Multifunctional molecules comprising soluble TCR fragments and uses thereof

Abstract

The present invention is in the field of immunotherapy, in particular cancer immunotherapy and relates to multifunctional molecules comprising at least a soluble TCR fragment, a CD3 agonist, a CD2 agonist and optionally, an antibody Fc region. Furthermore, the invention relates to methods of making the multifunctional molecule according to the invention, as well as therapeutic uses thereof.

Description

[0001] New PCT-Patent Application

[0002] Engimmune Therapeutics AG c / o Switzerland Innovation Park Basel Area AG

[0003] Vossius Ref.: AJ3536 PCT BS

[0004] MULTIFUNCTIONAL MOLECULES COMPRISING SOLUBLE TCR FRAGMENTS AND USES THEREOF

[0005] The present invention is in the field of immunotherapy, in particular cancer immunotherapy and relates to multifunctional molecules comprising at least a soluble TCR fragment, a CD3 agonist, a CD2 agonist and, optionally, an antibody Fc region. Furthermore, the invention relates to methods of making the multifunctional molecule according to the invention, as well as therapeutic uses thereof.

[0006] BACKGROUND INFORMATION

[0007] CD2 is a surface glycoprotein broadly expressed by human T cells, with naive T cells (i.e., antigen naive) displaying the lowest levels of surface CD2 and memory T cells (i.e., antigen experienced) expressing the highest. A notable exception are CD8+ T cells from the tumour infiltrating lymphocyte (TILs) compartment, which are T cell subset with high expression of exhaustion markers arising from recurrent antigen exposure, suggesting that repeated antigen stimulation may lead to a reduction in CD2 expression (Demetriou et al., Nature Immunology, 2020, 21 (10): 1232-43). Clinical observations indicating a role of CD2 in promoting antitumour responses include a correlation between increased T cell exhaustion levels and lower CD2 expression in CD8+ TILs isolated from colorectal, lung and liver tumours (Demetriou et al., Nature Immunology, 2020, 21 (10): 1232-43), and more importantly, a strong correlation between CD58 (i.e., the major ligand of CD2, also known as LFA-3) expression on tumour cells and response to CAR-T cell therapy targeted to CD19 in lymphoma (Majzner et al., Blood, 2020, 136 (Supplement 1): 53-54).

[0008] In vitro studies using CD58 knockout cells have shown that CD2 co-ligation in the context of repeated TCR / CD3 stimulation results in a more sustained T cell proliferation, thus providing strong evidence that the CD2:CD58 axis fulfils a role in mitigating T cell exhaustion (Shen et al., Journal for Immunotherapy of Cancer, 2022, 10 (3). https: / / doi.org / 10.1136 / jitc-2021- 004348). CD2 plays an important role in the formation of the immune synapse (IS) created by TCR recognition of peptide-MHC antigen on target cells. IS formation relies on clustering of multiple receptors in localised microdomains at the site of TCR recognition of peptide-MHC target, which are referred to as supramolecular activation clusters (SMAC). The IS is organised into distinct concentric regions with the TCR / CD3:pMHC at its centre, and has the following components (from TCR proximal to TCR distal) (Binder et al., Frontiers in Immunology, 2020, 11 (June):1090.):

[0009] • Central SMAC (cSMAC): incorporates the TCR / CD3 complex, CD4 or CD8 co-receptors and additional activating (e.g., CD2, CD28) and inhibitory receptors (e.g., PD-1) that tune the strength of TCR signalling by the action of their intracellular domains (ICD) in promoting or preventing phosphorylation of key residues within TCR / CD3 signalling domains.

[0010] • Peripheral SMAC (pSMAC): dominated by interactions between the adhesion molecule ICAM-1 on T cells with LFA-1 on target cells.

[0011] • CD2:CD58 corolla: this recently reported structure has been proposed to fulfil the role of further stabilising the IS through F-actin modulation

[0012] • Distal SMAC (dSMAC): enriched with excluded CD45 inhibitory receptor

[0013] Related to its role within the cSMAC, CD2 has been shown to co-elute with the CD3-zeta subunit of the TCR / CD3 complex in co-immunoprecipitation studies, but the nature of this interaction remains to be fully elucidated. The intracellular domain of CD2 has been shown to interact with both Fyn and Lek kinases which function to transmit T cell activation signals, ultimately supporting the killing of engaged target cells through secretion of granzymes and perforin. Furthermore, the CD2 ICD has been reported to interact indirectly with F-actin through CMS (Cas ligand with multiple Src homology (SH) 3 domains), which docks on its fourth SH3-binding domain, thus suggesting a role for CD2 in cytoskeleton stabilisation during the formation of the IS, most likely through the formation of the recently reported CD2 corolla.

[0014] Soluble T cell receptor (TCR) engagers are an emerging therapeutic modality for the treatment of advanced solid tumours. This class of biopharmaceuticals features an affinity-enhanced TCR arm recognising an antigen presented by major histocompatibility complex (MHC) molecules on the surface of tumour cells and an effector arm that engages T cells to kill the targeted tumour cell, through the creation of an artificial immune synapse. Soluble TCR engagers undergoing clinical development include those targeting the tumour antigens gplOO, MAGE- A4 / 8 and PRAME. Currently, all soluble TCR engagers in clinical development incorporate a single engager arm acting as an agonist of the CD3E or CD3s6 subunits of the TCR-CD3 complex present on the surface of T cells.

[0015] The only approved soluble TCR engager, tebentafusp for the treatment of metastatic uveal melanoma (mUM), targets the melanocyte antigen gplOO on melanoma cells through an affinity-enhanced TCR fragment and engages CD3E6 on T cells with an agonistic antibody fragment (single-chain fragment variable or scFv). Treatment with tebentafusp leads to increased infiltration of uveal melanoma tumours by T cells, thus supporting the mechanism of action of T cell redirection into tumours (Carvajal et al., Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology, 2022, 40 (17): 1939

[0016] -48.).

[0017] Three-year overall survival in mUM patients treated with tebentafusp is reported at 27%, compared to 18% in a control group of patients treated with standard of care therapies (pembrolizumab, ipilimumab or dacarbazine). Furthermore, median progression-free survival is reported at 3.4 months for patients treated with tebentafusp compared to 2.9 months in the control group (Hassel et al., The New England Journal of Medicine, 2023, 389 (24): 2256- 66). Despite prolonging survival in a historically intractable indication and its ability to redirect T cells into tumours, tebentafusp treatment elicits a modest objective response rate (i.e., partial and complete responses) of 11% (compared to 5% for standard of care). Similar objective response rates (ORR) have been reported in clinical trials of brenetafusp, a soluble TCR engager targeting PRAME and engaging CD3E6 on T cells (Hamid et al., Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 2024, 42 (16_suppl): 9507-9507). Recently, a strong correlation between ORR and the expression levels of markers CD28, TESPA1 and GPR183 by patient T cells prior to therapy was identified (Sacco et al., Annals of Oncology, 2024, 35 (September):S239-40). These markers are involved in T cell activation (CD28, TESPA1) and migration (GPC183) and have been proposed to provide a gene expression signature reflecting T cell fitness. In turn, this gene expression signature is correlated with the expression of IL7R, FOXO1 and TCF7, all of which are associated with T cell sternness (i.e., the ability of a T cell to remain functional despite repeated antigen challenge, see below) (Chan et al., Nature, 2024, 629 (8010): 201-10).

[0018] Strategies designed to improve the potency and, ultimately, the clinical response to soluble TCR engager treatment include half-life prolongation, multivalent TCR targeting and incorporation of additional engager arms to enhance T cell activation. It should be noted that repeated antigen stimulation can lead to T cell exhaustion, whereby T cells become less responsive and, in the case of cytotoxic T cells, gradually lose their ability to kill target cells. Importantly, T cell exhaustion is significantly higher when T cell stimulation occurs in the absence of co-stimulation (i.e., signal 2) (Chi, Pepper, and Thomas, Cell, 2024, 187 (9): 2052- 78. 2024). A hallmark of T cell exhaustion is the elevated expression of inhibitory receptors on their surface, such as PD-1 and LAG-3, which are also known as immune checkpoints. When engaged by their ligands, immune checkpoint molecules transmit inhibitory signals that attenuate T cell activation (Wherry and Kurachi, Nature Reviews Immunology, 2016, 15 (8): 486-99). Blockade of these interactions with monoclonal antibody drugs (i.e., immune checkpoint blockade) has resulted in unprecedented clinical benefit in a subset of advanced malignant conditions, such as cutaneous melanoma, liver cancer, and non-small cell lung cancer (Sharma et al., Cell, 2023, 186 (8): 1652-69.).

[0019] Despite these recent advances, there is still a need for improved TCR-based therapeutics. Therefore, it is an objective of the present invention to provide soluble TCR fragments that have been modified to improve their potency.

[0020] SUMMARY OF THE INVENTION

[0021] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0022] 1. A multifunctional molecule comprising i) a soluble T cell receptor (TCR) fragment; ii) a CD3 agonist; and iii) a CD2 agonist.

[0023] 2. The multifunctional molecule according to embodiment 1, wherein the soluble TCR fragment is a heterodimeric TCR fragment comprising a first and a second polypeptide chain, in particular wherein the soluble TCR fragment is an a|3-heterodimeric TCR fragment comprising extracellular fragments of a TCR a-chain and a TCR p-chain.

[0024] 3. The multifunctional molecule according to embodiment 1 or 2, wherein the soluble TCR fragment has been engineered for increased affinity, specificity and / or stability.

[0025] 4. The multifunctional molecule according to any one of embodiments 1 to 3, wherein the soluble TCR fragment has been engineered to comprise one or more artificial disulphide bonds.

[0026] 5. The multifunctional molecule according to any one of embodiments 1 to 4, wherein the soluble TCR fragment specifically binds to MAGE-A3, EBNA-1, GPC3, KRAS protooncogene neoantigens, TCF-1, AFP or PSA.

[0027] 6. The multifunctional molecule according to any one of embodiments 1 to 5, wherein the CD3 agonist is an anti-CD3 antibody, or an antigen-binding fragment thereof.

[0028] 7. The multifunctional molecule according to embodiment 6, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv fragment or a nanobody, or wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment. 8. The multifunctional molecule according to any one of embodiments 1 to 7, wherein the CD2 agonist is a CD2 ligand, an anti-CD2 antibody, an antigen-binding fragment thereof, or a non-antibody binding scaffold.

[0029] 9. The multifunctional molecule according to any one of embodiments 1 to 8, wherein the CD2 agonist is a CD58 ectodomain.

[0030] 10. The multifunctional molecule according to embodiment 9, wherein the CD58 ectodomain comprises or consists of (i) an amino acids sequence as set forth in any one of SEQ ID NOs:l-4, or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:l-4, wherein the CD58 domain remains the ability to engage CD2 on the surface of a T cell.

[0031] 11. The multifunctional molecule according to any one of embodiments 1 to 10, wherein the soluble TCR fragment is linked via a first polypeptide chain comprised in the soluble TCR fragment to a polypeptide chain comprised in the CD3 agonist.

[0032] 12. The multifunctional molecule according to any one of embodiments 1 to 11, wherein the CD3 agonist is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0033] 13. The multifunctional molecule according to embodiment 12, wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment, or a single chain CD3 agonist, in particular an scFv; preferably wherein the CD3 agonist is an scFv.

[0034] 14. The multifunctional molecule according to embodiment 12 or 13, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv, and wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

[0035] 15. The multifunctional molecule according to embodiment 14, wherein the CD2 agonist is linked: a) to the N-terminal end of the single chain CD3 agonist; b) to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment; or c) to a C-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment. 16. The multifunctional molecule according to embodiment 12 or 13, wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment, and wherein an N- terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

[0036] 17. The multifunctional molecule according to embodiment 16, wherein the CD2 agonist is linked: a) to an N-terminal end of a second polypeptide chain comprised in the heterodimeric CD3 agonist; b) to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist; c) to an N-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment; or d) to a C-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

[0037] 18. The multifunctional molecule according to any one of embodiments 1 to 17, wherein the molecule further comprises an antibody Fc region.

