 59, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 60, 61 and 62, or functionally equivalent variants thereof;

[0152] (d) the sequence set forth in SEQ ID NO: 64, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 65, 66 and 67, or functionally equivalent variants thereof;

[0153] (e) the sequence set forth in SEQ ID NO: 69, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 70, 71 and 72, or functionally equivalent variants thereof;

[0154] (f) the sequence set forth in SEQ ID NO: 74, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 75, 76 and 77, or functionally equivalent variants thereof;

[0155] (g) the sequence set forth in SEQ ID NO: 79, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 80, 81 and 82, or functionally equivalent variants thereof;

[0156] (h) the sequence set forth in SEQ ID NO: 84, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 85, 86 and 87, or functionally equivalent variants thereof;

[0157] (i) the sequence set forth in SEQ ID NO: 89, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 90, 91 and 92, or functionally equivalent variants thereof;

[0158] (j) the sequence set forth in SEQ ID NO: 94, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 95, 96 and 97, or functionally equivalent variants thereof;

[0159] (k) the sequence set forth in SEQ ID NO: 99, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 100, 101 and 102, or functionally equivalent variants thereof;

[0160] (l) the sequence set forth in SEQ ID NO: 104, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 105, 106 and 107, or functionally equivalent variants thereof;

[0161] (m)the sequence set forth in SEQ ID NO: 109, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 110, 111 and 112, or functionally equivalent variants thereof;

[0162] (n) the sequence set forth in SEQ ID NO: 114, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 115, 116 and 117, or functionally equivalent variants thereof;

[0163] (o) the sequence set forth in SEQ ID NO: 119, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 120, 121 and 122, or functionally equivalent variants thereof;

[0164] (p) the sequence set forth in SEQ ID NO: 124, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 125, 126 and 127, or functionally equivalent variants thereof; or

[0165] (q) the sequence set forth in SEQ ID NO: 4, or functionally equivalent variants thereof, wherein the CDR1, CDR2 and CDR3 regions comprise, respectively, the sequences set forth in SEQ ID NO: 1, 2 and 3, or functionally equivalent variants thereof. In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the anti-TIM3 SdAb comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of the sequences set forth in SEQ ID NO: 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, 124, or 4.

[0166] Anti-TIM3 antibodies have been described in the prior art, such as Cobolimab (WO2018129553A1), APE5121 (US10508149B2), Sabatolimab (W02015117002A1), ABTIM3- hum03 (W02015117002A1), Verzistobart (WO2017205721A1), LY-3321367

[0167] (W02018039020A1), Surzebiclimab (WO2018036561A1), Tim3-0438 (EP3632935A1), Tim3- 0443 (EP3632935A1) and Sabestomig (WO2025088496A1).

[0168] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the a nti-TI M 3 antibody or antibody fragment comprises the VH-CDRs and the VL-CDRs, the VH-FRs and the VL-FRs or the VH and VL regions of an antibody selected from the group consisting of Cobolimab, APE5121, Sabatolimab, ABTIM3-hum03, Verzistobart, LY-3321367, Surzebiclimab, Tim3-0438, Tim3-0443 and Sabestomig.

[0169] The SEQ ID NOs of the VH-CDRs and the VL-CDRs, the VH-FRs and the VL-FRs or the VH and VL region sequences of the a nti-TI M3 antibody or antibody fragments of Cobolimab, APE5121, Sabatolimab, ABTIM3-hum03, Verzistobart, LY-3321367, Surzebiclimab, Tim3-0438, Tim3- 0443 and Sabestomig are disclosed in Table 1. le 1: SEQ ID NOs of the VH-CDRs and the VL-CDRs, the VH-FRs and the VL-FRs or the VH and VL region sequences of the anti-TIM3 antibody or ibody fragments of Cobolimab, APE5121, Sabatolimab, ABTIM3-humO3, Verzistobart, LY-3321367, Surzebiclimab, Tim3-0438, Tim3-0443 and estomig.

[0170] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the a nti-TI M 3 antibody or antibody fragment is an a nti-TI M3 antibody construct, preferably a scFv.

[0171] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the VH region of the scFv is located at the N-terminal or at the C-terminal with respect to the VL region. Preferably, the VH and VL regions of the scFv are connected by a linker region, more preferably wherein the linker region comprises the sequence set forth in SEQ ID NO: 314 to 334.

[0172] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the scFv comprises the VH and VL regions comprising the sequences selected from the group consisting of:

[0173] (a) the VH sequence set forth in SEQ ID NO: 150 and the VL sequence set forth in SEQ ID NO: 151, or functionally equivalent variants thereof;

[0174] (b) the VH sequence set forth in SEQ ID NO: 152 and the VL sequence set forth in SEQ ID NO: 153, or functionally equivalent variants thereof;

[0175] (c) the VH sequence set forth in SEQ ID NO: 160 and the VL sequence set forth in SEQ ID NO: 161, or functionally equivalent variants thereof;

[0176] (d) the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO: 164, or functionally equivalent variants thereof;

[0177] (e) the VH sequence set forth in SEQ ID NO: 171 and the VL sequence set forth in SEQ ID NO: 172, or functionally equivalent variants thereof;

[0178] (f) the VH sequence set forth in SEQ ID NO: 179 and the VL sequence set forth in SEQ ID NO: 180, or functionally equivalent variants thereof;

[0179] (g) the VH sequence set forth in SEQ ID NO: 185 and the VL sequence set forth in any one of SEQ ID NO: 186, or functionally equivalent variants thereof;

[0180] (h) the VH sequence set forth in SEQ ID NO: 193 and the VL sequence set forth in SEQ ID NO: 194, or functionally equivalent variants thereof;

[0181] (i) the VH sequence set forth in SEQ ID NO: 195 and the VL sequence set forth in SEQ ID NO: 194, or functionally equivalent variants thereof; and

[0182] (j) the VH sequence set forth in SEQ ID NO: 201 and the VL sequence set forth in SEQ ID NO: 202, or functionally equivalent variants thereof. In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the VH region comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of the sequences set forth in SEQ ID NO: 150, 152, 160, 163, 171, 179, 185, 193, 195 or 201.

[0183] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the VL region comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of the sequences set forth in SEQ ID NO: 151, 153, 161, 164, 172, 180, 186, 194 or 202.

[0184] The invention also relates to a nucleic acid encoding the replication competent oncolytic adenovirus of the first aspect of the invention. The invention further relates to a gene construct, an expression cassette or a vector comprising the nucleic acid encoding the replication competent oncolytic adenovirus of the first aspect of the invention. The invention further relates to a cell comprising the nucleic acid encoding the replication competent oncolytic adenovirus of the first aspect of the invention, or the gene construct, the expression cassette or the vector comprising the nucleic acid encoding the replication competent oncolytic adenovirus of the first aspect of the invention.

[0185] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-CD47 antibody or antibody fragment, optionally linked or fused to a Fc fragment. In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody or antibody fragment, optionally linked or fused to a Fc fragment, wherein said antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9, 11, 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9, 11, 13 or 15 thereto.

[0186] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 9, 11, 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9, 11, 13 or 15 thereto.

[0187] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 9, or 11, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9 or 11, thereto; preferably the nucleic acid sequence encoding the SdAb is any of the sequences SEQ ID NO: 10 or SEQ ID NO: 12, or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12.

[0188] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 13 or 15 thereto; preferably the nucleic acid sequence encoding the SdAb is any of the sequences SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 14 or SEQ ID NO:

[0189] 16.

[0190] In another particular embodiment, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein said antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain, comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [VPSGRIFSINRLG] (SEQ ID NO: 6), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 6 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [AVIDGGGSTN] (SEQ ID NO: 7), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 7 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [NALATYTGPARNY] (SEQ ID NO: 8), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 8 capable of specifically binding to an epitope of the human CD47 protein.

[0191] In another particular embodiment, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein said antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [VPSGRIFSINRLG] (SEQ ID NO: 6), b. HCDR2 consisting of [AVIDGGGSTN] (SEQ ID NO: 7); and c. HCDR3 consisting of [NALATYTGPARNY] (SEQ ID NO: 8).

[0192] In another particular embodiment according to the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein said antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises a heavy chain domain (VH) domain comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [VPSGRIFSINRLG] (SEQ ID NO: 6), b. HCDR2 consisting of [AVIDGGGSTN] (SEQ ID NO: 7); and c. HCDR3 consisting of [NALATYTGPARNY] (SEQ ID NO: 8).

[0193] In some embodiments, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide sequence set forth in sequences SEQ ID NO: 13 or 15 or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 14 or SEQ ID NO: 16; wherein said VH domain comprises at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides wherein: a. HCDR1 comprises, consists, or consists essentially of [VPSGRIFSINRLG] (SEQ ID NO: 6), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 6 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [AVIDGGGSTN] (SEQ ID NO: 7), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 7 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [NALATYTGPARNY] (SEQ ID NO: 8), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 8 capable of specifically binding to an epitope of the human CD47 protein.

[0194] In some embodiments, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13 or 15; or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 14 or SEQ ID NO: 16; wherein said VH domain comprises the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [VPSGRIFSINRLG] (SEQ ID NO: 6), b. HCDR2 consisting of [AVIDGGGSTN] (SEQ ID NO: 7); and c. HCDR3 consisting of [NALATYTGPARNY] (SEQ ID NO: 8).

[0195] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 13 or 15 thereto; or said single-domain antibody (SdAb) is encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 14 or SEQ ID NO: 16; wherein said SdAb comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [VPSGRIFSINRLG] (SEQ ID NO: 6), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 6 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [AVIDGGGSTN] (SEQ ID NO: 7), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 7 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [NALATYTGPARNY] (SEQ ID NO: 8), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 8 capable of specifically binding to an epitope of the human CD47 protein.

[0196] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 13 or 15 thereto; or said single-domain antibody (SdAb) is encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 14 or SEQ ID NO: 16; wherein said SdAb comprises or consists of a VH domain comprising all of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consists of [VPSGRIFSINRLG] (SEQ ID NO: 6); b. HCDR2 consists of [AVIDGGGSTN] (SEQ ID NO: 7); and c. HCDR3 consists of [NALATYTGPARNY] (SEQ ID NO: 8),

[0197] In another particular embodiment, the antibody or antibody fragment is an anti-CD47 antibody fragment, said antibody fragment is selected from the list consisting of a singledomain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain, comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [AASGFTFDDYAI] (SEQ ID NO: 32), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 32 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [SCISSSDGKTY] (SEQ ID NO: 33), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 33 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [AVGPYWVLTTTPEGESLDLPDGED] (SEQ ID NO: 34), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 34 capable of specifically binding to an epitope of the human CD47 protein.