[0038] 19. The multifunctional molecule according to embodiment 18, wherein the antibody Fc region is derived from a human IgG antibody heavy chain.

[0039] 20. The multifunctional molecule according to embodiment 18 or 19, wherein the antibody Fc region is a heterodimeric antibody Fc region.

[0040] 21. The multifunctional molecule according to any one of embodiments 18 to 20, wherein the antibody Fc region comprises one or more mutations that reduce immune effector functions.

[0041] 22. The multifunctional molecule according to any one of embodiments 18 to 21, wherein a first polypeptide chain comprised in the antibody Fc region is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment or the CD3 agonist.

[0042] 23. The multifunctional molecule according to any one of embodiments 18 to 22, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment. 24. The multifunctional molecule according to embodiment 23, wherein the CD3 agonist is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0043] 25. The multifunctional molecule according to embodiment 24, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv.

[0044] 26. The multifunctional molecule according to embodiment 24 or 25, wherein the CD2 agonist is linked: a) to an N-terminal end of a polypeptide chain comprised in the single chain CD3 agonist, in particular to the N-terminal end of the scFv; b) to an available N-terminal end of a polypeptide chain comprised in the soluble TCR fragment; or c) to an available C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0045] 27. The multifunctional molecule according to any one of embodiments 18 to 26, wherein: a) the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment; b) the CD3 agonist, in particular the scFv, is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment; and c) the CD2 agonist, in particular the CD58 ectodomain, is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

[0046] 28. A nucleic acid encoding the multifunctional molecule of the preceding embodiments.

[0047] 29. A cell comprising the nucleic acid according to embodiment 28.

[0048] 30. A method of producing the multifunctional molecule according to any one of embodiments 1 to 27, the method comprising a step of culturing the cell according to embodiment 29.

[0049] 31. A pharmaceutical composition comprising the multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid molecule according to embodiment 28 and / or the cell according to embodiment 29, and a pharmaceutically acceptable carrier. 32. The multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31 for use in medicine, in particular for use in the treatment of cancer, viral infections or autoimmune diseases.

[0050] 33. The multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31 for use in the treatment of cancer in a subject, wherein the subject's cancer is characterized by the presence of exhausted T-cells.

[0051] 34. The multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31 for use in the treatment of cancer in a subject, wherein the subject is refractory to, or has relapsed after, a prior immunotherapy.

[0052] 35. The multifunctional molecule for use according to embodiment 34, wherein the prior immunotherapy is a checkpoint inhibitor therapy.

[0053] 36. An ex vivo or in vitro method for reactivating a population of exhausted T-cells, the method comprising the step of contacting a population of T-cells comprising exhausted T-cells with the multifunctional molecule according to any one of embodiments 1 to 27.

[0054] 37. A population of reactivated T-cells obtainable by the method of embodiment 36.

[0055] 38. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31, wherein the subject has been identified as having exhausted T-cells prior to administration of the multifunctional molecule.

[0056] 39. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the multifunctional molecule according to any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31, wherein the subject has previously been treated with and failed to respond to a checkpoint inhibitor therapy. 40. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of the multifunctional molecule of any one of embodiments 1 to 27, the nucleic acid according to embodiment 28, the cell according to embodiment 29 or the pharmaceutical composition according to embodiment 31, wherein the administration of the molecule reactivates exhausted T-cells in the subject.

[0057] The present invention is based on the hypothesis that the synapse-dependent activity of CD2 is well aligned with the mechanism of action of soluble TCR engagers. Thus, the inventors sought to recapitulate a natural immune synapse for redirecting T cells to more potently kill tumour cells. To test the hypothesis that CD3xCD2 co-ligation can improve the potency of soluble TCR engagers, the inventors designed a panel of trispecific molecules incorporating an affinity-enhanced TCR, a CD3 agonist antibody, and the ectodomain of CD58 to engage CD2. To investigate the effect of IS dimension on activity, the inventors designed trispecific molecules using two types of parental soluble TCR scaffolds, namely a larger dual Fab-Fab scaffold (TCR-Fab, CD3 agonist Fab), and a more compact Fab-scFv scaffold (TCR-Fab, CD3 agonist scFv) (Fig. 1). Finally, given the importance of IS organisation on TCR signalling strength, the inventors incorporated the CD58 ectodomain at different sites of the tested scaffolds to assess the impact of molecular geometry on soluble TCR engager potency. With this approach, the inventors surprisingly found that the efficacy of existing bispecific TCR engagers can be markedly increased by covalently attaching the TCR engager to a CD2 agonist, as demonstrated in Example 3.

[0058] Furthermore, the inventors found that this increase in potency was not merely an incremental enhancement but a profound and unexpected improvement, particularly in clinically relevant contexts. The experiments demonstrated that the addition of the CD58 ectodomain, especially in a discrete molecular configuration, results in highly potent target-specific T cell killing and activation, with an approximately 10-fold higher activity than the bispecific control molecule.

[0059] Most surprisingly, the inventors found that the trispecific molecules of the invention are capable of potently reactivating exhausted T-cells, a cell population that is functionally inert and represents a major hurdle in cancer immunotherapy. In co-cultures using exhausted T- cells as effectors, the trispecific molecule demonstrated a substantial increase in activity, showing up to a 15-fold increase in potency for IFN-y secretion compared to its bispecific counterpart (FIG. 11). This potent reactivation of exhausted T-cells is a qualitatively different and non-obvious technical effect that goes far beyond a simple enhancement of TCR activity.

[0060] Importantly, this enhanced potency was not limited to a single TCR target. The inventors demonstrated that the inventive concept is a generalizable platform technology by successfully applying the trispecific format to TCRs targeting different antigens, including MAGE-A3 and GPC3 (FIG. 18, FIG. 19, and FIG. 21). In each case, the trispecific molecule incorporating the CD58 ectodomain showed superior activity compared to the corresponding bispecific molecule. This confirms that the specific molecular architecture is a broadly applicable solution for improving the efficacy of soluble TCR engagers.

[0061] Finally, this significant increase in on-target potency was achieved without a corresponding increase in off-target activity. In co-cultures with antigen-negative cells, the trispecific molecules displayed only low or negligible levels of T-cell activation, highlighting a safe and highly specific activity window for the molecules of the invention (FIG. 12, FIG. 13, FIG. 18, FIG. 19, and FIG. 21).

[0062] 1. Formats of multifunctional molecules

[0063] In a particular embodiment, the invention relates to a multifunctional molecule comprising (i) a soluble T cell receptor (TCR) fragment; and (ii) a CD3 agonist and, (iii) a CD2 agonist.

[0064] The present invention relates to a multifunctional molecule comprising at least a soluble TCR fragment, a CD3 agonist and a CD2 agonist. It was demonstrated that coupling a CD3 agonist and a CD2 agonist to a soluble TCR fragment directs the CD3 agonist and CD2 agonist to the target site of the soluble TCR fragment, thereby enhancing activation and co-stimulation of T cells directly at the target site of the soluble TCR fragment.

[0065] Molecules comprising a soluble TCR fragment and a CD3 agonist are known in the art and can act as a bridge between T cells and target cells expressing an antigen recognized by the soluble TCR fragment, such as cancer cells or virus-infected cells. Besides this bridging function, CD3 agonists also have an immune activating function. Eventually, this will result in an enhanced immune response against cells expressing the target antigen, such as cancer cells.

[0066] Further incorporating a CD2 agonist into a TCR engager resulted in significantly higher potency. Without wishing to be bound by theory, this increased potency over conventional TCR engagers may be due to the formation of an immune synapse that more closely mimics the natural immune synapse (see Fig. 5).

[0067] In the context of this application, the term "multifunctional" refers to a molecule that possesses multiple distinct biological activities within a single entity. Specifically, the multifunctional molecule comprises a soluble TCR fragment, a CD3 agonist, and a CD2 agonist. This design allows the molecule to engage and activate T cells through the TCR and CD3 receptors while providing co-stimulatory signals via the CD2 receptor, thereby enhancing the overall therapeutic efficacy in targeting and eliminating tumour cells. Within the present invention, the multifunctional molecule comprising a soluble TCR fragment, a CD3 agonist, and a CD2 agonist may also be referred to as a multispecific molecule or a trispecific molecule.

[0068] The individual components of the multifunctional molecule of the invention may be assembled in any possible way. Preferably, the individual components are assembled such that each of the components retains its biological activity. Preferred embodiments of the multifunctional molecule according to the invention are described herein below.

[0069] The soluble TCR fragment comprised in the multifunctional molecule according to the invention is preferably a heterodimeric TCR fragment comprising a first and a second polypeptide chain. That is, the soluble TCR fragment is preferably a heterodimer comprising two polypeptide chains (a|3 or y6), as defined in more detail elsewhere herein.

[0070] In a preferred embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the soluble TCR fragment is a heterodimeric TCR fragment comprising a first and a second polypeptide chain, wherein the soluble TCR fragment is an a|3- heterodimeric TCR fragment comprising extracellular fragments of a TCR a-chain and a TCR |3- chain.

[0071] The soluble TCR fragment may be linked to the CD3 agonist and / or the CD2 agonist in any suitable way. That is, the CD3 agonist and / or the CD2 agonist may be linked, without limitation, to the N-terminal or the C-terminal end of the first or second polypeptide chain comprised in the soluble TCR fragment.

[0072] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the soluble TCR fragment is linked via a first polypeptide chain comprised in the soluble TCR fragment to a polypeptide chain comprised in the CD3 agonist.

[0073] More preferably, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is linked to an N-terminal end, or a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0074] The CD3 agonist may be any CD3 agonist known in the art or disclosed herein. It is however preferred herein that the CD3 agonist is a fragment of an anti-CD3 antibody, such as a Fab fragment or an scFv fragment derived from an anti-CD3 antibody.

[0075] The multifunctional molecule according to the invention further comprises a CD2 agonist in addition to the soluble TCR fragment and the CD3 agonist. The CD2 agonist comprised in the multifunctional molecule according to the invention may be any suitable CD2 agonist, as defined elsewhere herein. In certain embodiments, the CD2 agonist may be a natural or engineered CD2 ligand, an anti-CD2 antibody, an antigen-binding fragment thereof, or a nonantibody binding scaffold. In a preferred embodiment, the CD2 agonist is a CD58 molecule, more preferably a CD58 ectodomain.

[0076] In a more preferred embodiment, the CD58 ectodomain comprises or consists of (i) an amino acids sequence as set forth in any one of SEQ ID NOs:l-4, or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:l-4, wherein the CD58 domain remains the ability to engage CD2 on the surface of a T cell.

[0077] The CD2 agonist may be linked to any suitable position of the soluble TCR fragment or the CD3 agonist, as discussed in more detail herein below.

[0078] 1.1 Multifunctional molecules comprising a soluble TCR a single chain CD3

[0079] In certain embodiments, the CD3 agonist consists of a single polypeptide chain, such as an scFv or a nanobody. In such embodiments, the single chain CD3 agonist may be fused to an N- terminal end of a polypeptide chain comprised in the soluble TCR fragment via its C-terminal end. Alternatively, the single chain CD3 agonist may be fused to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment via its N-terminal end.

[0080] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist, in particular the single chain CD3 agonist, is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment. In certain embodiments, the CD3 agonist linked to an N-terminal end of a polypeptide chain comprised in a soluble TCR fragment may be a Fab fragment or an scFv.

[0081] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a single chain CD3 agonist, such as an scFv, and wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

[0082] That is, in certain embodiments, a single chain CD3 agonist, preferably an scFv fragment, may be linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment, as shown in FIG.l, top right panel. It is to be understood that FIG.l (top panel) only shows the part of the multifunctional molecule of the invention comprising the soluble TCR fragment and the CD3 agonist, and that the final molecule further comprises a CD2 agonist, preferably in the positions marked with an X.