[0198] In another particular embodiment, the antibody or antibody fragment is an anti-CD47 antibody fragment, said antibody fragment is selected from the list consisting of a singledomain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain, comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [AASGFTFDDYAI] SEQ ID NO: 32; b. HCDR2 consisting of [SCISSSDGKTY] SEQ ID NO: 33; and c. HCDR3 consisting of [AVGPYWVLTTTPEGESLDLPDGED] f SEQ ID NO: 34.

[0199] In another particular embodiment according to the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein said antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody fragment, optionally linked or fused to a Fc fragment, comprises a VH domain comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [AASGFTFDDYAI] SEQ ID NO: 32; b. HCDR2 consisting of [SCISSSDGKTY] SEQ ID NO: 33; and c. HCDR3 consisting of [AVGPYWVLTTTPEGESLDLPDGED] f SEQ ID NO: 34. In some embodiments, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9 or 11; or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 10 or SEQ ID NO: 12; and wherein said VH domain comprises at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [AASGFTFDDYAI] (SEQ ID NO: 32), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 32 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [SCISSSDGKTY] (SEQ ID NO: 33), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 33 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [AVGPYWVLTTTPEGESLDLPDGED] (SEQ ID NO: 34), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 34 capable of specifically binding to an epitope of the human CD47 protein.

[0200] In some embodiments, the nucleic acid coding for an antibody or antibody fragment operatively linked to a transcriptional control element codes for an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9 or 11; or the VH encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 10 or SEQ ID NO: 12; and wherein said VH domain comprises the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [AASGFTFDDYAI] SEQ ID NO: 32; b. HCDR2 consisting of [SCISSSDGKTY] SEQ ID NO: 33; and c. HCDR3 consisting of [AVGPYWVLTTTPEGESLDLPDGED] f SEQ ID NO: 34.

[0201] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a singledomain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9 or 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9 or 11 thereto; or said single-domain antibody (SdAb) is encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 10 or SEQ ID NO: 12 wherein said SdAb comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [AASGFTFDDYAI] (SEQ ID NO: 32), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 32 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [SCISSSDGKTY] (SEQ ID NO: 33), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 33 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [AVGPYWVLTTTPEGESLDLPDGED] (SEQ ID NO: 34), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 34 capable of specifically binding to an epitope of the human CD47 protein.

[0202] In some embodiments, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9 or 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9 or 11 thereto; or said single-domain antibody (SdAb) is encoded by a sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the nucleotide sequences SEQ ID NO: 10 or SEQ ID NO: 12; wherein said SdAb comprises or consists of a VH domain comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of [AASGFTFDDYAI] SEQ ID NO: 32; b. HCDR2 consisting of [SCISSSDGKTY] SEQ ID NO: 33; and c. HCDR3 consisting of [AVGPYWVLTTTPEGESLDLPDGED] f SEQ ID NO: 34.

[0203] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the antibody or antibody fragment is a neutralizing antibody or antibody fragment.

[0204] In an embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the adenovirus genome might further comprise a sequence encoding an immune cell stimulatory receptor agonist, preferably selected from an 0X40 (CD134) agonist and a 41BB (CD137) agonist, operatively linked to a transcriptional control element. In a preferred embodiment, the 0X40 (CD134) agonist is an 0X40 ligand (QX40L) preferably wherein the nucleic acid encoding QX40L encodes a polypeptide comprising or consisting of the amino acid sequence set forth in GenBank Accession Number NP_003317.1) or a sequence at least 95% identical thereto and more preferably wherein the nucleic acid encoding QX40L has the nucleic acid sequence of NCBI Reference Sequence: NM_003326.5 or a sequence at least 95% identical thereto, or is an antibody or fragment thereof against 0X40. In another preferred embodiment, the sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element encodes a 4-1BB ligand (41BBL), preferably wherein the nucleic acid encoding 4-1BBL encodes a polypeptide which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical thereto, and more preferably wherein the nucleic acid encoding 4-1BBL has the nucleic acid sequence set forth in SEQ ID NO: 31 (NCBI Reference Sequence: NM_003811.4 or a sequence at least 95% identical thereto, or is an antibody or fragment thereof against 4-1BB.

[0205] In an embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the replication competent oncolytic adenovirus is preferably further characterized by comprising a second heterologous nucleic acid (from herein referred to as second heterologous nucleic acid) inserted into a nonessential region of the virus genome encoding an immune cell stimulatory receptor agonist, preferably selected from 0X40 (CD134) agonist and a 4-1BB (CD137) agonist, operatively linked to a transcriptional control element. In a preferred embodiment the 0X40 (CD134) agonist is an 0X40 ligand (OX40L), preferably wherein the nucleic acid encoding OX40L encodes a polypeptide having the amino acid sequence set forth in GenBank Accession Number NP_003317.1 or a sequence at least 95% identical thereto and more preferably wherein the nucleic acid encoding OX40L has the nucleic acid sequence of NCBI Reference Sequence: NM_003326.5 or a sequence at least 95% identical thereto, or is an antibody or fragment thereof against 0X40. In another preferred embodiment, the sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element encodes a 4- lBBIigand (4-1BBL), preferably wherein the nucleic acid encoding 4-1BBL encodes a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical thereto, and more preferably wherein the nucleic acid encoding 4-1BBL has the nucleic acid sequence set forth in SEQ ID NO: 31 (NCBI Reference Sequence: NM_003811.4 ) or a sequence at least 95% identical thereto, or is an antibody or fragment thereof against 4-1BB.

[0206] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises: a) a transcriptional control element, preferably a promoter; b) preferably a sequence encoding an immune cell stimulatory receptor agonist operatively linked to the transcriptional control element, wherein the immune cell stimulatory receptor agonist is preferably selected from an 0X40 (CD134) agonist and a 4-11B (CD137) agonist; and c) a sequence encoding an antibody or antibody fragment, optionally linked or fused to a Fc fragment, operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N-terminus; and wherein sequences b) and c) are preferably linked directly or optionally through a linker.

[0207] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises: a) a transcriptional control element, preferably a promoter; b) preferably a sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element, wherein the 4-1BB (CD137) agonist is preferably a 4- 1BB ligand (4-1BBL); and c) a sequence encoding an antibody or antibody fragment, optionally linked or fused to a Fc fragment, operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N-terminus; and wherein sequences b) and c) are preferably linked directly or optionally through a linker.

[0208] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises: a) a sequence encoding an immune cell stimulatory receptor agonist operatively linked to a transcriptional control element, preferably a promoter, wherein the immune cell stimulatory receptor agonist is preferably selected from an 0X40 (CD134) agonist and a 4-11B (CD137) agonist; and b) a sequence encoding an antibody or antibody fragment, optionally linked or fused to a Fc fragment, operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N-terminus; and wherein sequences a) and b) are preferably linked directly or optionally through a linker.

[0209] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises: a) a sequence encoding a 4-1BB (CD137) agonist operatively linked to a transcriptional control element, preferably a promoter, wherein the 4-1BB (CD137) agonist is preferably a 4-1BB ligand (4-1BBL); and b) a sequence encoding an antibody or antibody fragment, optionally linked or fused to a Fc fragment, operatively linked to a further transcriptional control element, preferably a promoter, wherein the sequence further comprises a signal sequence at its N-terminus and wherein sequences a) and b) are preferably linked directly or optionally through a linker.

[0210] It is thus noted that the sequence encoding an immune cell stimulatory receptor agonist and the sequence encoding an antibody or antibody fragment can share or not the same transcriptional control element.

[0211] In another embodiment of the first aspect of the invention, optionally in combination with any of the specific embodiments of the first aspect of the invention, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises: a) a transcriptional control element, preferably a promoter; and b) a sequence encoding an antibody or antibody fragment, optionally linked or fused to a Fc fragment, operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N-terminus; wherein the replication competent oncolytic adenovirus is preferably further characterized by comprising a second heterologous nucleic acid (from herein referred to as second heterologous nucleic acid) inserted into a nonessential region of the virus genome encoding an immune cell stimulatory receptor agonist operatively linked to a transcriptional control element, wherein the immune cell stimulatory receptor agonist is preferably selected from an 0X40 (CD134) agonist and a 4-11B (CD137) agonist. Preferably the 4-1BB (CD137) agonist is a 4-1BB ligand (4-1BBL). Preferably, the sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element encodes a 4-1BBL, preferably wherein the nucleic acid encoding 4-1BBL encodes a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical thereto-, and more preferably wherein the nucleic acid encoding 4-1BBL has the nucleic acid sequence set forth in SEQ ID NO: 31 (NCBI Reference Sequence: NM_003811.4 ) or a sequence at least 95% identical thereto, or is an antibody or fragment thereof against 4-1BB.

[0212] It is noted that any of the above-mentioned sequences encoding a 4-1BB (CD137) agonist operatively linked to a transcriptional control element can be replaced by any sequence encoding any 0X40 (CD134) agonist, preferably an 0X40 ligand (OX40L), more preferably an OX40L polypeptide having the amino acid sequence set forth in GenBank Accession Number NP_003317.1 or a sequence at least 95% identical thereto and more preferably wherein the nucleic acid encoding OX40L has the nucleic acid sequence of NCBI Reference Sequence: NM_003326.3 or a sequence at least 95% identical thereto, or an antibody or fragment thereof against 0X40.

[0213] As already indicated repeatedly throughout the present specification, preferably, the sequence encoding an antibody or antibody fragment operatively linked to the transcriptional control element is selected from any of the sequences identified above for anti-TIM3 or anti- CD47. That is, any of the specific sequences encoding an antibody or antibody fragment disclosed throughout the present specification can be selected for the above mentioned embodiments.

[0214] Preferably, the signal sequence is the sequence set forth in SEQ ID NO 17, a signal peptide obtained from human Igk (Uniprot P01601). Any promoter known to those of ordinary skill in the art that would be active in a cell in a subject is contemplated as a promoter that can be applied in the methods and compositions of the present invention. In certain embodiments, for example, the promoter is a constitutive promoter, an inducible promoter, or a repressible promoter. The promoter can also be a tissue-selective promoter. A tissue selective promoter is defined herein to refer to any relatively more active promoter in specific tissue or cell types than others. Thus, promoters suitable for realizing the present invention include, but are not necessarily limited to, constitutive promoters such as derivatives of eukaryotic virus genomes such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, LTR regions of retroviruses, the immunoglobulin gene promoter, the actin gene promoter, the EF-lalpha gene promoter as well as inducible promoters wherein protein expression depends on the addition of a molecule or exogenous signal, such as tetracycline systems, the NFKB / UV light system, the Cre / Lox system and the heat shock genes promoter, the regulable RNA polymerase II promoters described in WO / 2006 / 135436 and tissue-specific promoters.

[0215] A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5 '-non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™ (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Naturally, it will be important to employ a promoter and enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally understand the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001), incorporated herein by reference. The promoter may be heterologous or endogenous.

[0216] The particular promoter employed to control the expression of the nucleic acid of interest is not believed to be critical so long as it can express the polynucleotide in the targeted cell at sufficient levels. Thus, where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human, mammalian or viral promoter.

[0217] More preferably, the heterologous nucleic acid inserted into a nonessential region of the virus genome, preferably of the adenovirus genome, comprises, consists of or consists essentially of any one of the nucleic acid sequences comprising, consisting essentially of or consisting of SEQ. ID NO: 18 to SEQ ID NO: 27.

[0218] As indicated for the previous embodiments, the heterologous nucleic acid encoding an antibody or antibody fragment, in particular a SdAb, and optionally one or more immune cell stimulatory receptor agonists, is meant to include any genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence can be transcribed. It is noted that if one or more immune cell stimulatory receptor agonists are present, said one or more immune cell stimulatory receptor agonists can be encoded in the heterologous nucleic acid encoding an antibody or antibody fragment or in a different heterologous nucleic acid (second heterologous nucleic acid). It is further noted that if one or more immune cell stimulatory receptor agonists are present and said one or more immune cell stimulatory receptor agonists are encoded in the heterologous nucleic acid encoding an antibody or antibody fragment, the nucleic acid encoding said one or more immune cell stimulatory receptor agonists can be placed in any order with respect to the nucleic acid encoding an antibody or antibody fragment; in addition, the nucleic acid encoding said one or more immune cell stimulatory receptor agonists can be operatively linked to the same transcriptional element or to a different transcriptional element to the one operatively linked to the nucleic acid encoding an antibody or antibody fragment.

[0219] For the heterologous nucleic acid to effectively express a transcript, the nucleic acid encoding gene will be under the transcriptional control of a promoter as defined above.

[0220] Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have very similar modular organization.

[0221] Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a gene.

[0222] Initiation Signals - A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0223] IRES - In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene or polycistronic messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap-dependent translation and begin translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well as an IRES from a mammalian message. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages (see U.S. Pat. Nos. 5,925,565 and 5,935,819). Additionally, "self-cleavage" peptides P2A / T2A family elements to two proteins from a single mRNA and others.

[0224] Multiple Cloning Sites - Expression cassettes can include a multiple cloning site (MCS), a nucleic acid region containing various restriction enzyme sites, which can be used in conjunction with standard recombinant technology to digest the vector.

[0225] Transcription termination elements," a broader category that includes pA signals but also other termination elements that are independent of pA machinery, such as WPRE.

[0226] Other heterologous nucleic acid Components - In certain embodiments of the invention, cells infected by the adenoviral vector may be identified in vitro by including a reporter gene in the heterologous nucleic acid.

[0227] On the other hand, it is noted that oncolytic adenoviruses, such as the ones described for the present invention, are attractive delivery systems, in particular for use in the treatment of brain tumors. Therefore, a second aspect of the invention refers to a pharmaceutical composition comprising a replication-competent oncolytic adenovirus according to the first aspect of the invention or any of its particular and preferred embodiments, and optionally a pharmaceutically acceptable carrier. The present invention thus also provides a pharmaceutical composition comprising any composition of the present invention, and a pharmaceutically acceptable earner or carrier. The present invention also provides a vaccine composition comprising any composition of the present invention. The vaccine composition may further comprise at least one adjuvant.

[0228] The replication-competent oncolytic viruses and compositions of the present invention may be used in medicine and therapy.

[0229] The replication-competent oncolytic viruses and compositions of the present invention may be used to treat any cancer of the brain and any type of other solid tumors. In one particular embodiment, the tumor is a glioma. In a more particular embodiment, the tumor is a diffuse midline glioma (DMG).

[0230] Therefore, a third aspect of the invention refers to a replication-competent oncolytic adenovirus according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the present invention, including any of its particular and preferred embodiments or any combination thereof, for use as a medicament.

[0231] A fourth aspect of the invention refers to a replication-competent oncolytic adenovirus according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention, including any of its particular and preferred embodiments or any combination thereof, for use in a method of treatment of cancer in a subject in need thereof. Preferably, wherein the subject has a cancer selected from any solid tumours, primary or secondary (metastatic) brain cancer, preferably glioma, head and neck cancer, spinal cord cancer, melanoma, metastases, adenocarcinoma, thyoma, lymphoma, sarcoma, lung cancer, chordoma, Ewing's Sarcoma, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, malignant vascular tumors, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, and pancreatic cancer, preferably wherein the patient has a low-grade or high-grade brain tumor -pediatric or adult- and wherein the adenovirus is administered preferably but not limited to, intratumorally, intravascularly, or in a neuronal or mesenchymal stem cell carrier, preferably wherein the adenovirus is administered intratumorally.

[0232] In a particular embodiment, the subject is a pediatric patient or an adolescent or young adult (aya).

[0233] According to the invention, replication-competent oncolytic viruses or compositions may be administered locally or systemically. For example, without limitation, oncolytic viruses or compositions according to the invention, can be administered intravascularly (intraarterially or intravenously), intratumorally, intramuscularly, intradermally, intraperitoneally, subcutaneously, orally, parenterally, intranasally, intratracheally, percutaneously, intraspinal, ocularly, or intracranially. In preferred embodiments, an adenovirus or composition of the invention is administered intravascularly or intratumorally.

[0234] The quantity to be administered, both according to a number of treatments and dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Furthermore, a fifth aspect of the invention refers to an anti-TIM3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a singledomain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [SGSISSI I AM A] (SEQ ID NO: 1), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 capable of specifically binding to an epitope of the human TIM3 protein; b. HCDR2 comprises, consists, or consists essentially of [IHTGGSTH] (SEQ ID NO: 2), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 capable of specifically binding to an epitope of the human TIM3 protein; and c. HCDR3 comprises, consists, or consists essentially of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 capable of specifically binding to an epitope of the human TIM3 protein.

[0235] In another particular embodiment of the fifth aspect of the invention, the antibody or antibody fragment is an anti-TIM3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises a heavy chain domain (VH) comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. a HCDR1 consisting of [SGSISSI I AMA] (SEQ ID NO: 1); b. a HCDR2 consisting of [IHTGGSTH] (SEQ ID NO: 2); and c. a HCDR3 consisting of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3).

[0236] In another particular embodiment of the fifth aspect of the invention, the antibody or antibody fragment is an anti-TIM3 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises a heavy chain domain (VH) comprising the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. a HCDR1 consisting of [SGSISSI I AMA] (SEQ ID NO: 1); b. a HCDR2 consisting of [IHTGGSTH] (SEQ ID NO: 2); and c. a HCDR3 consisting of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3).

[0237] In another embodiment of the fifth aspect of the invention, optionally in combination with any of the specific embodiments of the fifth aspect of the invention, the antibody or antibody fragment is an anti-TIM3 antibody fragment, wherein the antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 4 or 29 or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4 or 29 thereto.

[0238] In some embodiments of the fifth aspect of the invention, optionally in combination with any of the specific embodiments of the fifth aspect of the invention, said antibody or antibody fragment comprises or consists of a VH domain, wherein the VH domain comprises a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide sequence set forth in SEQ ID NO: 4 or SEQ ID NO 29; and wherein said VH domain comprises at least one or more, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides, wherein a) HCDR1 comprises, consists, or consists essentially of [SGSISSI I AMA] (SEQ ID NO: 1), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 capable of specifically binding to an epitope of the human TIM3 protein; b) HCDR2 comprises, consists, or consists essentially of [IHTGGSTH] (SEQ ID NO: 2), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 capable of specifically binding to an epitope of the human TIM3 protein; and c) HCDR3 comprises, consists, or consists essentially of [DRDADDLLGFSSKRLRY] (SEQ ID NO: 3), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 capable of specifically binding to an epitope of the human TIM3 protein.

[0239] It is noted that the anti-TIM3 antibody or antibody fragment of the fifth aspect of the invention can be additionally defined as characterized in any of the specific embodiments of the first aspect of the invention.

[0240] A sixth aspect of the invention refers to an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [VPSGRIFSINRLG] (SEQ ID NO: 6), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [AVIDGGGSTN] (SEQ ID NO: 7), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [NALATYTGPARNY] (SEQ ID NO: 8), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8 capable of specifically binding to an epitope of the human CD47 protein.

[0241] In another embodiment of the sixth aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody or antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the SEQ ID NO.: 13 or SEQ ID NO.: 15; wherein said SdAb comprises at least one, preferably all, of the

[0242] HCDR1, HCDR2 and HCDR3 polypeptides, wherein a. HCDR1 comprises, consists, or consists essentially of [VPSGRIFSINRLG] (SEQ ID NO: 6), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [AVIDGGGSTN] (SEQ ID NO: 7), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [NALATYTGPARNY] (SEQ ID NO: 8), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 8 capable of specifically binding to an epitope of the human CD47 protein.

[0243] In another embodiment of the sixth aspect of the invention, optionally in combination with any of the specific embodiments of the sixth aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13 or 15 thereto.

[0244] An alternative sixth aspect of the invention refers to an anti-CD47 antibody or antibody fragment, wherein the antibody fragment is selected from the list consisting of a singledomain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment, optionally linked or fused to a Fc fragment, comprises or consists of a VH domain comprising at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 comprises, consists, or consists essentially of [AASGFTFDDYAI] (SEQ ID NO: 32), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [SCISSSDGKTY] (SEQ ID NO: 33), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 33 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [AVGPYWVLTTTPEGESLDLPDGED] (SEQ ID NO: 34), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34 capable of specifically binding to an epitope of the human CD47 protein.