[0083] Non-limiting examples of multifunctional molecules comprising a soluble TCR fragment, a single chain CD3 agonist and a CD2 agonist are provided herein below:

[0084] In certain embodiments, the CD3 agonist is a single chain CD3 agonist, preferably an scFv, wherein the C-terminal end of the single chain CD3 agonist is linked to the N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to the N-terminal end of the scFv (see FIG.l, construct 374).

[0085] The polypeptide chain of the soluble TCR fragment to which the single chain CD3 agonist and the CD2 agonist are linked may be the alpha or beta chain of a soluble a|3 TCR fragment. Thus, in certain embodiments, the TCR fragment may be an a|3-heterodimeric TCR fragment and the CD3 agonist may be a single chain CD3 agonist, such as an scFv fragment. In such embodiments, the CD2 agonist, preferably the CD58 ectodomain, may be linked via its C- terminal end to the N-terminal end of the single chain CD3 agonist, preferably the scFv, and the single chain CD3 agonist may be linked via its C-terminal end to the N-terminal end of the alpha or beta polypeptide chain comprised in the soluble TCR fragment. Preferably, the polypeptide chain of the soluble TCR fragment to which the single chain CD3 agonist and the CD2 agonist are linked is the beta chain of a soluble a|3 TCR fragment.

[0086] Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is an scFv, wherein the C-terminal end of the scFv is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to the N-terminal end of the scFv.

[0087] In a particularly preferred embodiment, the CD3 agonist is a single chain CD3 agonist, preferably an scFv, wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment (see FIG.l, construct 376).

[0088] That is, the CD3 agonist and the CD2 agonist may be linked to the N-terminal ends of different polypeptide chains comprised in the soluble TCR fragment. In such embodiments, the soluble TCR fragment is preferably an a|3-heterodimeric TCR fragment and the CD3 agonist is preferably a single chain CD3 agonist, such as an scFvfragment. The CD2 agonist may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and the single chain CD3 agonist may be linked via its C-terminal end to the N-terminal end of the other one of the alpha or beta chain of the TCR fragment.

[0089] Preferably, the CD2 agonist is linked via its C-terminal end to the N-terminal end of the alpha chain of the TCR fragment and the single chain CD3 agonist is linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment.

[0090] Alternatively, the CD2 agonist may be linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment and the single chain CD3 agonist may be linked via its C- terminal end to the N-terminal end of the alpha chain of the TCR fragment.

[0091] In a specific embodiment, the invention relates to the multifunctional molecule according to the invention, wherein a CD58 ectodomain is linked to the N-terminal end of a first polypeptide comprised in the soluble TCR fragment, preferably the alpha chain of an a|3- heterodimeric TCR fragment, and the single chain CD3 agonist, preferably an scFv, is linked to the N-terminal end of a second polypeptide comprised in the soluble TCR fragment, preferably the beta chain of an a|3-heterodimeric TCR fragment.

[0092] Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is an scFv, wherein the C-terminal end of the scFv is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

[0093] In certain embodiments, the CD3 agonist is a single chain CD3 agonist, preferably an scFv, wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to a C-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment (see FIG.1, construct 375 and 378).

[0094] In such embodiments, the TCR fragment is preferably an a|3-heterodimeric TCR fragment and the CD3 agonist is preferably a single chain CD3 agonist, such as an scFv fragment. In such embodiments, the single chain CD3 agonist, preferably an scFv, may be linked via its C- terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C- terminal end of the alpha or beta chain of the TCR fragment.

[0095] In certain embodiments, the single chain CD3 agonist, preferably the scFv, may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C-terminal end of the same polypeptide chain of the TCR fragment as the CD3 agonist.

[0096] In certain embodiments, the single chain CD3 agonist, preferably the scFv, may be linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C- terminal end of the beta chain of the TCR fragment.

[0097] Alternatively, the single chain CD3 agonist, preferably the scFv, may be linked via its C-terminal end to the N-terminal end of the alpha chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C-terminal end of the alpha chain of the TCR fragment.

[0098] In certain embodiments, the single chain CD3 agonist, preferably the scFv, may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C-terminal end of the other one of the of the alpha or beta chain of the TCR fragment.

[0099] In certain embodiments, the single chain CD3 agonist, preferably the scFv, may be linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C- terminal end of the alpha chain of the TCR fragment.

[0100] Alternatively, the single chain CD3 agonist, preferably an scFv, may be linked via its C-terminal end to the N-terminal end of the alpha chain of the TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked via its N-terminal end to the C-terminal end of the beta chain of the TCR fragment.

[0101] Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is an scFv, wherein the C-terminal end of the scFv is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist is linked to a C-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment.

[0102] While it is preferred herein that the single chain CD3 agonist, preferably the scFv, is linked to the N-terminal end of a polypeptide chain comprised in the soluble TCR fragment, the present invention also encompasses embodiments where a single chain CD3 agonist, such as an scFv, is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment. That is, the single chain CD3 agonist, preferably the scFv, may be linked to the C-terminal end of the first or second polypeptide chain of a soluble TCR fragment. In certain embodiments, the single chain CD3 agonist, preferably the scFv, may be linked to the C-terminal end of the alpha or beta chain of a soluble a|3 TCR fragment. In such embodiments, the CD2 agonist, preferably the CD58 ectodomain, may be linked to any suitable position in the soluble TCR fragment or the CD3 agonist.

[0103] Preferred constructs comprising a soluble TCR fragment, a single chain CD3 agonist and a CD2 agonist are depicted in Fig.l bottom panel (formats 374, 375, 376 and 378).

[0104] 1.2 Multifunctional molecules comprising a soluble TCR a heterodimeric CD3

[0105] In other embodiments, the CD3 agonist may be a heterodimeric CD3 agonist comprising two polypeptide chains, such as a Fab fragment derived from an anti-CD3 antibody. The heterodimeric CD3 agonist may be linked to a C-terminal or N-terminal end of a polypeptide chain comprised in the soluble TCR fragment. However, it is preferred herein that the heterodimeric CD3 agonist, preferably the Fab fragment, is linked to the C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0106] Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment, and wherein an N-terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

[0107] In certain embodiments, the heterodimeric CD3 agonist, preferably the Fab fragment, is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, as shown in FIG.l top left. It is to be understood that FIG.l (top panel) only shows a part of the multifunctional molecule of the invention and that the final molecule further comprises a CD2 agonist, preferably in the positions marked with an X.

[0108] In certain embodiments, the heterodimeric CD3 agonist, in particular the Fab fragment, may be linked via the N-terminal end of the heavy or light chain comprised in the heterodimeric CD3 agonist to the C-terminal end of the alpha or beta chain of an a|3-heterodimeric TCR fragment. It is, however, preferred that the heterodimeric CD3 agonist, in particular the Fab fragment, is linked via the N-terminal end of the heavy chain of the heterodimeric CD3 agonist to the C-terminal end of the alpha chain of an a|3-heterodimeric TCR fragment. In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment, wherein an N-terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to an N-terminal end of a second polypeptide chain comprised in the heterodimeric CD3 agonist (see FIG.l, construct 369).

[0109] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to a C-terminal end of the alpha or beta chain of the soluble TCR fragment and the N-terminal end of the second polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the CD2 agonist, preferably the CD58 ectodomain.

[0110] In certain embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the TCR fragment and the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the CD2 agonist, preferably the CD58 ectodomain.

[0111] In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the TCR fragment and the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the CD2 agonist, preferably the CD58 ectodomain.

[0112] In certain embodiments, the N-terminal end of the light or heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the TCR fragment and the N-terminal end of the other one of the light or heavy chain of the Fab fragment may be linked to the C- terminal end of the CD2 agonist, preferably the CD58 ectodomain.

[0113] In certain embodiments, the N-terminal end of the light or heavy chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the TCR fragment and the N-terminal end of the other one of the light or heavy chain of the Fab fragment may be linked to the C- terminal end of the CD2 agonist, preferably the CD58 ectodomain.

[0114] In a preferred embodiment, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the TCR fragment and the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the CD2 agonist, preferably the CD58 ectodomain. In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist (see FIG.l, construct 368 and 370).

[0115] In certain embodiments, the soluble TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, and the CD2 agonist, preferably the CD58 ectodomain, is linked to the C-terminal end of the same polypeptide chain comprised in the heterodimeric CD3 agonist (see FIG.l, construct 370).

[0116] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist.

[0117] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist.

[0118] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a Fab fragment. In such embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C- terminal end of the heavy chain of the Fab fragment.

[0119] In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the light chain of the Fab fragment. In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the same polypeptide chain of the Fab fragment.

[0120] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the same polypeptide chain of the Fab fragment.

[0121] In a preferred embodiment, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the heavy chain of the Fab fragment.

[0122] In certain embodiments, the soluble TCR fragment is linked to an N-terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, and the CD2 agonist, preferably the CD58 ectodomain, is linked to the C-terminal end of a second polypeptide chain comprised in the heterodimeric CD3 agonist (see FIG.l, construct 368).

[0123] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the second polypeptide chain comprised in the heterodimeric CD3 agonist.

[0124] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the second polypeptide chain comprised in the heterodimeric CD3 agonist.

[0125] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a Fab fragment. In such embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C- terminal end of the light chain of the Fab fragment. In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of heavy light chain of the Fab fragment.

[0126] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the heavy or light chain of the Fab fragment.

[0127] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the heavy or light chain of the Fab fragment.

[0128] In a preferred embodiment, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the light chain of the Fab fragment.

[0129] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to an N-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment (see FIG.1, construct 371 and 373).

[0130] That is, in certain embodiments, the soluble TCR fragment is linked via a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment to an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist, and the CD2 agonist, preferably the CD58 ectodomain, is linked to the N-terminal end of the first polypeptide chain comprised in the soluble TCR fragment (see FIG.l, construct 371).

[0131] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the same polypeptide chain of the soluble TCR fragment.

[0132] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the alpha chain of the soluble TCR fragment.

[0133] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the beta chain of the soluble TCR fragment.

[0134] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a Fab fragment. In such embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the same polypeptide chain of the soluble TCR fragment.

[0135] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the alpha chain of the soluble TCR fragment.

[0136] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the beta chain of the soluble TCR fragment.

[0137] In certain embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the same polypeptide chain of the soluble TCR fragment.

[0138] In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the same polypeptide chain of the soluble TCR fragment.

[0139] In a preferred embodiment, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the alpha chain of the soluble TCR fragment.

[0140] In certain embodiments, the soluble TCR fragment is linked via a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment to an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist, and the CD2 agonist, preferably the CD58 ectodomain, is linked to the N-terminal end of the second polypeptide chain comprised in the soluble TCR fragment (see FIG .1, construct 373).

[0141] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the other polypeptide chain of the soluble TCR fragment.

[0142] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the beta chain of the soluble TCR fragment.

[0143] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the alpha chain of the soluble TCR fragment.

[0144] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a Fab fragment. In such embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the other polypeptide chain of the soluble TCR fragment. In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the beta chain of the soluble TCR fragment.

[0145] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the alpha chain of the soluble TCR fragment.

[0146] In certain embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the other polypeptide chain of the soluble TCR fragment.

[0147] In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the other polypeptide chain of the soluble TCR fragment.

[0148] In a preferred embodiment, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the beta chain of the soluble TCR fragment.

[0149] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to the C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to the C-terminal end of the second polypeptide chain comprised in the soluble TCR fragment (see FIG.1, construct 372).

[0150] That is, in certain embodiments, the soluble TCR fragment is linked via a C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment to an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, and the CD2 agonist, preferably the CD58 ectodomain, is linked to the C-terminal end of a second polypeptide chain comprised in the soluble TCR fragment. In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a heterodimeric CD3 agonist, preferably a Fab fragment. In such embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the alpha or beta chain of the soluble TCR fragment.

[0151] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment.

[0152] In certain embodiments, the N-terminal end of the first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably the Fab fragment, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment.