[0245] In another alternative embodiment of the sixth aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody or antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9 or 11 or a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the SEQ ID NO.: 9 or SEQ ID NO.: 11; wherein said SdAb comprises at least one, preferably all, of the HCDR1, HCDR2 and HCDR3 polypeptides, wherein a. HCDR1 comprises, consists, or consists essentially of [AASGFTFDDYAI] (SEQ ID NO: 32), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32 capable of specifically binding to an epitope of the human CD47 protein; b. HCDR2 comprises, consists, or consists essentially of [SCISSSDGKTY] (SEQ ID NO: 33), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33 capable of specifically binding to an epitope of the human CD47 protein; and c. HCDR3 comprises, consists, or consists essentially of [AVGPYWVLTTTPEGESLDLPDGED] (SEQ ID NO: 34), or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34 capable of specifically binding to an epitope of the human CD47 protein. In another alternative embodiment of the sixth aspect of the invention, optionally in combination with any of the specific embodiments of the sixth aspect of the invention, the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 9 or 11 or a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9 or 11 thereto.

[0246] It is noted that the anti-CD47 antibody or antibody fragment of the sixth aspect of the invention can be additionally defined as characterized in any of the specific embodiments of the first aspect of the invention.

[0247] In another embodiment of the fifth or sixth aspects of the invention, optionally in combination with any of the specific embodiments of the fifth or sixth aspects of the invention, the antibody or antibody fragment is a neutralizing antibody or antibody fragment.

[0248] A seventh aspect refers to a pharmaceutical composition comprising an antibody or antibody fragment as defined in any of the fifth or sixth aspects of the invention and a pharmaceutically acceptable carrier.

[0249] Another aspect of the present invention relates to a polynucleotide encoding any of the sequences of the fifth or sixth aspects of the invention, from hereinafter the polynucleotides of the invention.

[0250] A further aspect of the present invention relates to a vector comprising any of the polynucleotides according to the invention, from hereinafter the vector of the invention.

[0251] Yet another aspect of the present invention relates to a host cell comprising the polynucleotide according to the invention or the vector according to the invention.

[0252] Antibodies or antibodies fragments according to any of the fifth or sixth aspects of the invention and compositions of the seventh aspect of the invention may be used in medicine. Antibodies or antibodies fragments according to any of the fifth or sixth aspects of the invention and compositions of the seventh aspect of the invention, further including polynucleotides, vectors or host cells of the invention, may be used to treat any cancer of the brain and any type of other solid tumors. In one particular embodiment, the tumor is a glioma. In a more particular embodiment, the tumor is a diffuse midline glioma (DMG).

[0253] Therefore, an eight aspect of the invention refers to an antibody or antibody fragment according to any of the fifth or sixth aspects of the invention or the pharmaceutical composition according to the seventh aspect of the invention, for use as a medicament.

[0254] A further aspect of the present invention refers to an antibody or antibody fragment according to any of the fifth or sixth aspects of the invention or the pharmaceutical composition according to the seventh aspect of the invention, for use as a medicament for treating cancer.

[0255] An antibody or antibody fragment according to any of the fifth or sixth aspects of the invention or the pharmaceutical composition according to the seventh aspect of the invention for use as a medicament for treating cancer according to precedent paragraph, wherein the patient has a cancer selected from any solid tumours, primary or secondary (metastatic) brain cancer, preferably glioma, head and neck cancer, spinal cord cancer, melanoma, metastases, adenocarcinoma, thyoma, lymphoma, sarcoma, lung cancer, chordoma, Ewing's Sarcoma, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, malignant vascular tumors, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, and pancreatic cancer, preferably wherein the patient has a low-grade or high-grade pediatric or adult brain tumors, and wherein the antibody or antibody fragment is preferably administered intratumorally, intravascularly, or in a neuronal or mesenchymal stem cell carrier, preferably wherein the antibody or antibody fragment is administered intratumorally.

[0256] The antibody or antibody fragment according to any of the fifth or sixth aspects of the invention or the composition of the seventh aspect of the invention may be administered locally or systemically. For example, without limitation said antibodies or antibodies fragments or said compositions, according to the invention, can be administered intravascularly (intraarterially or intravenously), intratumorally, intramuscularly, intradermally, intraperitoneally, subcutaneously, orally, parenterally, intranasally, intratracheally, percutaneously, intraspinal, occularly, or intracranially. In preferred embodiments, an antibody or antibody fragment of the invention or a composition of the invention is administered intravascularly or intratumorally. The quantity to be administered, both according to a number of treatments and dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

[0257] In a particular embodiment, the subject is a mammal, preferably the subject is a human.

[0258] In a more particular embodiment, the subject is a pediatric patient or an adolescent or young adult (aya).

[0259] The following examples and figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0260] Table 2: Sequences names and their corresponding SEQ ID NOs.

[0261]

[0262] Examples

[0263] Oncolytic virus armed with / or without immune agonist and a SdAbs against TIM3

[0264] We conducted the first-in-human Phase I trial of the oncolytic adenovirus Delta-24-RGD (DNX-2401; NCT03178032) for newly diagnosed DIPGs (Diffuse intrinsic pontine gliomas). This trial showed that the combination of Delta-24-RGD and RT is non-toxic and results in prolonged survival (Fig. 1).

[0265] In our efforts to amplify the antitumor immune response to enhance the efficacy of oncolytic viroimmunotherapy for DMGs (Diffuse Midline Glioma) we have shown that arming Delta-24-RGD with T-cell costimulatory ligand 4-1BBL (CD137L) is a promising strategy. This virus, called Delta-24-ACT, significantly increases the survival of mice bearing DMG tumors compared with the parental Delta-24-RGD or the standard of care (RT), maintains a safe profile, and remodels the T cell immune landscape (Fig. 2A-D). Although this armed virus generates a protective antitumor- adaptive memory results are still insufficient to be curative for 100% of subjects due to countermeasures at the TME such as the activation of suppressor T-regs that are known to express high-levels of the 4-1BBL receptor (Fig. 2D) indicating that there is room for therapeutic improvement.

[0266] TIM-3 is an immunosuppressive pleiotropic molecule found on the cell surface of various immune populations in TME (T cells, DCs, macrophages). We have demonstrated the relevance of TIM-3 expression in human DI PG cells and TME and its blockade mediates potent anti-tumor responses in DIPG mouse models (Fig.3A-B). Of relevance, TIM-3 is upregulated in DIPG TME after RT (the standard of care) or oncolytic adenovirus treatment, underscoring the potential synergy between TIM-3 inhibitors and Delta-24-RGD derivatives to boost virotherapy for DIPG (Fig. 3C).

[0267] In this invention, we show that treatment of an extremely aggressive orthotopic model of DIPG (26C-7) with the combination of one dose of intratumoral Delta-24-RGD virus and 3 systemic doses of anti-TIM-3 antibody increased survival slightly compared to monotherapies (Fig. 4A-B). Considering that in previous laboratory results intratumoral anti-TIM-3 treatment had worked better than systemic treatment, we decided to give both virus and antibody intratumorally (Fig. 4C). Importantly, the intratumoral combination significantly improved the therapeutic outcome compared to the other treatments, even significantly improving the therapeutic benefit over the previous combination with the antibody systemically (Fig. 4D).

[0268] These data demonstrated that the combination of both intratumoral treatments was a great therapeutic advantage. So, we decided to clone the antibody into the oncolytic virus so that a constant production of the antibody would occur each time the virus replicated. However, the sequence of an antibody is too large to insert into the genome of the virus, so we began to develop a single-domain antibody (SdAbs) or single-chain antibody (Nb) that could be inserted into the oncolytic virus. The mTIM-3 ectodomain was purified and used as immunogen in llamas (immunization carried out in Uruguay). Mouse TIM-3-SdAbs were identified and characterized in vitro using different experimental approaches, including a phage display platform. Our best candidate (called Nb5) was able to bind to TIM-3 recombinant protein (Fig. 5A) and TIM-3-expressing cells in a similar fashion to a TIM3 Ab (Fig. 5B) without "off target" targeting. In addition, our SdAbs are able to effectively bind at up to tenfold lower concentrations (weight) to the antibody (Fig. 5C).

[0269] We not only wanted to demonstrate that our mNb5 TIM-3 binds to TIM-3, but also that it does so in a functional manner. To do so, we treated ex vivo T cells from C57 mice that had been activated with anti-CD3 / CD28 beads and taken to exhaustion. We showed that our mNb5 was able to re-activate these T cells in the same way as the anti-TIM-3 antibody, measuring markers of activation and proliferation such as CD25, GrzB and Ki67 in both CD4 (Fig. 6A) and CD8 T cells (Fig. 6B). We repeated this experiment, this time using T-cells from pmel mice and re-stimulating them against tumor cells expressing gplOO. Our mNb5 and anti- TIM-3 Ab were able to significantly re-stimulate these T-cells again when compared to an antibody and a control single-domain antibody (Fig. 6C-D).

[0270] Finally, we tested whether our mNb5 TIM-3 could have a similar therapeutic effect in an orthotopic model of DIPG in vivo. For this, we injected XFM cells and treated intratumorally with control IgG [25pg], anti-TIM-3 [25pg] or our mNb5 [2.5pg] at ten times lower concentration (Fig 7A). We show that our mNb5 has a very similar effect to the anti-TIM-3

[0271] Next, we engineered the single-domain antibody mNb5 to express an Fc domain as well (Figure 8). We wanted to assess whether this FC portion will give the single domain antibody a functional advantage for the therapeutic effect. Below, we herein provide the following sequences, the sequence of the single-domain mNb5 (SdAbsTIM-3) set forth in SEQ ID NO 5 and the sequence of the single-domain SdAb TIM3-Fc set forth in SEQ ID NO 30.

[0272] To evaluate whether the addition of the Fc portion could result in a better therapeutic effect or safer profile, we evaluated several parameters after injecting these two molecules either intratumorally (IT) or intraperitoneally (IP) and the amount of SdAbs in the tumor and the rest of the mouse's body.

[0273] We observed that SdAb TIM3-Fc was retained at a slightly higher concentration in the tumor. Importantly, from a safety and security point of view, the SdAb-TIM3-Fc diffused less in the body and was found at higher concentrations in the different ganglia (Table 3).

[0274] Table 3: Percentage of SdAb-mNb5 (Tim3) in different locations after intratumoral or intraperitoneal administration. mNb5-Fc: TIM-3 single domain antibody-Fc. mNb5: TIM3 single domain antibody. IT: Intratumoral; IP: Intraperitoneal. Tumor, Deep Cerv. LN: Deep Cervical lymph nodes, Superf. Cerv. LN: Superficial Cervical lymph nodes, Cortex and Spleen.

[0275] All these previous data corroborated that our single-domain antibody was as good as the commercial antibody that had previously shown such good results in pre-clinical orthotopic models of DIPG. So, we decided to clone it into the genome of our oncolytic virus (Fig. 9).

[0276] Evaluation of the therapeutic effect of the different viruses uncovered D24-ACT-mNb5- Fc as the best candidate (Median overall survival undefined) compared to the standard of care RT (OS=21, 5) or the other viruses D23-RGD (OS=24.45), D24-ACT (OS=48,5) or the D24- ACT-mNb5 (OS=87 days). Nevertheless, all the viruses were significantly better than the standard of care in a model of Diffuse Midline Glioma (H3 mutant) (see Fig 10). It is important to note that neither of the new oncolytic viruses D24-ACT-mNb5 and D24- ACT-mNb5-Fc showed any toxicity in healthy mice, either at the neurocognitive level (see Fig. 20) or systemically in different important organs (see Fig. 21).

[0277] Finally, we decided to expand our virus to other high-risk solid tumors. TIM-3 expression analysis in tumor tissue compared to healthy tissue in a variety of organs demonstrated that TIM-3 is significantly overexpressed in many of them during cancer development (see Fig. 22). Importantly, evaluation of the efficacy of Delta-24-ACT-SdAbTIM3 (Delta-24-RGD-41BBL- single-domain antibody anti-TIM3) in an immunocompetent model of GBM (GL261), pediatric osteosarcoma (K7M2), lung metastasis, breast cancer (4T1), and colorectal cancer (MC38) demonstrated a significantly therapeutic effect compared with the parental virus and control group (Fig 23). These data revealed that this new asset can be applied and extended in a promising way to other types of tumors in addition to gliomas.

[0278] CD47 SdAbs block CD47-SIRPa binding and induce prophagocytic effects

[0279] The inhibition of CD47-SIRPa axis could be a therapeutic approach to treat tumors by increasing tumor cell phagocytosis. Hence, we studied CD47 SdAbs potential to block this axis by binding to CD47 expressed in tumor cell membranes and promoting an "eat me" signal.

[0280] The first step to generate hCD47 SdAbs was immunizing a llama with hCD47 protein and hCD47 peptide region recognized by SIRPa, to boost immune response against that specific motif (Fig. 11).

[0281] During the weeks of the immunization process, hCD47 antibody levels in llama serum were measured by ELISA (Fig. 12A). Comparing the antibody titer before the beginning of immunization, where there was observed no titer, an increase in levels was seen at week 6 (1:62500), reaching maximum levels at week 10 (1:1875000). Antibody titers decreased at week 14 (1:62500) compared to week 10, but an adequate immune response was still being elicited, allowing to move to hCD47 SdAbs library construction.

[0282] PBMCs isolated from llama fresh blood collected at weeks 10 and 14 were used to generate the hCD47 SdAbs library. After RNA extraction and cDNA synthesis, SdAbs gene products were cloned into pADL-20c phagemid vector for phage display biopanning. Five rounds of phage display were conducted to obtain SdAbs clones with hCD47 binding capacity. Following each panning round, hCD47 binding enrichment was checked by 10-fold dilution dot assay (Fig. 12B). For all rounds, an enrichment of the pool of phages binding to hCD47 was observed with respect to the BSA negative control. Once the clone panning rounds were completed, five different SdAbs clones were identified as hCD47-binders: 1A1, 1B1, 2A1, 2C1 and 2G1.

[0283] Next, to test which of these clones had the ability to block the binding of hCD47 to SIRPa, a BLI competition assay was performed. In this assay, once the SdAbs clones had been loaded into the sensor, if they could block the binding of SIRPa to CD47, loading SIRPa into the sensor resulted in no signal detected. Among the five identified clones, only 1A1 and 1B1 clones inhibited the interaction between CD47 and SIRPa (Fig. 13A). Hence, both clones were selected for further testing on inducing prophagocytic effects.

[0284] Expression, cassettes of both 1A1 and 1B1 SdAbs, were cloned in the plasmid pAB26 RGD and transfected in HEK293 cultures to generate CM. That CM was used on a phagocytosis assay using HSJD-DIPG-007-mCherry tumor cells and BMDM in a 2:1 ratio. Both SdAbs increased phagocytosis in a human DIPG cell line, but the efficacy of clone 1B1 was remarkably higher than 1A1 (52,75% and 24,96% phagocytosis, respectively) (Fig. 13B).

[0285] Once we had a good candidate. We went ahead and produced the virus with the 1B1 clone. After generating the new viruses, we first demonstrated that introducing the new transgenes into the genome of the oncolytic adenovirus Delta-24-ACT does not affect its cytopathic and replication capacity. To do this, we infected tumor cells with the Delta-24-ACT- Nb virus compared to the parental Delta-24-ACT virus. As demonstrated in Figure 2, the new transgenes introduced into the virus genome do not affect its replicative capacity (Figure 14A) or its cytolytic capacity (Figure 14B).

[0286] After this, we demonstrated that our 2 transgenes are produced efficiently in the new oncolytic adenovirus Delta-24-ACT-SdAb CD47. We infected tumor cells with this virus and measured the expression of both 41BBL in the membrane and the SdAbs. In this way, we demonstrate that the new oncolytic virus is capable of producing both proteins efficiently (Figure 15). To determine the expression of 41BBL in the new viruses, we infected two tumor lines with both a virus that does not express 41BBL (Delta-24-RGD), and with the parental one that expresses 41BBL (Delta-24-ACT) and the new oncolytic virus. (Delta-24-ACT-NbCD47). We found no difference in the number of 41BBL+ cells between the two viruses that carry the transgene for 41BBL. For the expression of the SdAbs, we used 3 viruses that carry 3 SdAbs sequences for different targets. As we can see, we demonstrate that in all cases, these viruses are capable of producing both the SdAb and other viral proteins (E1A and Fiber) (Figure 15).

[0287] After demonstrating that the transgene proteins are produced properly, we wanted to demonstrate whether the SdAb were produced functionally. Therefore, we decided to do experiments to validate its functional / therapeutic capacity, which we had previously corroborated before cloning the sequences within the virus. To do this, in this case, we use an oncolytic virus that carries a transgene to produce SdAb capable of increasing phagocytosis. In this case, we co-culture bone marrow-derived macrophages with tumor cells infected with a control, the parental virus (Delta-24-ACT) or the virus modified with the SdAb (Delta-24-ACT- NbCD47). The virus carrying the SdAbs can improves phagocytosis compared to the other two groups (Figure 16), demonstrating that our modified virus can produce functional therapeutic SdAbs. Amino acid sequence of 1B1 and 1A1 set forth in SEQ ID NO: 13 and SEQ ID NO: 9, respectively.

[0288] All the sequences within the expression cassette:

[0289] CMV + Signal Peptide + SdAb CD47.1 + BGH polyA (amino acid sequence set forth in SEQ ID NO: 35 and nucleic acid sequence set forth in SEQ ID NO: 36).

[0290] Nano CD47 1A1 (amino acid sequence set forth in SEQ ID NO: 9).

[0291] CMV + Signal Peptide + SdAb CD47.1 + BGH polyA (amino acid sequence set forth in SEQ ID NO: 37 and nucleic acid sequence set forth in SEQ ID NO: 38).

[0292] Evaluation of the Fc contribution to the effect.

[0293] To demonstrate whether it would be therapeutically beneficial to attach SdAb to a Fc to increase their efficacy both for use alone and for carrying them within a virus, we generated the human CD47 SdAb with and without Fc (see material and methods).

[0294] First, we analyzed the capacity of these two SdAb to induce phagocytosis in the context of human cells from diffuse trunk tumors, which express the mCherry fluorochrome and which we confronted with macrophages derived from bone marrow of NSG mice (which have the capacity to recognize human SIRPa). The human CD47 SdAb is superior in its phagocytic capacity compared to a human CD47 antibody. It should be noted that the CD47 SdAb linked to an Fc is significantly superior in inducing phagocytosis than the one without it. Therefore, the data indicate that using it together with the Fc (Figure 17) results in a more effective phagocytic effect and, therefore, a better therapeutic effect. First, we evaluated the therapeutic effect of the Delta-24-CD47-Fc (Delta-24-EATME-FC, which lacks the 4-1BB ligand). This experiment revealed that the D24-CD47-Fc (median overall survival not achieved) was significantly better than Delta-24-RGD (OS=49,5) (Figure 18). We are currently evaluating the effect of the Delta-24-41BBL-EATME-Fc virus.

[0295] Material and methods

[0296] 1. Design and generation of Delta-24-RGD-Nb (D24RGD-Nb) and Delta-24-ACT-Nb (D24ACT- Nb) oncolytic viruses.

[0297] 1.1. D24RGD-Nb and D24ACT-Nb construction

[0298] The E3 adenoviral gene in D24RGD-Nb and D24ACT-Nb was replaced with transgenes encoding SdAbs (with or without Fc) against CD47 or TIM3, or SdAb together with 4-1BBL. This resulted in eight virus constructs: D24RGD-Nb CD47, D24RGD-Nb CD47-Fc, D24RGD-Nb TIM3, D24RGD-Nb TIM3-FC, D24ACT-Nb CD47, D24ACT-Nb CD47-Fc, D24ACT-Nb TIM3, and D24ACT- Nb TIM3-Fc, all with 4-1BBL co-expression in the D24ACT-Nb constructs. A P2A sequence was introduced between 4-1BBL and the SdAb in D24ACT-Nb to allow co-expression, and both SdAb (with and without Fc) were engineered with a signal peptide to drive their secretion (Figure 19). To generate the modified viruses, expression cassettes containing the transgenes flanked with the cytomegalovirus (CMV) promoter and bovine growth hormone polyadenylation (BGHpolyA) sequences were synthesized and cloned into the shuttle vector pAB26-RGD at the Clal / BamHI site (GenScript). The pAB26 derivatives were digested with EcoRV, and the fragment containing the expression cassettes was co-transformed with pVK- 500C-A24, a plasmid containing the entire A24-RGD genome, previously linearized with Swal, into BJ5183 cells (Ref#16398, Addgene) by electroporation (Gene Pulser X Cell; Biorad). This process facilitated the subcloning of the expression cassettes into pVK-500C-A24 via homologous recombination, ultimately generating the genome of the modified viruses. For viral rescue, the obtained plasmid was linearized with Pad and transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen), and subsequently amplified in A549 cells and purified by double CsCI gradient ultracentrifugation. Gene Pulser X Cell electroporator (Ref#2238 BIO RAD)

[0299] 1.2. Viral replication assay 2.5xl05NP53 and XFM murine, and TP54 and SU-DIPG-IV human cell lines were seeded in a six-well plate. The following day, the cells were infected with the different adenovirus at a MOI of 100 and 10 in murine and human cell lines, respectively. After 16 and 72 hours, complete cell cultures were collected and freeze-thawed three times, and total infectious titers were determined by serial dilutions in HEK293 cells by hexon staining.