[0153] In certain embodiments, the TCR fragment is an a|3-heterodimeric TCR fragment and the CD3 agonist is a Fab fragment. In such embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the alpha or beta chain of the soluble TCR fragment.

[0154] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment.

[0155] In certain embodiments, the N-terminal end of the heavy or light chain of the Fab fragment may be linked to the C-terminal end of the beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment.

[0156] In certain embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the alpha or beta chain of the soluble TCR fragment. In certain embodiments, the N-terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the alpha or beta chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the other one of the alpha or beta chain of the soluble TCR fragment.

[0157] In certain embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the alpha chain of the soluble TCR fragment and the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the beta chain of the soluble TCR fragment.

[0158] While it is preferred herein that the heterodimeric CD3 agonist, such as a Fab fragment, is linked to the C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, the present invention also encompasses embodiments where a heterodimeric CD3 agonist, preferably a Fab, is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment. That is, the heterodimeric CD3 agonist, preferably the Fab, may be linked to the N-terminal end of the first or second polypeptide chain of a soluble TCR fragment. In certain embodiments, the heterodimeric CD3 agonist, preferably the Fab, may be linked to the N-terminal end of the alpha or beta polypeptide chain of a soluble a|3 TCR fragment. In such embodiments, the CD2 agonist, preferably the CD58 ectodomain, may be linked to any suitable position in the soluble TCR fragment or the CD3 agonist.

[0159] Preferred constructs comprising a soluble TCR fragment, a heterodimeric CD3 agonist and a CD2 agonist are depicted in Fig.l middle panel (formats 368, 369, 370, 371, 372 and 373).

[0160] 1.3 Multifunctional molecules further comprising an antibody Fc region

[0161] In certain embodiments, the multifunctional molecule of the invention further comprises an antibody Fc region.

[0162] Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the molecule further comprises an antibody Fc region.

[0163] Attaching an antibody Fc region to a multifunction molecule may increase serum half-life of the multifunction molecule. Further advantages of attaching an antibody Fc region to a multifunction molecule may include improved manufacturability and additional immune effector functions. The multifunctional molecule comprising the soluble TCR fragment, the CD3 agonist and the CD2 agonist may be linked to the antibody Fc region in any suitable way. However, it is preferred herein that the multifunctional molecule comprising the soluble TCR fragment, the CD3 agonist and the CD2 agonist is linked to an N-terminal end of a polypeptide chain comprised in the antibody Fc region. Preferably, the antibody Fc region is linked via an N- terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment or the CD3 agonist.

[0164] Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein a first polypeptide chain comprised in the antibody Fc region is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment or the CD3 agonist.

[0165] Any of the multifunctional molecules disclosed herein above may be linked to an antibody Fc region, preferably to an N-terminal end of a polypeptide chain comprised in an antibody Fc region. In particular, any of the formats 368, 369, 370, 371, 372, 373, 374, 375, 376 or 378 disclosed herein above may be linked to an antibody Fc region, preferably to an N-terminal end of a polypeptide chain comprised in an antibody Fc region.

[0166] To avoid lengthy repetition, the constructs described herein below are described in a more generic way. However, any specific combination of a soluble TCR fragment, a CD3 agonist and a CD2 agonist disclosed herein above may be combined with an antibody Fc region, preferably wherein the antibody Fc region is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment or the CD3 agonist.

[0167] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0168] The soluble TCR fragment is preferably a soluble a|3 TCR fragment and the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of the alpha or beta chain of the soluble TCR fragment. In a preferred embodiment, the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to the C-terminal end of the alpha chain of the soluble TCR fragment. In such embodiments, the CD3 agonist is preferably linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment. Thus, in a particular embodiment, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD3 agonist is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0169] More preferably, the CD3 agonist that is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment is a single chain CD3 agonist, preferably an scFv.

[0170] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, wherein the CD3 agonist, preferably an scFv, is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to an N-terminal end of a polypeptide chain comprised in the CD3 agonist, in particular to the N-terminal end of the scFv.

[0171] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, wherein the CD3 agonist, preferably an scFv, is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

[0172] That is, both the CD3 agonist and the CD2 agonist may be linked to N-terminal ends of different polypeptide chains comprised in the soluble TCR fragment, whereas the antibody Fc region may be linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0173] In a particularly preferred embodiment, the CD3 agonist is a single chain CD3 agonist, preferably an scFv, wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, wherein the CD2 agonist is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment, and wherein the antibody Fc region is linked to the C-terminal end of the first or second polypeptide chain comprised in the soluble TCR fragment.

[0174] In certain embodiments, the soluble TCR fragment is preferably an a|3-heterodimeric TCR fragment and the CD3 agonist is preferably a single chain CD3 agonist, such as an scFv fragment. In such embodiments, the CD2 agonist may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment, the single chain CD3 agonist may be linked via its C-terminal end to the N-terminal end of the other one of the alpha or beta chain of the TCR fragment, and the antibody Fc region may be linked to the C-terminal end of the alpha or beta chain of the TCR fragment.

[0175] In a specific embodiment, the invention relates to the multifunctional molecule according to the invention, wherein a CD58 ectodomain is linked to the N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, preferably the alpha chain, the single chain CD3 agonist, preferably an scFv, is linked to the N-terminal end of a second polypeptide comprised in the soluble TCR fragment, preferably the beta chain, and the antibody Fc region is linked to the C-terminal end of the alpha or beta chain of the TCR fragment, preferably the alpha chain of the TCR fragment.

[0176] In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein: a) the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment; b) the CD3 agonist, in particular the scFv, is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment; and c) the CD2 agonist is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

[0177] A preferred construct comprising an antibody Fc region is depicted in Fig.9.

[0178] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, wherein the CD3 agonist, preferably an scFv, is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, is linked to an available C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

[0179] That is, in certain embodiments, a first polypeptide chain comprised in the soluble TCR fragment is linked via its N-terminal end to the CD3 agonist, preferably an scFv, and via its C- terminal end to the antibody Fc region, whereas the second polypeptide chain comprised in the soluble TCR fragment is linked via its C-terminal end to the CD2 agonist. In certain embodiments, a first polypeptide chain comprised in the soluble TCR fragment is linked via its N-terminal end to the CD3 agonist, preferably an scFv, and via its C-terminal end to the CD2 agonist, whereas the second polypeptide chain comprised in the soluble TCR fragment is linked via its C-terminal end to the antibody Fc region.

[0180] Alternatively, the CD3 agonist comprised in the multifunctional molecule comprising an antibody Fc region may be a heterodimeric CD3 agonist, such as a Fab fragment. In such embodiments, the heterodimeric CD3 agonist is preferably linked to an N-terminal end of a polypeptide chain comprised in an antibody Fc region, and the soluble TCR fragment in linked to an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist. The CD2 agonist may be linked to any suitable position in the heterodimeric CD3 agonist or the soluble TCR fragment.

[0181] In certain embodiments, the antibody Fc region may be linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the first polypeptide chain comprised in the Fab fragment may be linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N- terminal end of the second polypeptide chain comprised in the Fab fragment (see format 369 in Fig.10).

[0182] In certain embodiments, the antibody Fc region may be linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, wherein an N-terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, may be linked to an available C-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist.

[0183] That is, in certain embodiments, a first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, may be linked via its N-terminal end to a polypeptide chain comprised in the soluble TCR fragment and via its C-terminal end to a polypeptide chain comprised in the antibody Fc region, whereas the second polypeptide chain comprised in the heterodimeric CD3 agonist may be linked via its C-terminal end to the CD2 agonist, preferably the CD58 ectodomain (see format 368 in Fig.10).

[0184] In certain embodiments, a first polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, may be linked via its N-terminal end to a polypeptide chain comprised in the soluble TCR fragment and via its C-terminal end to the CD2 agonist, preferably the CD58 ectodomain, whereas the second polypeptide chain comprised in the heterodimeric CD3 agonist may be linked via its C-terminal end to a polypeptide chain comprised in the antibody Fc region (see format 370 in Fig.10).

[0185] In certain embodiments, the antibody Fc region may be linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, wherein an N-terminal end of a polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, may be linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment (see format 371 in Fig.10).

[0186] In certain embodiments, the antibody Fc region may be linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the a polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, may be linked to the C-terminal end of the second polypeptide chain comprised in the soluble TCR fragment (see format 372 in Fig.10).

[0187] In certain embodiments, the antibody Fc region may be linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist, preferably a Fab fragment, wherein the N-terminal end of the a polypeptide chain comprised in the heterodimeric CD3 agonist may be linked to the C-terminal end of a first polypeptide chain comprised in the soluble TCR fragment, and wherein the CD2 agonist, preferably the CD58 ectodomain, may be linked to the N-terminal end of the second polypeptide chain comprised in the soluble TCR fragment (see format 373 in Fig.10).

[0188] The antibody Fc region may be a homodimeric antibody Fc region comprising two identical heavy chain fragments. In such embodiments, both polypeptide chains of the antibody Fc region may be linked to a multifunctional construct comprising a soluble TCR fragment, a CD3 agonist and a CD2 agonist.

[0189] However, the antibody Fc region may also be a heterodimeric antibody Fc comprising two different heavy chain fragments. In such embodiments, only one of the polypeptide chains comprised in the antibody Fc region may be linked to a multifunctional construct comprising a soluble TCR fragment, a CD3 agonist and a CD2 agonist. Heterodimeric antibody Fc regions are described in more detail elsewhere herein.

[0190] 2. The soluble TCR fragment

[0191] The multifunctional molecule of the invention comprises one or more soluble TCR fragments through which the multifunctional molecule of the invention can engage with a TCR ligand, such as a cognate peptide-MHC (pMHC) ligand. However, encompassed herein are also soluble TCR fragments that engage with other TCR ligands, such as lipids or small molecules presented by molecules such as CD1 or MR1 (van Rhijn, Nat Rev Immunol. 2015 Sep 21;15(10):643-654).

[0192] As used herein, the term "soluble T cell receptor fragment" preferably refers to heterodimeric truncated variants of native TCRs, which preferably comprise extracellular portions of the TCR a-chain and p-chain linked by a disulphide bond, but which lack the transmembrane and cytosolic domains of the native protein.

[0193] The terms "soluble T cell receptor a-chain sequence and soluble T-cell receptor p-chain sequence" refer to TCR a-chain and p-chain sequences that lack the transmembrane and cytosolic domains. The sequence (amino acid or nucleic acid) of the soluble TCR a-chain and P-chains may be identical to the corresponding sequences in a native TCR or may comprise variant soluble TCR a-chain and p-chain sequences, as compared to the corresponding native TCR sequences. The term "soluble T cell receptor" as used herein encompasses soluble TCRs with variant or non-variant soluble TCR a-chain and p-chain sequences. The variations may be in the variable or constant regions of the soluble TCR a-chain and p-chain sequences and can include, but are not limited to, amino acid deletion, insertion, substitution mutations as well as changes to the nucleic acid sequence, which do not alter the amino acid sequence. Soluble TCRs of the invention preferably retain the binding functionality of their parent molecules. Alternatively, soluble TCR fragments may comprise the extracellular portions of the TCR y- chain and the TCR 6-chain.

[0194] Within the present invention, reference is made to the first and second polypeptide chain comprised in the soluble TCR fragment. In certain embodiments, the first polypeptide chain comprised in the soluble TCR fragment is derived from the TCR a-chain and the second polypeptide chain comprised in the soluble TCR fragment is derived from the TCR p-chain, or vice versa. In certain embodiments, the first polypeptide chain comprised in the soluble TCR fragment is derived from the TCR y-chain and the second polypeptide chain comprised in the soluble TCR fragment is derived from the TCR 6-chain, or vice versa. In certain embodiments, the soluble TCR fragment may be a single chain TCR, for example as disclosed by Aggen et al. (Gene Ther. 2012, 19(4): 365-374).

[0195] The soluble TCR fragment may have been derived from any TCR. However, it is preferred herein that the soluble TCR fragment has been derived from a genetically engineered TCR. Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the soluble TCR fragment has been engineered for increased affinity, avidity, specificity and / or stability.