[0300] 1.3. Cell viability assays

[0301] 2,000 Np53 or XFM mouse DIPG cells were infected with the adenoviruses at different MOIs ranging from 0 to 10,000. Five days later, cell viability was evaluated using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (G3581, Promega) and measured using a SPECTROstar Nano reader (BMG Labtech).

[0302] 1.4. Characterization of viral proteins and transgenes by western blot and flow cytometry

[0303] For western blot analysis. 1 million A549 cells were seeded in a plOO plate, and 24 hours later, the cells were mock infected or infected with the different adenoviruses at MOIs of 10. The cells were collected 48 hours after viral infection. Protein extracts were prepared from the cells by incubation with lOOpL of lysis buffer (PBS+0.1% Triton and a protease inhibitor cocktail) for 30 minutes on ice. The samples were then centrifuged at 10,000 and 4°C for 15 min, and the supernatant was collected. All protein extracts were quantified using a colorimetric assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions and measured using a SPECTROstar Nano reader (BMG Labtech). Forty micrograms of protein were loaded on and separated in a 10% SDS- polyacrylamide gel under denaturing conditions. Afterward, the proteins were transferred to a nitrocellulose membrane and incubated with the following antibodies: anti-ElA (1:1000, Sc- 430 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Fiber (1:1000, NB600-541 Novus Biologica Is, Englewood, CO, USA), anti-Grb2 (1:1000, 610112 BD), anti-Vinculin (1:1000, #4650 CS), anti-VHH (1:1000, A02020 GenScript) and anti-4-lBBL (1:1000, AF1246 R&D Systems). The protein bands were detected by enhanced chemiluminescence using a ChemiDoc MP imaging system (Bio-Rad).

[0304] To detect correct expression of 4-1BBI in the cell membrane, 500,000 A549 cells were seeded in a plOO plate, and 24 hours later, the cells were mock infected or infected with the different adenoviruses at MOIs of 10. The cells were collected 48 hours after viral infection and stained with anti-41BBL (Biolegend, 107105). After this, 41BBL expression was measured by flow cytometry (CytoflexLX, Beckman Coulter).

[0305] 1.5. Phagocytosis studies with adenoviruses expressing CD47 SdAbs

[0306] Phagocytosis assays were conducted to evaluate the functionality of the anti-CD47 singledomain antibody. Bone marrow-derived macrophages (BMDMs) were differentiated from NSG mice over 5-6 days in the presence of M-CSF. Once differentiated, BMDMs were coincubated with mCherry+ tumor cells at a 1:2 ratio, along with conditioned media from virus- infected cultures. The percentage of macrophages (F4 / 80+) that had engulfed tumor cells (mCherry+) was then assessed by flow cytometry.

[0307] 1.6. Animal studies

[0308] Ethical approval for the animal studies was granted by the Animal Ethical Committee of the University of Navarra (CEEA; Comite Etico de Experimentacion Animal). All animal studies were performed at the veterinary facilities of the Center for Applied Medical Research in accordance with institutional, regional, and national laws and ethical guidelines for experimental animal care. The orthotopic DIPG model was established by injection into the brainstem using a guide-screw system (Taconic Farms, Inc.).

[0309] For adenoviruses expressing CD47 SdAbs, 250,000 UC-BL6-C7 cells in 3 pl of uncomplemented DMEM were implanted in C57BI6 mice. lxlO7pfu of D24RGD-Nb (CD47), or D24RGD as the control group, were administered intratumorally 7 and 14 days after cell implantation.

[0310] 1.7. Binding assays

[0311] To study the binding of the single-domain antibody to the recombinant protein, an ELISA was used in which the different proteins were coated on the plate (lh 37^C). The single-domain antibody was run at different concentrations for lh at 250rpm. CD27 and EDA were chosen as control antigens because they have been used previously to immunize the same llama from which the TIM3 Nb derived response. For the binding study of single-domain antibody to cells, the cells were incubated with different concentrations for 30' at 4^C. After that, an anti-HA PE (Tag) was used to detect the single-domain antibody by flow cytometry.

[0312] 1.8. Ex vivo T-cells re-estimulation T-cells were extracted from spleens of C57 and pmel mice. These lymphocytes were selected and activated in both cases by 48h of treatment with anti-CD3 / CD28 beads. After this they were maintained for 4 days with 50U of IL-2 maintaining proliferation until they reached exhaustion. C57 T-cells were re-stimulated with anti-CD3 / CD28 beads and treated with [lOpg / ml] of each treatment for 48h. After this, the different markers of activation and cytotoxicity were analyzed. In the case of T-Cells from pmel mice, they were re-stimulated with DIPG tumor cells expressing the gplOO antigen and treated in the same way as in the previous experiment for 24h. After this, the markers were also analyzed by cytometry.

[0313] 1.9. Animal models

[0314] Murine DMG tumors were developed by injecting XFM (IxlO3) cells into the pons of BALB / c mice, and 26C-7 (2.5xl05) cells into C57BL / 6 mice. The cells were implanted in 3 pL of uncomplemented media. Then, the animals were randomly assigned to each experimental group. Treatments were done intratumorally in 3 pL or systemically (lOmg / kg per mice) on the indicated days.

[0315] 2. Single-domain CD47 antibody development

[0316] The goal here is to obtain therapeutic single-domain antibodies (SdAbs) to promote tumor cell phagocytosis by inhibiting the anti-phagocytic axis CD47-SIRPa. Therefore, we have designed a protocol tailored to generate a list of VHH sequences that can bind human CD47 (hCD47) and impede its interaction with SIRPa.

[0317] Briefly, the strategy consisted of the following steps. Step 1: Immunizing a llama with the extracellular domain of the hCD47 and / or peptide corresponding to the CD47 motif interacting with SIRPa to enrich the library in clones targeting this specific region (Fig. 19A). Step 2: Peripheral blood mononuclear cells (PBMCs) were extracted from llama blood serum to build a SdAbs library (Fig. 19B, C). Step 3: Several rounds of panning against hCD47 were conducted to select hCD47-binding SdAb (Fig. 19D). Step 4: Following panning selection, SdAb clones blocking SIRPa binding were identified by biolayer interferometry (BLI) technique (Fig. 19E). Step 5: Finally, assessment of SdAb pro-phagocytic properties was evaluated through phagocytosis assays (Fig. 19E). The steps 1 to 4 were outsourced to the company Abcore (project 799L). 2.1. Camelid immunization schedule

[0318] Llamas were immunized with 0.5mg of recombinant hCD47 protein combined with Freund's Complete Adjuvant (CFA) at week 0 and with Freund's Incomplete Adjuvant (IFA) at weeks 2, 4, and 12. Additionally, llamas were also immunized with the peptide region recognized by SIRPa (SEQ ID NO: 39) fused to a KLH carrier at weeks 0, 2, 4, 8 and 12. This peptide increased the odds of obtaining inhibitory SdAbs, since it boosted the interaction of the CD47 fragment with its natural receptor (SIRPa) potentiating those sequences targeting this peptide.

[0319] ELISA tests were performed with 10 ml of serum taken before immunization and at weeks 6, 8 and 14 to check the levels of antibodies in the blood. Finally, 500ml of whole blood was extracted at weeks 10 and 14 to begin the production of the SdAbs. The entire process is detailed in table 4:

[0320] CFA: Complete Freund's adjuvant; IFA: Incomplete Freund's adjuvant

[0321] 2.2. Serum titer ELISA

[0322] Antibody titers of sera were determined by ELISA. CD47 protein was coated to the ELISA plate, followed by serum dilutions of the pre-immune bleed and production bleeds. Two negative controls were included to confirm the specificity of the titers: 1) sera were applied to wells not containing CD47 antigen, and 2) wells were coated with antigen but no serum was applied. A llama IgG coated well (no serum added) served as a positive control.

[0323] 2.3. PBMC isolation

[0324] When an adequate immune response was elicited, PBMCs from the llama (animal 2657L) were isolated from fresh blood within 4 hours of the blood-draw to generate a SdAbs library. Cells were counted under the microscope with trypan blue staining. Less than 5% cell death were required to pass quality control.

[0325] 2.4. Library Construction Build

[0326] 2.4.1. RNA Extraction

[0327] Total RNA was isolated using RNeasy Maxi Kit (Qiagen). The isolated RNA concentration was measured at Abs260. Quality was determined by Abs260 / 280 ratio with an expected ratio >1.9. RNA integrity was determined by agarose gel electrophoresis, visualizing distinct bands for 18s and 28s RNA and no apparent degradation.

[0328] 2.4.2. Library Cloning

[0329] Based on the serum titer results (Figure 2A), PBMCs from the bleeds at weeks 10 and 14 (production bleed) of the llama were selected for library construction. Proprietary animalspecific primers were used for cDNA synthesis and subsequent 2-step PCR. Products were examined by agarose gel electrophoresis. Distinct bands with the expected sizes for the SdAb gene products were isolated and cloned into pADL-20c phagemid vector using Sfil restriction sites. Ligated vectors were transformed into TGI cells, which are electrocompetent cells usually used for phage display screening.

[0330] 2.5. Library Panning Build

[0331] To eliminate enrichment of clones that bind to the negative antigen, the phage pool was first pre-absorbed on wells coated with the negative antigen. The phage pool was panned on hCD47 to get single-domain antibodies using standard panning procedures. In table 5 there is a brief description of all the rounds of panning:

[0332] 2.5.1. Monitoring for Enrichment of Binders After Each Round of Panning A dilution series of the eluted phage after each round of panning demonstrated enrichment by comparing the total phage pool binding on hCD47 antigen (+) compared to BSA (-).

[0333] 2.6. Library Screening Build

[0334] After each panning round that showed enrichment in the dot assay, 94 individual clones were expressed and tested in an off-phage initial screen for specific binding to hCD47 protein.

[0335] Screening of monoclones in an off-phage format was performed by transforming the phagemid pool into a non-amber-suppressor strain of E. coli and picking random colonies. The SdAbs proteins were recovered in the periplasmic fraction by performing an osmotic shock. Periplasmic fractions containing His-tagged SdAbs were tested for binding to the target proteins. Bound SdAbs were detected with a polyclonal goat anti-alpaca VHH domain antibody. Each clone was also tested for non-specific binding to BSA in parallel. The ELISA values obtained on BSA were subtracted from the ELISA values obtained on target proteins.