[0196] That is, in certain embodiments, the soluble TCR fragment comprised in the multifunctional molecule according to the invention has been engineered for increased affinity, (functional / structural) avidity, specificity and / or stability.

[0197] The term "affinity" as used herein means the strength of a single interaction between a TCR ligand, for example a peptide (presented in the context of an MHC), and a TCR. Affinity is measured in a cell-free context, for example, where the TCR ligand, such as the MHC- presented peptide, is immobilized on a solid interface and the TCR of interest is in solution.

[0198] TCR affinity is measured by determining its equilibrium binding constant (KD) to its ligands. KD can be determined using biophysical methods, such as surface plasmon resonance and biolayer interferometry, to quantify the association (Ka, in M l-s1) and dissociation (Kd, in s1) rates of a TCR in solution to an immobilised TCR ligand ("M" is concentration in molar units and "s" is time in seconds). KD in molar units is then determined by dividing Kd over Ka, with a small KD (e.g., due to a slow Kd and a fast Ka) reflecting a high affinity.

[0199] In a preferred embodiment, the soluble TCR fragment has been engineered to bind to a TCR ligand with an equilibrium binding constant KD of between about 10“7M and 10“12M.

[0200] The term "avidity" as used herein refers to a measure of multiple affinities and reflects the overall binding strength or the observed functional response between a TCR ligand, such as a target peptide-MHC of interest, and a TCR. In this context, the TCR ligand, such as the peptide- MHC, and the TCR can be presented on the surface of a cell or may be provided as soluble binding reagents (e.g., monomers or multimers). Preferably, the TCR is presented on the surface of the cell according to the invention, and the TCR ligand, such as the peptide-MHC, is in the form of a soluble reagent (i.e., peptide-MHC multimers) or presented by an APC.

[0201] The term "functional avidity" as used herein is the concentration of a soluble TCR engager required to achieve 50% of maximal response in a functional assay. For each functional assay, the maximal response is determined. Functions measured include for example T cell signalling as described in the invention, cytokine secretion and lysis of target cells (i.e., cytotoxicity). The maximal response is obtained when T cells are maximally stimulated. Therefore, maximal responses depend on the functional capabilities of given T cell(s) and can for example be expressed as EC50 in nM or other molar units.

[0202] The term "structural avidity" or "binding avidity" as used herein, which can be expressed as EC50 in pg / mL or other concentration units, is the concentration of an antigen (e.g. a peptide bound by an MHC multimer) required to achieve half-maximal antigen staining (e.g. multimer staining) of a cell-bound TCR or TCR fragment. Functional and structural avidity can for example be calculated with software known in the art such as GraphPad prism 6.

[0203] Functional avidity can be measured for example using an enzyme-linked immunospot (ELISpot) assay and measuring, for example, interferon-gamma (IFNy) and / or IL-2 production and / or a combination thereof. Structural avidity can be measured by staining TCR-expressing cells, optionally T cells or the cells according to the invention expressing a TCR, using graded concentrations of a TCR ligand, such as peptide-MHC multimers. In some embodiments, avidity may be assessed by flow cytometric analysis for specific TCR binding to fluorochrome- labelled multimeric synthetic peptide-MHC complexes, and / or functional assessment with peptide-pulsed APCs and screening for IFN-gamma production using standard enzyme-linked immunosorbent assay (ELISA) or ELISpot assays. In some embodiments, T cell avidity may be detected via a dual parameter cell sorting protocol that detects cells bound to the fluorescently labelled multimer in conjunction with, indicating that the cell was activated by the recognition of the TCR-multimer complex.

[0204] In certain embodiments, the soluble TCR fragment has been engineered to exhibit increased specificity for a desired TCR ligand, such as a pMHC ligand. Increased specificity for a desired ligand may be achieved by increasing the affinity / avidity for said ligand and / or by reducing the affinity / avidity for an off-target ligand.

[0205] Methods for increasing the affinity, avidity and / or specificity of a TCR have been described in detail in WO 2021 / 074249, which is incorporated by reference herein.

[0206] In certain embodiments, the soluble TCR fragment comprised in the multifunctional molecule of the invention has been engineered for increased stability. Strategies for increasing the stability of a soluble TCR fragment are known in the art and include, without limitation, the introduction of stabilizing mutations into the constant region of the soluble TCR fragment, for example as summarized by Robinson et al. (FEBS J, 2021, 288(21) :6159-6173).

[0207] In certain embodiments, the engineered soluble TCR fragment may comprise one or more of the mutations disclosed in WO 2021 / 046072, which is fully incorporated herein by reference. That is, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a T cell receptor (TCR) alpha constant domain (Ca) comprising at least one of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a TCR beta constant domain (CP) comprising at least one of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Kabat numbering).

[0208] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a T cell receptor (TCR) alpha constant domain (Ca) comprising at least one of the following residues: leucine or tryptophane at position 178, asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a TCR beta constant domain (CP) comprising at least one of the following residues: threonine at position 128, tryptophan at position 150, glutamate at position 170, or histidine at position 184 (residues numbered according to Kabat numbering).

[0209] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: glutamate at position 156, tyrosine at position 169, leucine or tryptophane at position 178, or serine at position 190 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: threonine at position 128, glutamate at position 170, or histidine at position 184 (residues numbered according to Kabat numbering).

[0210] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: leucine or tryptophane at position 178, and / or serine at position 190 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: glutamate at position 170, and / or histidine at position 184 (residues numbered according to Kabat numbering).

[0211] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: leucine at position 178 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: glutamate at position 170 (residues numbered according to Kabat numbering). In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: tryptophane at position 178 and serine at position 190 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: glutamate at position 170 (residues numbered according to Kabat numbering).

[0212] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: tryptophane at position 178 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: glutamate at position 170 (residues numbered according to Kabat numbering).

[0213] In certain embodiments, the engineered soluble TCR fragment may comprise a first polypeptide chain comprising a Ca domain comprising at least one of the following residues: tryptophane at position 178 (residues numbered according to Kabat numbering); and / or a second polypeptide chain comprising a C|3 domain comprising at least one of the following residues: histidine at position 184 (residues numbered according to Kabat numbering).

[0214] In certain embodiments, the TCR Ca domain and the TCR CP domain comprised in the soluble TCR fragment are linked by an inter-chain disulphide bond formed between two native cysteine residues. Thus, in a particular embodiment, first and second polypeptide chain of the soluble TCR fragment are linked by an inter-chain disulphide bond between a cysteine residue at position 213 of the TCR Ca domain and a cysteine residue at position 247 of the TCR CP domain (residues numbered according to Kabat numbering).

[0215] In a particular embodiment, the soluble TCR fragment may comprise one or more artificial disulphide bonds. Soluble TCRs comprising artificial disulphide bonds have been disclosed, inter alia, by Boulter et al., (Protein Engineering, Design and Selection, Volume 16, Issue 9, September 2003, Pages 707-711) and in WO 2003 / 020763, both of which are incorporated herein by reference in their entirety.

[0216] In some embodiments, the Ca domain comprised in the soluble TCR fragment may further comprise a cysteine residue at position 166 (residue numbered according to Kabat numbering), and the CP domain comprised in the soluble TCR fragment may further comprise a cysteine residue at position 173 (residue numbered according to Kabat numbering), wherein the first polypeptide chain and the second polypeptide chain are linked by an inter-chain disulphide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of cp. In a preferred embodiment, the first and second polypeptide chains comprised in the soluble TCR fragment are linked together by an artificial inter-chain disulphide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of C|3 and by a naturally occurring inter-chain disulphide bond between the cysteine residue at position 213 of Ca and the cysteine residue at position 247 of cp.

[0217] In certain embodiments, the soluble TCR fragment only comprises a single inter-chain disulphide bond formed between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of cp. In such embodiments, it is preferred that the C-terminal ends of the TCR alpha and beta constant domains are truncated such that the native inter-chain disulphide bond between the cysteine residue at position 213 of Ca and the cysteine residue at position 247 of cp can no longer be formed.

[0218] In a preferred embodiment, the soluble TCR fragment comprises an artificial disulphide bond, preferably between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of cp, and one or more of the stabilizing mutations disclosed herein above.

[0219] The TCR variable regions may bind any tumour or viral antigen, including but not limited to, a viral antigen, a neoantigen (the antigens expressed only in cancer cells but not in normal cells), a tumour-associated antigen (the processed fragments of proteins that are expressed at low levels in normal cells, but are overexpressed in cancer cells), or a cancer / testis (CT) antigen (derived from proteins usually only expressed by reproductive tissues, e.g. testes, fetal ovaries, and placenta, and have limited / no expression in all other adult tissues) (see Pritchard, et al., BioDrugs, 2018, 32:99-109). In some embodiments, the TCR variable region may bind an antigen selected from any one of the following: ERBB2, CD19, NY-ESO-1, MAGE (e.g., MAGE-A1, A2, A3, A4, A6, A10, A12), gplOO, MART-l / Melan-A, gp75 / TRP-l, TRP-2, Tyrosinase, BAGE, CAMEL, SSX-2, b-Catenin, Caspase-8, CDK4, MUM-2 (TRAPPCI), MUM-3, MART-2, OS-9, P14ARF (CDKN2A), GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, PRDX5, CLPP, PPP1R3B, EF2 (see Pritchard, et al., BioDrugs, 2018, 32:99-109; and Wang, et al., Cell Research, 2017, 27:11-37).

[0220] Preferably, the TCR variable regions may bind to an antigenic peptide derived from any one of the antigens MAGE-A3, EBNA-1, GPC3, KRAS proto-oncogene neoantigens, TCF-1, AFP or PSA. That is, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the soluble TCR fragment specifically binds to MAGE-A3, EBNA-1, GPC3, KRAS proto-oncogene neoantigens, TCF-1, AFP or PSA. In a particularly preferred embodiment, the soluble TCR fragment comprised in the multifunctional molecule of the invention specifically binds to MAGE-A3. That is, the soluble TCR fragment specifically binds to a pMHC ligand comprising an antigenic peptide derived from MAGE-A3. In certain embodiments, the antigenic peptide derived from MAGE-A3 is EVDPIGHLY (SEQ ID NO:5). In certain embodiments, the antigenic peptide EVDPIGHLY (SEQ ID NO:5) is presented by Human HLA-A*01:01. In certain embodiments, the soluble TCR fragment comprised in the multifunctional molecule of the invention has been engineered to exhibit increased affinity, avidity and / or specificity for an antigenic peptide derived from MAGE-A3, preferably the peptide EVDPIGHLY (SEQ ID NO:5). In certain embodiments, the soluble TCR fragment comprised in the multifunctional molecule of the invention is derived from any one of the engineered MAGE-A3 specific TCRs disclosed in WO 2021 / 074249.

[0221] In another particularly preferred embodiment, the soluble TCR fragment comprised in the multifunctional molecule of the invention specifically binds to glypican-3 (GPC3). GPC3 is an oncofetal antigen that is an attractive target for cancer immunotherapy. The soluble TCR fragment may specifically bind to a pMHC ligand comprising an antigenic peptide derived from GPC3, preferably presented by an HLA-A*02 molecule. In certain embodiments, the soluble TCR fragment comprised in the multifunctional molecule of the invention has been engineered to exhibit increased affinity, avidity and / or specificity for an antigenic peptide derived from GPC3.

[0222] In yet another particularly preferred embodiment, the soluble TCR fragment comprised in the multifunctional molecule of the invention specifically binds to gplOO. That is, the soluble TCR fragment specifically binds to a pMHC ligand comprising the YLEPGPVTA peptide (SEQ ID NO:23) derived from gplOO, preferably presented by an HLA-A*02:01 molecule. In a specific embodiment, the soluble TCR fragment comprises a TCR variable alpha (Va) chain comprising the amino acid sequence of SEQ ID NO: 27 and a TCR variable beta (V|3) chain comprising the amino acid sequence of SEQ ID NO: 26. In a further specific embodiment, the soluble TCR fragment comprises a V|3 chain comprising a CDRip of SEQ ID NO: 28, a CDR2|3 of SEQ ID NO: 29, and a CDR3|3 of SEQ ID NO: 30; and a Va chain comprising a CDRla of SEQ ID NO: 31, a CDR2a of SEQ ID NO: 32, and a CDR3a of SEQ ID NO: 33. In a particularly preferred embodiment, the multifunctional molecule comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 24 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 25.