[0336] 2.7. Repeat Screening of Positive Hits Build

[0337] The hits identified in the initial screen of the phage pools were re-tested for binding to hCD47. Periplasmic fractions were prepared again by performing osmotic shock. Periplasmic fractions containing His-tagged SdAbs were tested for binding to hCD47. In this assay, bound SdAb was detected with an anti-VHH antibody. Each clone was also tested for non-specific binding to BSA in parallel. The ELISA values obtained on BSA were subtracted from the ELISA values obtained on hCD47.

[0338] 2.8. Sequences of hCD47-binding SdAb clones

[0339] During panning rounds, anti-CD47 candidates were interrogated to select those sequences that inhibit the interaction between human SIRPa and CD47 recombinant proteins. Finally, after several rounds of panning five different SdAbs clones were identified as hCD47-binders: 1A1, 1B1, 2A1, 2C1 and 2G1. The amino acid sequences of hCD47 1A1, hCD47 1B1, hCD47 2A1, hCD47 2C1 and hCD47 2G1 are set forth in SEQ ID NOs: 9, 13, 40, 41 and 42, respectively.

[0340] 2.9. Identification of hCD47-SIRPa blocking clones

[0341] A competition assay through BLI was used to screen which of the identified SdAb clones could block CD47-SIRPa binding. On an Octet RH8 equipment, CD47-Avi-His (Acrobio CD7-H82E9) was immobilized on streptavidin sensors at 7 pg / ml. Following an equilibrium step in running buffer, clones were loaded on sensor until saturation. Following another equilibrium step, sensors were dipped into SIRPa-His (Acrobio SIA-H5225) at 20 nM and monitored for an increase in signal. Among the five identified clones, only 1A1 and 1B1 clones inhibited the interaction between CD47 and SIRPa

[0342] 3. Single-domain antibodies validation

[0343] 3.1. Single-domain antibodies-Fc design

[0344] A single-domain antibody-Fc fusion protein was designed to enhance its pro-phagocytic properties giving rise to hCD47 lAl-Fc and hCD47 lBl-Fc. The amino acid sequences of hCD47 lAl-Fc and hCD47 lBl-Fc are set forth in SEQ ID NO: 43 and SEQ ID NO: 44, respectively.

[0345] 3.2. Functional studies

[0346] A codon-optimized cDNA sequence encoding hCD47 lAl-Fc or hCD47 lBl-Fc was synthesized (Genscript) and introduced in an expression cassette containing the CMV promoter and BGH poly A regulatory sequences. Additionally, the mouse Igk signal peptide (SEQ ID NO: 45) was included upstream of the single-domain antibody sequence to allow its secretion to the supernatant, obtaining the amino acid sequence of the signal peptide + hCD47 lAl-Fc, set forth in SEQ ID NO: 46, and the amino acid sequence of the signal peptide + hCD47 lBl-Fc, set forth in SEQ ID NO: 47.

[0347] The expression cassettes were cloned in the plasmid pAB26-RGD and transfected in HEK293 cultures, and the conditioned media (CM) were collected 48 hours after transfection. Then, we evaluated phagocytosis on human tumor cells (HSJD-DIPG-007) as explained above.

[0348] Both SdAbs induced human DIPG phagocytosis, but the efficacy of clone 1B1 was remarkably higher than 1A1 (Fig. 3B).

[0349] 3.3. Animal studies

[0350] Ethical approval for the animal studies was granted by the Animal Ethical Committee of the University of Navarra (CEEA; Comite Etico de Experimentacion Animal) under protocol number CEEA / 004-21. All animal studies were performed at the veterinary facilities of the Center for Applied Medical Research in accordance with institutional, regional, and national laws and ethical guidelines for experimental animal care. The orthotopic DIPG model was established by injection into the pons using a guide-screw system (Taconic Farms, Inc.).

[0351] 1,000 XFM cells in 3 pl of uncomplemented DMEM were implanted in Balb / c mice. The XFM cell line was generated in the same way that NP53 from tumors developed in a mouse model driven by PDGF-B signaling and Ink4a and ARF loss with H3 wild-type. Ink4a-ARF deletions are more common in secondary DIPGs (induced by RT). SdAbs, mAb (as positive control), or saline as the control group, were administered intratumorally in 3 pl (2.5pg SdAbs, 25pg mAb) and then injected two times intraperitoneally (25pg SdAbs, 250pg mAb) at 4, and 8 days after cell implantation, respectively in the hybrid schedule. In addition, we performed an independent experiment with only one intratumoral dose of the treatment 3 days after tumor implantation. Mice were sacrificed when symptoms were visible. In the murine models, in which growth kinetics were very fast, we considered long-term survivors to be those animals that lived at least three times longer than the median survival time of control animals.

Claims

1. 86CLAIMS1. A replication-competent oncolytic adenovirus comprising a heterologous nucleic acid inserted into a nonessential region of the adenovirus genome, said heterologous nucleic acid comprising a nucleic acid sequence encoding an antibody or antibody fragment operatively linked to a transcriptional control element, wherein the antibody fragment is selected from the list consisting of a single-domain antibody (SdAb), scFv F(ab')2, Fab and scFab, preferably the antibody fragment is a single-domain antibody (SdAb); and wherein the adenovirus is a Delta-24 or Delta-24-RGD virus.

2. The replication-competent oncolytic adenovirus according to claim 1, wherein the replication-competent oncolytic adenovirus further comprises a sequence encoding a 4-1BB ligand (4-1BBL) operatively linked to a transcriptional control element, preferably wherein the nucleic acid encoding a 4-1BBL encodes a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 95% identical thereto.

3. The replication-competent oncolytic adenovirus according to any one of the precedent claims, wherein said nucleic acid comprising a nucleic acid sequence encoding an antibody or antibody fragment further encodes at the N-terminus of the antibody or fragment thereof a signal sequence, preferably said signal sequence is of SEQ ID NO: 17 or a sequence at least 95% identical thereto.

4. The replication competent oncolytic adenovirus according to any one of the precedent claims, wherein the antibody or antibody fragment is an anti-TIM3 antibody, an anti- TIM3 antibody fragment, an anti-TIM3 antibody construct, or an anti-TIM3 singledomain antibody (SdAb).

5. The replication competent oncolytic adenovirus according to claim 4, wherein said anti-TIM3 antibody or antibody fragment is an anti-TIM3 single-domain antibody (SdAb).

6. The replication competent oncolytic adenovirus according to claim 5, wherein said anti-TIM3 SdAb comprises:(a) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 50, 51 and 52, or functionally equivalent variants thereof;87(b) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 55, 56 and 57, or functionally equivalent variants thereof;(c) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 60, 61 and 62, or functionally equivalent variants thereof;(d) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 65, 66 and 67, or functionally equivalent variants thereof;(e) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 70, 71 and 72, or functionally equivalent variants thereof;(f) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 75, 76 and 77, or functionally equivalent variants thereof;(g) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 80, 81 and 82, or functionally equivalent variants thereof;(h) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 85, 86 and 87, or functionally equivalent variants thereof;(i) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 90, 91 and 92, or functionally equivalent variants thereof;(j) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 95, 96 and 97, or functionally equivalent variants thereof;(k) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 100, 101 and 102, or functionally equivalent variants thereof;(l) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 105, 106 and 107, or functionally equivalent variants thereof;(m)the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 110, 111 and 112, or functionally equivalent variants thereof;(n) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 115, 116 and 117, or functionally equivalent variants thereof;(o) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 120, 121 and 122, or functionally equivalent variants thereof;88(p) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 125, 126 and 127, or functionally equivalent variants thereof; or(q) the CDR1, CDR2 and CDR3 regions comprising, respectively, the sequences set forth in SEQ ID NO: 1, 2 and 3, or functionally equivalent variants thereof.

7. The replication competent oncolytic adenovirus according to claim 6, wherein said anti-TIM3 SdAb comprises:(a) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(a) comprising, respectively, the sequences set forth in SEQ ID NO: 203, 204, 205 and 206, or functionally equivalent variants thereof;(b) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(b) comprising, respectively, the sequences set forth in SEQ ID NO: 207, 208, 209 and 206, or functionally equivalent variants thereof;(c) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(c) comprising, respectively, the sequences set forth in SEQ ID NO: 210, 211, 212 and 206, or functionally equivalent variants thereof;(d) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(d) comprising, respectively, the sequences set forth in SEQ ID NO: 213, 214, 215 and 206, or functionally equivalent variants thereof;(e) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(e) comprising, respectively, the sequences set forth in SEQ ID NO: 216, 217, 218 and 206, or functionally equivalent variants thereof;(f) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(f) comprising, respectively, the sequences set forth in SEQ ID NO: 219, 220, 221 and 206, or functionally equivalent variants thereof;(g) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(g) comprising, respectively, the sequences set forth in SEQ ID NO: 222, 220, 223 and 206, or functionally equivalent variants thereof;(h) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(h) comprising, respectively, the sequences set forth in SEQ ID NO: 224, 225, 226 and 206, or functionally equivalent variants thereof;89(i) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(i) comprising, respectively, the sequences set forth in SEQ ID NO: 219, 225, 227 and 228, or functionally equivalent variants thereof;(j) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(j) comprising, respectively, the sequences set forth in SEQ ID NO: 229, 230, 212 and 228, or functionally equivalent variants thereof;(k) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(k) comprising, respectively, the sequences set forth in SEQ ID NO: 231, 225, 232 and 206, or functionally equivalent variants thereof;(l) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(1) comprising, respectively, the sequences set forth in SEQ ID NO: 233, 234, 235 and 206, or functionally equivalent variants thereof;(m)the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(m) comprising, respectively, the sequences set forth in SEQ ID NO: 236, 225, 237 and 238, or functionally equivalent variants thereof;(n) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(n) comprising, respectively, the sequences set forth in SEQ ID NO: 239, 225, 240 and 206, or functionally equivalent variants thereof;(o) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(o) comprising, respectively, the sequences set forth in SEQ ID NO: 241, 225,242 and 238, or functionally equivalent variants thereof;(p) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(p) comprising, respectively, the sequences set forth in SEQ ID NO: 224, 225,243 and 238, or functionally equivalent variants thereof; or(q) the FR1, FR2, FR3 and FR4 regions within the anti-TIM3 SdAb as defined in claim 6(q) comprising, respectively, the sequences set forth in SEQ ID NO: 299, 300, 301 and 302, or functionally equivalent variants thereof.