[0223] In another embodiment, the soluble TCR fragment that binds specifically to gplOO is an affinity-enhanced T-cell receptor. Methods for engineering TCRs to exhibit increased affinity are known in the art. For example, specific sequences for affinity-enhanced TCRs targeting the gplOO antigen, as well as methods for their generation, are disclosed in International Publication No. WO 2011 / 001152, the entire contents of which are hereby expressly incorporated by reference for all purposes. Accordingly, in a specific embodiment, the soluble TCR fragment comprises any one of the gplOO-specific TCR sequences disclosed therein.

[0224] In certain embodiments, the multifunctional molecule of the invention comprises two different soluble TCR fragments or more. In certain embodiments, a first soluble TCR fragment specifically binds to a first pMHC ligand comprising an antigenic peptide derived from an antigen and a second soluble TCR fragment specifically binds to a second pMHC ligand comprising another antigenic peptide derived from the same antigen. In certain embodiments, a first soluble TCR fragment specifically binds to a first pMHC ligand comprising an antigenic peptide derived from a first antigen and a second soluble TCR fragment specifically binds to a second pMHC ligand comprising an antigenic peptide derived from a second antigen.

[0225] 3. The CD3 agonist

[0226] The multifunctional molecule of the invention further comprises a CD3 agonist. A CD3 agonist is a molecule that interacts with CD3 on the surface of T cells and induces T cell activation. Preferably, the CD3 agonist is a protein. Even more preferably, the CD3 agonist is an agonist anti-CD3 antibody or is derived from an agonist anti-CD3 antibody. Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD3 agonist is an anti-CD3 antibody, or an antigen-binding fragment thereof.

[0227] That is, in certain embodiments, the CD3 agonist may be a full length anti-CD3 antibody. In certain embodiments, the CD3 agonist may be an antigen-binding fragment derived from an anti-CD3 antibody.

[0228] As used herein, "antibody" refers to immunoglobulin, a structure of four-peptide chains connected together by disulphide bonds between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and rank orders, hence present different kinds of antigenicity. Accordingly, immunoglobulin can be divided into five categories, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with heavy chain p, 6, y, a and E, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulphide bonds, the same type of Ig can be divided into different sub-categories. For example, IgG can be divided into IgGl, lgG2, lgG3, and lgG4. Light chains can be divided into K or X chain, due to different constant regions. Each IgG among the five types has K or X chain. The sequence of about 110 amino acids closest to the N-terminus of the antibody heavy and light chains is commonly referred to as the variable region (Fv region). The sequence of amino acids closest to the C-terminus is commonly referred to as the constant region. The variable region comprises three hypervariable regions (HVR) and four framework regions (FR) having relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) comprises three CDR regions and four FR regions. Sequentially ordered from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. The three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3. The number and location of the CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen binding fragment herein comply with known Kabat numbering criteria (e.g., LCDR1-3, HCDR2-3), or comply with Kabat and Chothia numbering criteria (e.g., HCDR1).

[0229] The term "Fc-region" denotes the C-terminal region of an immunoglobulin. The Fc-region is a dimeric molecule comprising two disulphide-linked antibody heavy chain fragments (heavy chain Fc-region polypeptide chains). An Fc-region can be generated by papain digestion, or IdeS digestion, or trypsin digestion of an intact (full length) antibody or can be produced recombinantly.

[0230] The Fc-region obtainable from a full length antibody or immunoglobulin comprises at least residues 226 (Cys) to the C-terminus of the full length heavy chain and, thus, comprises a part of the hinge region and two or three constant domains, i.e. a CH2 domain, a CH3 domain, and an additional / extra CH4 domain on IgE and IgM class antibodies

[0231] In certain embodiment, the antibody may be a human or human derived antibody. The terms "human antibody" and "human derived antibody" are used interchangeably and refer to an antibody comprising one or more variable and constant regions derived from a human immunoglobulin sequence. In a preferred embodiment of the invention, all of the variable and constant regions are derived from human immunoglobulin sequences, i.e., "fully human derived antibody" or "fully human antibody". These antibodies can be obtained in a variety of ways, including antibodies obtained by using phage display technology, including isolating B cells from human PBMC, spleen, lymph node tissue and constructing natural single-stranded phage human antibody library, or by immunizing transgenic mice expressing human antibody light and heavy chain and screening.

[0232] In certain embodiments, the antibody may be a murine antibody. The term "murine antibody" used in the present invention refers to a monoclonal antibody against human CD3 prepared according to the knowledge and skill in the art. During preparation, the test subject is injected with a CD3 antigen, and then the hybridoma expressing antibodies having desired sequences or functional properties are isolated. In a preferred embodiment of the invention, the murine anti-CD3 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or a variant thereof, or further comprises a heavy chain constant region of murine IgGl, lgG2, lgG3 or variants thereof.

[0233] In certain embodiments, the antibody may be a chimeric antibody. The term "chimeric antibody" is an antibody which is formed by fusing the variable region of a murine antibody with the constant region of a human antibody, so as to alleviate the murine antibody-induced immune response. To establish a chimeric antibody, a hybridoma secreting a specific murine monoclonal antibody is established and a variable region gene is cloned from the murine hybridoma cells. Then a desired constant region gene of a human antibody is cloned and connected with the murine variable region genes to form a chimeric gene which can be subsequently inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in eukaryotic or prokaryotic system. In a preferred embodiment of the present invention, the light chain of the anti-CD3 chimeric antibody further comprises a light chain constant region derived from the human kappa, lambda chain or a variant thereof. The heavy chain of the anti-CD3 chimeric antibody further comprises a heavy chain constant region derived from human IgGl, lgG2, lgG3 or lgG4 or a variant thereof.

[0234] In certain embodiments, the antibody may be a humanized antibody. The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences into a variable region framework of a human antibody (i.e., antibodies produced within different types of human germline antibody framework sequences). A humanized antibody overcomes the heterologous response induced by a chimeric antibody that carries a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in e.g., "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), as well as found in Kabat, E A, et al, 1991 Sequences of Proteins of Immunological Interest, 5th edition. The CDR graft can reduce the affinity of the anti-CD3 antibody or antigen-binding fragment thereof to the antigen, due to the framework residues that are in contact with the antigen. Such interaction can be the result of hyper-mutation in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids onto the framework of humanized antibodies. Amino acid residues from a non-human anti-CD3 antibody or antigen-binding fragment thereof which are involved in antigen binding can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be relevant. If the closest germline cannot be determined, the sequence can be compared with the common sequence of a subtype or the sequence of the murine with a high similarity percentage. Rare framework residues are thought to be the result of somatic hyper-mutation and thus play an important role in binding.

[0235] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD3).

[0236] Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the anti-CD3 antibody, or the antigen-binding fragment thereof, is a nanobody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a single-chain antibody (scFv), a dimerized V region (diabody), a disulphide-stabilized V region (dsFv), or a CDR-containing peptide.

[0237] In a particular embodiment, the CD3 agonist may be a single-chain CD3 agonist. A single-chain CD3 agonist is an antibody fragment designed to bind specifically to CD3. These agonists consist of a single polypeptide chain, typically making them smaller, easier to produce, and more effective at tissue penetration compared to full-length antibodies. Examples include nanobodies, single-chain antibodies (scFv), and CDR-containing peptides.

[0238] The term "nanobody", as used herein, refers to a single-domain antibody. The single-domain antibody 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).

[0239] It should be noted that the term "nanobody," as used herein in its broadest sense, is not limited to a specific biological source or to a specific method of preparation. For example, the nanobodies hereof can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and, in particular, from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0240] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in prior art consist of repeated GGGGS (SEQ ID NO:21) amino acid sequence or variants thereof, for example a variant having 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0241] The scFv of the present invention can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three- dimensional structure thereof; constructing a DNA encoding the scFv; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said scFv.

[0242] The CDR-containing peptide is constructed by one or more regions of CDRs of VH or VL. Peptides comprising several CDRs can be linked directly or via a suitable peptide linker.

[0243] The CDR-containing peptide of the present invention can be produced by the following steps: constructing a DNA encoding CDRs of the VH and the VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said peptide. The CDR-containing peptide can also be produced by chemical synthesis methods such as Fmoc method or tBoc method.

[0244] A heterodimeric CD3 agonist is a type of antibody fragment composed of two different polypeptide chains that together form a functional unit capable of binding to the CD3 receptor on T cells, thereby activating them. These agonists typically involve the pairing of variable regions from different antibody fragments, which can enhance binding specificity and functional activity. Examples of heterodimeric CD3 agonists include, without limitation: Fab fragments, F(ab')2 fragments, Fab' fragments, dimerized V regions (diabodies) and disulphide- stabilized V regions (dsFv)

[0245] Fab is an antibody fragment having a molecular weight of about 50,000 and having antigenbinding activity, such fragments are obtained by treating an IgG antibody molecule with protease papain (cleaving amino acid residue at position 224 of H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound by disulphide bond. The Fab of the present invention may be produced by treating a monoclonal antibody with papain. Furthermore, the Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab. The Fab fragment may be linked to another component of the multifunctional molecule, i.e., a soluble TCR fragment, an antibody Fc region or an CD2 agonist, via the polypeptide chain that comprises the heavy or light chain variable region.

[0246] F(ab')2 is an antibody fragment obtained by digesting the lower part of two disulphide bonds in IgG hinge region with pepsin. It has a molecular weight of about 100,000 and antigenbinding activity, and comprises two Fab regions linked at the hinge position.

[0247] The F(ab')2 of the present invention can be produced by treating the monoclonal antibody of the present invention, which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof, with pepsin. Furthermore, the F(ab')2 can be produced by linking the Fab' described below with a thioether bond or a disulphide bond.

[0248] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigenbinding activity. It is obtained by cleaving the disulphide bond in the hinge region of the F(ab')2 mentioned above. The Fab' of the present invention may be produced by treating the F(ab')2 of the present invention, which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof, with a reducing agent (such as dithiothreitol).

[0249] Furthermore, the Fab' can be produced by inserting a DNA encoding a Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab'. A diabody is an antibody fragment in which the scFv is dimerized. Diabodies may have bivalent antigen-binding activity.

[0250] The diabody of the present invention can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; constructing a DNA encoding scFv such that the length of the linker peptide is 8 or less amino acid residues; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express the diabody.

[0251] The dsFv is obtained by substituting one amino acid residue in each of the VH and the VL with cysteine residue, and then linking the polypeptides via disulphide bond between the two cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody in accordance with known methods (Protein Engineering, 7, 697 (1994)).

[0252] The dsFv of the present invention can be produced by the following steps: obtaining the cDNA encoding the VH and the VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; constructing a dsFv-encoding DNA; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said dsFv.

[0253] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (CDR LI, CDR L2, CDR L3 or LI, L2, L3), as well as CDR2 and CDR3 of heavy chain variable domain (CDR H2, CDR H3 or H2, H3).

[0254] The term "antibody framework" as used herein, refers to a portion of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

[0255] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on CD3 molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

[0256] The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10“7M, such as approximately less than about 10“8M, 10“9M or 10“10M or less.

[0257] To reduce the risk of immunogenicity towards the multifunctional molecule of the invention, it is preferred herein that the CD3 agonist is a human or humanized anti-CD3 antibody or an antigen-binding fragment thereof. In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody OKT3 or a humanized antigen-binding fragment thereof, as described by Adair et al. (Hum Antibodies Hybridomas, 1994, 5(l-2):41-7), which is fully incorporated herein by reference.

[0258] In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from the anti-CD3 antibody OKT3. In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from a humanized variant of the anti-CD3 antibody OKT3.

[0259] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody SP34 or a humanized antigen-binding fragment thereof, as disclosed in W02016020444, which is fully incorporated herein by reference.

[0260] In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from the anti-CD3 antibody SP34. In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from a humanized variant of the anti-CD3 antibody SP34.

[0261] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody UCHT1 or a humanized antigen-binding fragment thereof. Such variants may be designed for tuned affinity. UCHT1 was initially described by Beverley and Callard (Eur J Immunol, 1981, ll(4):329-34), which is fully incorporated herein by reference. Preferably, the humanized variant of UCHT1 comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:16 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:17.

[0262] In a further preferred embodiment, the humanized antigen-binding fragment of UCHT1 comprises a heavy chain variable region comprising a heavy chain complementarity- determining region 1 (CDR-H1) having the amino acid sequence GYSFTGYT (SEQ ID NO: 34), a CDR-H2 having the amino acid sequence INPYKGVS (SEQ ID NO: 35), and a CDR-H3 having the amino acid sequence ARSGYYGDSDWYFDV (SEQ ID NO: 36). In another preferred embodiment, the humanized antigen-binding fragment of UCHT1 comprises a light chain variable region comprising a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence QDIRNY (SEQ ID NO: 37), a CDR-L2 having the amino acid sequence YTS, and a CDR-L3 having the amino acid sequence QQGNTLPWT (SEQ ID NO: 38). In a particularly preferred embodiment, the CD3 agonist is an antigen-binding fragment comprising the six CDRs set forth in SEQ ID NOs: 34 to 38.

[0263] In other preferred embodiments, the CD3 agonist is an affinity-tuned humanized UCHT1 antigen-binding fragment. In a specific embodiment, such a fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39 (UCHTl_VL_Alal3), which itself comprises a CDR-L1 having the sequence QDIRNY (SEQ ID NO: 37), a CDR-L2 having the sequence YTS, and a CDR-L3 having the sequence QQGNTAPWT (SEQ ID NO: 40). This light chain variable region is paired with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 (UCHT1_VH_A5). This heavy chain variable region itself comprises a CDR-H1 having the sequence GESFTGYT (SEQ ID NO: 42), a CDR-H2 having the sequence INPYKVVS (SEQ ID NO: 43), and a CDR-H3 having the sequence ARSGYYGDSDWYFDV (SEQ ID NO: 36).

[0264] In further embodiments, the CD3 agonist comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39 (UCHTl_VL_Alal3), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 (UCHT1_VH). In a particularly preferred embodiment, such an antigen-binding fragment comprises a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 40, a CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36.

[0265] In another embodiment, the affinity-tuned humanized UCHT1 fragment comprises the light chain variable region of SEQ ID NO: 39 paired with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44 (UCHT1_VH_C3). This heavy chain variable region comprises a CDR-H1 having the sequence GYWFTGYT (SEQ ID NO: 45), a CDR-H2 having the sequence INPYKGRS (SEQ ID NO: 46), and a CDR-H3 having the sequence ARSGYYGQSDWYFDV (SEQ ID NO: 47).

[0266] In further embodiments, the CD3 agonist comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (UCHT1_VL), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44 (UCHT1_VH_C3). In a particularly preferred embodiment, such an antigen-binding fragment comprises a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 38, a CDR-H1 of SEQ ID NO: 45, a CDR-H2 of SEQ ID NO: 46, and a CDR-H3 of SEQ ID NO: 47.

[0267] In a further embodiment, the affinity-tuned humanized UCHT1 fragment comprises the light chain variable region of SEQ ID NO: 39 paired with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 (UCHT1_VH_B4). This heavy chain variable region comprises a CDR-H1 having the sequence GYSFVGYT (SEQ ID NO: 49), a CDR-H2 having the sequence INPYKGYS (SEQ ID NO: 50), and a CDR-H3 having the sequence ARSGYYGDSDWLFDV (SEQ ID NO: 51).

[0268] In further embodiments, the CD3 agonist comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (UCHT1_VL), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 (UCHT1_VH_B4). In a particularly preferred embodiment, such an antigen-binding fragment comprises a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 38, a CDR-H1 of SEQ ID NO: 49, a CDR-H2 of SEQ ID NO: 50, and a CDR-H3 of SEQ ID NO: 51.

[0269] In yet another embodiment, the affinity-tuned humanized UCHT1 fragment comprises the light chain variable region of SEQ ID NO: 39 paired with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52 (UCHT1_VH_G5). This heavy chain variable region comprises a CDR-H1 having the sequence GYSFTGFT (SEQ ID NO: 53), a CDR- H2 having the sequence INLYKGVS (SEQ ID NO: 54), and a CDR-H3 having the sequence ARSGYYGDSDWYFDG (SEQ ID NO: 55).

[0270] In further embodiments, the CD3 agonist comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (UCHT1_VL), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52 (UCHT1_VH_G5). In a particularly preferred embodiment, such an antigen-binding fragment comprises a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 38, a CDR-H1 of SEQ ID NO: 53, a CDR-H2 of SEQ ID NO: 54, and a CDR-H3 of SEQ ID NO: 55.

[0271] In another embodiment, the affinity-tuned humanized UCHT1 fragment comprises the light chain variable region of SEQ ID NO: 39 paired with a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 (UCHT1_VH_A3). This heavy chain variable region comprises a CDR-H1 having the sequence GYRFTGYT (SEQ ID NO: 57), a CDR-H2 having the sequence INPLKGVS (SEQ ID NO: 58), and a CDR-H3 having the sequence ARSGYYGDSDWYFDV (SEQ ID NO: 36). In further embodiments, the CD3 agonist comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (UCHT1_VL), and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 (UCHT1_VH_A3). In a particularly preferred embodiment, such an antigen-binding fragment comprises a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 38, a CDR-H1 of SEQ ID NO: 57, a CDR-H2 of SEQ ID NO: 58, and a CDR-H3 of SEQ ID NO: 36.

[0272] In another aspect, the invention provides the novel, affinity-tuned humanized UCHT1 antigenbinding fragments themselves. These fragments demonstrate unique binding properties and are valuable as building blocks for a variety of immunotherapeutic constructs.

[0273] In preferred embodiments, these fragments comprise one mutated variable region paired with its corresponding wild-type variable region. In one such embodiment, the antigenbinding fragment comprises a mutated light chain variable region having the amino acid sequence of SEQ ID NO: 39, paired with a wild-type heavy chain variable region having the amino acid sequence of SEQ ID NO: 16. This fragment is thus characterized by comprising a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, a CDR-L3 of SEQ ID NO: 40, a CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36.

[0274] In other preferred embodiments, the antigen-binding fragment comprises a wild-type light chain variable region having the amino acid sequence of SEQ ID NO: 17, paired with a mutated heavy chain variable region. The mutated heavy chain variable region is selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, and SEQ ID NO: 56. These fragments are thus characterized by comprising a CDR-L1 of SEQ ID NO: 37, a CDR-L2 having the sequence YTS, and a CDR-L3 of SEQ ID NO: 38, paired with a set of heavy chain CDRs selected from the group consisting of: i) a CDR-H1 of SEQ ID NO: 42, a CDR-H2 of SEQ ID NO: 43, and a CDR-H3 of SEQ ID NO: 36; ii) a CDR-H1 of SEQ ID NO: 45, a CDR-H2 of SEQ ID NO: 46, and a CDR-H3 of SEQ ID NO: 47; iii) a CDR-H1 of SEQ ID NO: 49, a CDR-H2 of SEQ ID NO: 50, and a CDR-H3 of SEQ ID NO: 51; iv) a CDR-H1 of SEQ ID NO: 53, a CDR-H2 of SEQ ID NO: 54, and a CDR-H3 of SEQ ID NO: 55; and v) a CDR-H1 of SEQ ID NO: 57, a CDR-H2 of SEQ ID NO: 58, and a CDR-H3 of SEQ ID NO: 36.

[0275] The invention also relates to an isolated antigen-binding fragment that specifically binds to human CD3, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 39, paired with a heavy chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, and SEQ ID NO: 56. The invention also encompasses fragments comprising the six CDRs corresponding to these VL / VH pairings. These antigen-binding fragments are particularly useful as CD3 agonists for incorporation into multispecific molecules, including but not limited to the trispecific molecules described herein.

[0276] In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from the anti-CD3 antibody UCHT1. In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from a humanized variant of the anti-CD3 antibody UCHT1.

[0277] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody BMA031 a humanized antigen-binding fragment thereof. BMA031 was initially described by Borst et al. (Hum Immunol, 1990, 29(3):175-88), which is fully incorporated herein by reference.

[0278] In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from the anti-CD3 antibody BMA031. In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from a humanized variant of the anti-CD3 antibody BMA031.

[0279] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody 12F6 or a humanized antigen-binding fragment thereof, as described by Li et al. (Immunology, 2005, 116(4): 487-498), which is fully incorporated herein by reference. Preferably, the humanized variant of 12F6 comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:18 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:19.

[0280] In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from the anti-CD3 antibody 12F6. In certain embodiments, the CD3 agonist may be a Fab fragment or an scFv fragment derived from a humanized variant of the anti-CD3 antibody 12F6.

[0281] In a particularly preferred embodiment, the CD3 agonist may be a humanized variant of the anti-CD3 antibody UCHT1 or a humanized antigen-binding fragment thereof.

[0282] It is further preferred herein that the CD3 agonist comprised in the multifunctional molecule of the invention is a human or humanized Fab fragment, nanobody or scFv fragment.

[0283] 4. The CD2 agonist

[0284] The multifunctional molecule of the invention further comprises a CD2 agonist. A CD2 agonist is a molecule that specifically binds to the CD2 receptor on the surface of T cells and other immune cells, thereby activating or enhancing their immune response through costimulation. By engaging the CD2 receptor, CD2 agonists can promote the formation of an immune synapse, provide co-stimulatory signals, enhance T cell activation, and improve the immune system's ability to target and eliminate infected or malignant cells.

[0285] The CD2 agonist may be a natural ligand of CD2 or an engineered variant thereof. Alternatively, the CD2 agonist may be an anti-CD2 antibody, an antigen-binding fragment thereof, or a mimetic thereof.

[0286] Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD2 agonist is a CD2 ligand, an anti-CD2 antibody or an antigen-binding fragment thereof, or a non-antibody binding scaffold.

[0287] Preferably, the CD2 agonist is a human CD58 ectodomain or an engineered variant thereof.

[0288] CD58, also known as LFA-3 (lymphocyte function-associated antigen 3), is a cell surface glycoprotein that plays a critical role in the immune system by mediating cell adhesion and enhancing immune responses. CD58 is a ligand of CD2.

[0289] In certain embodiments, the CD2 agonist is a human CD58 ectodomain comprising the amino acid sequence set forth in SEQ IS NO:1 to 4:

[0290] Human CD58 ectodomain (SEQ ID NO:1)

[0291] FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSD EDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRN STSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHRY

[0292] Human CD58 ectodomain (SEQ ID NO:2)

[0293] FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSD EDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRN STSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHR

[0294] Human CD58 ectodomain (SEQ ID NO:3)

[0295] FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSD EDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRN STSIYFKMENDLPQKIQCTLSNPLENTTSSIILTTCIPSSGHSRHRY Human CD58 ectodomain (SEQ ID NO:4) FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSD EDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRN STSIYFKMENDLPQKIQCTLSNPLENTTSSIILTTCIPSSGHSRHR

[0296] The CD2 agonist may also be a sequence variant of a CD58 ectodomain. Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD58 ectodomain comprises or consists of (i) an amino acid sequence as set forth in any one of SEQ ID NOs:l-4, or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:l-4, wherein the CD58 domain remains the ability to engage CD2 on the surface of a T cell.

[0297] The term "sequence identity" as used herein refers to the degree of similarity between two amino acid or nucleotide sequences, expressed as a percentage. Sequence identity is determined by aligning the sequences for optimal comparison and calculating the percentage of identical residues (amino acids or nucleotides) at corresponding positions. The alignment can be performed using standard algorithms and software known in the art, such as BLAST or ClustalW.