8. The replication competent oncolytic adenovirus according to any one of claims 6 or 7, wherein:(a) the anti-TIM3 SdAb according to claim 6(a) comprises the sequence set forth in SEQ ID NO: 49, or functionally equivalent variants thereof;(b) the anti-TIM3 SdAb according to claim 6(b) comprises the sequence set forth in SEQ ID NO: 54, or functionally equivalent variants thereof;90(c) the anti-TIM3 SdAb according to claim 6(c) comprises the sequence set forth in SEQ ID NO: 59, or functionally equivalent variants thereof;(d) the anti-TIM3 SdAb according to claim 6(d) comprises the sequence set forth in SEQ ID NO: 64, or functionally equivalent variants thereof;(e) the anti-TIM3 SdAb according to claim 6(e) comprises the sequence set forth in SEQ ID NO: 69, or functionally equivalent variants thereof;(f) the anti-TIM3 SdAb according to claim 6(f) comprises the sequence set forth in SEQ ID NO: 74, or functionally equivalent variants thereof;(g) the anti-TIM3 SdAb according to claim 6(g) comprises the sequence set forth in SEQ ID NO: 79, or functionally equivalent variants thereof;(h) the anti-TIM3 SdAb according to claim 6(h) comprises the sequence set forth in SEQ ID NO: 84, or functionally equivalent variants thereof;(i) the anti-TIM3 SdAb according to claim 6(i) comprises the sequence set forth in SEQ ID NO: 89, or functionally equivalent variants thereof;(j) the anti-TIM3 SdAb according to claim 6(j) comprises the sequence set forth in SEQ ID NO: 94, or functionally equivalent variants thereof;(k) the anti-TIM3 SdAb according to claim 6(k) comprises the sequence set forth in SEQ ID NO: 99, or functionally equivalent variants thereof;(l) the anti-TIM3 SdAb according to claim 6(1) comprises the sequence set forth in SEQ ID NO: 104, or functionally equivalent variants thereof;(m)the anti-TIM3 SdAb according to claim 6(m) comprises the sequence set forth in SEQ ID NO: 109, or functionally equivalent variants thereof;(n) the anti-TIM3 SdAb according to claim 6(n) comprises the sequence set forth in SEQ ID NO: 114, or functionally equivalent variants thereof;(o) the anti-TIM3 SdAb according to claim 6(o) comprises the sequence set forth in SEQ ID NO: 119, or functionally equivalent variants thereof;(p) the anti-TIM3 SdAb according to claim 6(p) comprises the sequence set forth in SEQ ID NO: 124, or functionally equivalent variants thereof; or(q) the anti-TIM3 SdAb according to claim 6(q) comprises the sequence set forth in SEQ ID NO: 4, or functionally equivalent variants thereof.

9. The replication competent oncolytic adenovirus according to claim 4, wherein said anti-TIM3 antibody or antibody fragment comprises the VH-CDRs and the VL-CDRs, the VH-FRs and the VL-FRs or the VH and VL regions of an antibody selected from the group91 consisting of Cobolimab, APE5121, Sabatolimab, ABTIM3-hum03, Verzistobart, LY- 3321367, Surzebiclimab, Tim3-0438, Tim3-0443 and Sabestomig.

10. The replication competent oncolytic adenovirus according to any one of claims 4 or 9, wherein said anti-TIM3 antibody or antibody fragment is an anti-TIM3 antibody construct.

11. The replication competent oncolytic adenovirus according to claim 10, wherein said a nti-TI M3 antibody construct is a scFv.

12. The replication competent oncolytic adenovirus according to claim 11, wherein the VH region of the scFv is located at the N-terminal or at the C-terminal with respect to the VL region.

13. The replication competent oncolytic adenovirus according to any one of claims 11 or 12, wherein the VH and VL regions of the scFv are connected by a linker region.

14. The replication competent oncolytic adenovirus according to claim 13, wherein the linker region comprises the sequence set forth in SEQ ID NO: 314 to 334.

15. The replication competent oncolytic adenovirus according to any one of claims 11 to 14, wherein the scFv comprises the VH and VL regions comprising the sequences selected from the group consisting of:(a) the VH sequence set forth in SEQ ID NO: 150 and the VL sequence set forth in SEQ ID NO: 151, or functionally equivalent variants thereof;(b) the VH sequence set forth in SEQ ID NO: 152 and the VL sequence set forth in SEQ ID NO: 153, or functionally equivalent variants thereof;(c) the VH sequence set forth in SEQ ID NO: 160 and the VL sequence set forth in SEQ ID NO: 161, or functionally equivalent variants thereof;(d) the VH sequence set forth in SEQ ID NO: 163 and the VL sequence set forth in SEQ ID NO: 164, or functionally equivalent variants thereof;(e) the VH sequence set forth in SEQ ID NO: 171 and the VL sequence set forth in SEQ ID NO: 172, or functionally equivalent variants thereof;(f) the VH sequence set forth in SEQ ID NO: 179 and the VL sequence set forth in SEQ ID NO: 180, or functionally equivalent variants thereof;(g) the VH sequence set forth in SEQ ID NO: 185 and the VL sequence set forth in SEQ ID NO: 186, or functionally equivalent variants thereof;(h) the VH sequence set forth in SEQ ID NO: 193 and the VL sequence set forth in SEQ ID NO: 194, or functionally equivalent variants thereof;92(i) the VH sequence set forth in SEQ ID NO: 195 and the VL sequence set forth in SEQ ID NO: 194, or functionally equivalent variants thereof; and(j) the VH sequence set forth in SEQ ID NO: 201 and the VL sequence set forth in SEQ ID NO: 202, or functionally equivalent variants thereof.

16. A nucleic acid encoding the replication competent oncolytic adenovirus according to any one of claims 1 to 15.

17. A gene construct, an expression cassette or a vector comprising the nucleic acid according to claim 16.

18. A cell comprising the nucleic acid according to claim 16, or the gene construct, the expression cassette, or the vector according to claim 17.

19. The replication competent oncolytic adenovirus according to any one of claims 1 to 3, wherein the antibody or antibody fragment is an anti-CD47 antibody or antibody fragment, preferably an anti-CD47 single-domain antibody (SdAb).

20. The replication competent oncolytic adenovirus according to claim 19, wherein said anti-CD47 antibody or antibody fragment is selected from the list consisting of a singledomain antibody (SdAb), scFv, F(ab')2, Fab and scFab, and said antibody or antibody fragment comprises or consists of a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 9, 11, 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9, 11, 13 or 15 thereto.

21. The replication competent oncolytic adenovirus according to claim 20, wherein said anti-CD47 antibody or antibody fragment is a single-domain antibody (SdAb).

22. The replication competent oncolytic adenovirus according to claim 19, wherein the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO. 13 or 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 13 or 15 thereto, wherein said SdAb comprises or consists of a VH domain comprising all of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of SEQ ID NO: 6; b. HCDR2 consisting of SEQ ID NO: 7; and c. HCDR3 consisting of SEQ ID NO: 8.9323. The replication competent oncolytic adenovirus according to claim 19, wherein the antibody or antibody fragment is an anti-CD47 antibody fragment, wherein the antibody fragment is a single-domain antibody (SdAb), and said single-domain antibody (SdAb) is a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 9 or 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to any one of SEQ ID NO: 9 or 11 thereto, wherein said SdAb comprises or consists of a VH domain comprising all of the HCDR1, HCDR2 and HCDR3 polypeptides set forth below: a. HCDR1 consisting of SEQ ID NO: 32; b. HCDR2 consisting of SEQ ID NO: 33; and c. HCDR3 consisting of SEQ ID NO: 34.

24. The replication competent oncolytic adenovirus according to any one of claims 1 to 15 and 19 to 23, wherein the heterologous nucleic acid inserted into a nonessential region of the adenovirus genome comprises: a. a transcriptional control element, preferably a promoter; b. a sequence encoding a 4-1BB (CD137) agonist operatively linked to the transcriptional control element; wherein the 4-1BB (CD137) agonist is preferably a 4-1BB ligand (4-1BBL); and c. a sequence encoding an antibody or antibody fragment as defined in any one of claims 4 to 15 and 19 to 23 operatively linked to the transcriptional control element, wherein the sequence further comprises a signal sequence at its N- terminus; and wherein sequences b) and c) are preferably linked directly or optionally through a linker.

25. The replication competent oncolytic adenovirus according to claim 24, wherein i. the sequence encoding a 4-1BBL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 (GenBank Accession Number NP_003802.1) or a sequence at least 90% identical hereto; ii. the signal sequence is the sequence set forth in SEQ ID NO: 17 or a sequence at least 95% identical thereto; andiii. the sequence encoding an antibody or antibody fragment is the sequence of an anti-TIM3 antibody or antibody fragment as defined in any one of claims 4 to 15 or an anti-CD47 antibody or antibody fragment as defined in any one of claims 19 to 23.

26. The replication competent oncolytic adenovirus according to any one of the precedent claims, wherein the heterologous nucleic acid inserted into a nonessential region of the adenovirus genome comprises or consists of any one of the sequences set forth in SEQ. ID NO: 132 to 143 or SEQ ID NO 22 to 27.

27. A pharmaceutical composition comprising a replication competent oncolytic adenovirus according to any one of claims 1 to 15 or 19 to 26 and pharmaceutically acceptable carrier.

28. The replication competent oncolytic adenovirus according to any one of claims 1 to 15 or 19 to 26 or a pharmaceutical composition according to claim 27, for use as a medicament.

29. The replication competent oncolytic adenovirus according to any one of claims 1 to 15 or 19 to 26 or a pharmaceutical composition according to claim 27 for use in a method of treatment of cancer in a subject in need thereof, wherein the subject has a cancer selected from a solid tumour, including primary or secondary brain cancer, preferably glioma, head and neck cancer, spinal cord cancer, melanoma, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, chordoma, Ewing's Sarcoma, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, malignant vascular tumors, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, and pancreatic cancer.

30. The replication competent oncolytic adenovirus for use according to claim 29, wherein the subject has a low-grade or high-grade glioma.

31. The replication competent oncolytic adenovirus for use according to any one of claims 29 or 30, wherein the adenovirus is administered intratumorally, intravascularly, or in a neuronal or mesenchymal stem cell carrier.

32. The replication competent oncolytic adenovirus for use according to claim 31, wherein the adenovirus is administered intratumorally.

Images