[0298] For the purposes of this application, a sequence is considered to have a certain percentage of sequence identity to a reference sequence if, when optimally aligned, it shares at least that percentage of identical residues with the reference sequence. For example, a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a reference sequence (such as SEQ ID NOs:l-4) means that the sequence must match the reference sequence at the specified percentage of positions.

[0299] Alternatively, the CD2 agonist may be an ectodomain of CD48. CD48 is another cell surface protein that belongs to the immunoglobulin superfamily and is expressed on various immune cells, including T cells, B cells, and dendritic cells. The ectodomain of CD48 can interact with the CD2 receptor on T cells, similar to CD58, and provide co-stimulatory signals that enhance T cell activation and immune responses.

[0300] In certain embodiments, the CD48 ectodomain may comprise or consist of an amino acid sequence as set forth in SEQ ID NQ:20:

[0301] QGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYIS

[0302] KVQKEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYG

[0303] DKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARS Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD2 agonist may be a natural CD2 ligand, such as CD58, CD48, or the ectodomain thereof.

[0304] In certain embodiments, the CD2 agonist may be an engineered variant of the CD58 ectodomain, preferably an engineered variant of any one of SEQ ID NOs: 1-4, or of the CD48 ectodomain. For example, the engineered variant of the human CD58 or CD48 ectodomain may be engineered for increased affinity, specificity and / or stability.

[0305] An engineered variant refers to a modified version of a protein, such as the ectodomain of CD58 or CD48 that has been intentionally altered through genetic or protein engineering techniques to enhance its properties. These modifications can be designed to improve the protein's performance in specific applications, such as increasing its binding affinity, specificity, or stability.

[0306] Affinity refers to the strength of the interaction between a protein (such as an antibody or receptor) and its specific ligand or target molecule. Higher affinity indicates a stronger and more stable binding interaction, which can enhance the effectiveness of the protein in recognizing and binding to its target.

[0307] Specificity refers to the ability of a protein to selectively bind to a particular ligand or target molecule among a mixture of different molecules. High specificity ensures that the protein interacts primarily with its intended target, minimizing off-target effects and improving the precision of its biological activity.

[0308] Stability refers to the ability of a protein to maintain its structural integrity and functional activity under various conditions, such as changes in temperature, pH, or the presence of denaturing agents. Increased stability enhances the protein's durability and effectiveness in therapeutic or diagnostic applications by preventing degradation or loss of function over time.

[0309] By engineering variants of the human CD58 or CD48 ectodomains for increased affinity, specificity, and / or stability, more effective CD2 agonists for use in immunotherapy and other medical applications may be obtained.

[0310] Accordingly, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD2 agonist is (a) a specificity-tuned CD2 ligand, in particular a specificity-tuned CD58, specificity-tuned CD48, or the ectodomain thereof; (b) an affinity-tuned CD2 ligand, in particular an affinity-tuned CD58, affinity-tuned CD48, or the ectodomain thereof; and / or (c) a stabilised CD2 ligand, in particular a stabilized CD58, stabilized CD48, or the ectodomain thereof.

[0311] In certain embodiments, the CD2 agonist may be an antigen-binding fragment of an anti-CD2 antibody. Non-limiting examples of antigen-binding antibody fragments are provided elsewhere herein. In a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the CD2 agonist is an scFv, a Fab, or a nanobody.

[0312] In certain embodiments, the CD2 agonist may be a non-antibody binding scaffold. A nonantibody binding scaffold is a type of engineered protein that is designed to bind to specific target molecules with high affinity and specificity, similar to antibodies, but is structurally distinct from the traditional antibody framework. These scaffolds are typically smaller, more stable, and easier to produce than antibodies. Non-antibody binding scaffolds can be engineered to recognize a wide range of targets, including proteins, peptides, and small molecules.

[0313] An example of a non-antibody binding scaffold is DARPins (Designed Ankyrin Repeat Proteins). DARPins are derived from natural ankyrin repeat proteins, which are common protein interaction motifs found in many different proteins. DARPins are engineered to have high binding affinity and specificity for their target molecules by varying the amino acid sequences within the repeat units. They are known for their small size, high stability, and ease of production in bacterial systems.

[0314] Thus, in certain embodiments, the CD2 agonist may be a DARPin specifically binding to CD2.

[0315] 5. The antibody Fc region

[0316] In certain embodiments, the multifunctional molecule of the invention may comprise an antibody Fc region. The term "Fc region" or "Fc domain" refers to the region(s) of an antibody constant region (e.g., IgGl, lgG2, lgG3, or lgG4) that is involved in the binding interaction of the Fc region to one or more Fey receptors (e.g., FcyRI (CD64), FcyRllb (CD32b) or FcyRllla (CD16). The locations of the regions or domains of IgG isotype constant regions are known in the art and are described, for example, in Shields et al., 2001, J. Biol. Chem. 276:6591-6604, and Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491 and Sondermann et al., 2000, Nature 406(6793): 267-73. The Fc regions or domains include, for example and not for limitation, the hinge region and the CH2 domain. That is, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region is derived from a human IgG antibody heavy chain, in particular a human IgGl antibody.

[0317] The antibody Fc region may be a naturally occurring antibody Fc region or may be an engineered variant of a naturally occurring antibody Fc region. For example, the antibody Fc region may comprise mutations that reduce immune effector functions. Exemplary mutations that may be comprised in the antibody region are summarized in Liu et al. (Antibodies, 2020, 9(4):64).

[0318] Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, wherein the antibody Fc region comprises one or more mutations that reduce immune effector functions.

[0319] In certain embodiments, the antibody Fc region may comprise one or more mutations that prolong serum half-life of an Fc fusion by promoting interaction with the neonatal Fc receptor (FcRn). Different combinations of mutations have been described in the art that prolong serum half-life of the Fc-containing molecule. Among the engineered Fc variants with improved pH-dependent FcRn interactions, the M252Y / S254T / T256E (YTE) and M428L / N434S (LS) variants have been the most studied (Zalevsky J, et al., Nat. Biotechnol., 2010;28:157-159 and Dall'Acqua WF, et al., J. Biol. Chem. 2006;281:23514-23524). More recently, the mutations Q311R / M428L were reported to result in improved serum half-life (Ko et al., Exp Mol Med. 2022 Nov l;54(ll):1850-1861).

[0320] Accordingly, in certain embodiments, the antibody Fc region is an antibody Fc region comprising the YTE mutations (M252Y / S254T / T256E).

[0321] In certain embodiments, the antibody Fc region is an antibody Fc region comprising the LS mutations (M428L / N434S).

[0322] In certain embodiments, the antibody Fc region is an antibody Fc region comprising the mutations Q311R / M428L.

[0323] Furthermore, the antibody Fc region may comprise mutations that allow heterodimerization of the antibody Fc region. Normally, the two polypeptide chains of the antibody Fc region have an identical sequence and are expressed from the same gene. Thus, in certain embodiments, both polypeptide chains comprised in the antibody Fc region are linked to the same components. In such embodiments, no modification of the antibody Fc region is required.

[0324] In some embodiments, the first polypeptide chain comprised in the antibody Fc region is linked to different components than the second polypeptide chain comprised in the antibody Fc region. For example, a first polypeptide chain comprised in the antibody Fc region may be linked to a construct comprising a soluble TCR fragment, a CD3 agonist and a CD2 agonist, while the second polypeptide chain comprised in the antibody Fc region may not. In certain embodiments, the first polypeptide chain of the antibody Fc region is linked to a multifunctional molecule comprising a soluble TCR fragment, a CD3 agonist and a CD2 agonist via the hinge region, whereas the second polypeptide chain of the antibody Fc region merely comprises the hinge region.

[0325] To allow heterodimerization of heavy chain fragments that are linked to different components, certain modifications have to be introduced into the heavy chain fragments. The most commonly applied strategy to achieve heterodimerization of heavy chain fragments is by introducing knobs-into-holes mutations into the antibody Fc region.

[0326] The term "knobs-into holes mutations" refers to mutations, including those in the CHS domain of an Fc region, that facilitate heterodimerization of the first and second polypeptide chains in an antibody Fc region. Exemplary mutations useful for this heterodimerization are described in Ridgway et al. (1996) Protein Engin. 9(7): 617- 21 , Atwell et al. (1997) J, Mol. Biol 270:26-35, and PCT Publication No. W02014 / 106015, which are each incorporated by reference herein in their entirety. For instance, electrostatic or hydrophobic interactions can be altered to create knobs and corresponding holes in the two polypeptide chains. For instance, a "protuberance" comprising one or more amino acid modifications may be added to one chain to increase the bulk (e.g., the total volume) taken up by the amino acids. For instance, smaller amino acids can be modified or replaced by those having larger side chains which projects from the interface of the first polypeptide chain and can therefore be positioned in a related cavity in the adjacent second polypeptide chain so as to stabilize the heterodimer, and thereby favour heterodimer formation over homodimer formation. The protuberance may exist in the original interface or may be introduced synthetically (e.g., by altering one or more nucleic acid encoding the amino acid(s) at the interface). In some embodiments, a protuberance is introduced by modifying the nucleic acid encoding at least one '"original" amino acid residue in the interface of the first polypeptide with a nucleic acid encoding at least one "engineered" amino acid residue which has a larger side chain volume than die original amino acid residue, it will be appreciated that there can be more than one original and corresponding engineered residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

[0327] In addition to a knob (protuberance) added to one chain, a "cavity"' (hole) may be added to the second chain, comprising to at least one amino acid side chain which is recessed from the interface of the first or second polypeptide chain and therefore accommodates a corresponding protuberance on the adjacent second polypeptide chain. The cavity may exist in the original interface or may be introduced synthetically (e.g., by altering one or more nucleic acid encoding the amino acid(s) at the interface). In some embodiments, a protuberance is introduced by modifying the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide with a nucleic acid encoding at least one "engineered" amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding engineered residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

[0328] 6. Linkage of the individual components

[0329] Within the present invention, the individual components of the multifunctional molecule of the invention (the soluble TCR fragment, the CD3 agonist and the CD3 agonist, and optionally, the antibody Fc region) are linked to each other. The individual components may be linked in any way known in the art. It is, however, preferred that the individual components are covalently linked.

[0330] Preferably, all components of the multifunctional molecule of the invention are proteinbased. Thus, the multifunctional molecule of the invention is preferably composed of one or more fusion proteins. That is, two or more polypeptide chains derived from the individual components are genetically fused together. Here, the term "genetic fusion" refers to a colinear, covalent linkage of two or more proteins or fragments thereof via their individual peptide backbones, through genetic expression of a polynucleotide molecule encoding those proteins. Genetic engineering methods that may be used for obtaining genetic fusions are well known in the art.

[0331] Any of the protein-based components of the multifunctional molecule of the invention may be linked with a peptide linker. The term "peptide linker", "linker" or "spacer" as used herein refers to a spacer acting as a hinge region between polypeptide domains, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains. In a preferred embodiment, all protein-based components of the multifunctional molecule according to the invention, i.e., the TCR fragment, the CD3 agonist, the CD2 agonist and, optionally, the antibody Fc region, are linked with a peptide linker.

[0332] The length of the spacer can vary; typically, the number of amino acids in the spacer is 100 or less amino acids, preferably 50 or less amino acids, more preferably 40 or less amino acids, still more preferably, 30 or less amino acids, or even more preferably 20 or less amino acids. Preferred ranges are from 5 to 50 amino acids. In preferred embodiments, said spacer is a peptide having structural flexibility (i.e., a flexible linking peptide or "flexible linker") and comprises 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. Preferably, wherein at least 65%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acids in said flexible peptide linker are selected from the group consisting of glycine, serine, alanine and threonine.

[0333] In certain embodiments, the flexible linker comprises the sequence (S)-(NxGGGGS)-G-(S) where N = 1,2, 3,4,5, or 6. That is, in certain embodiments, the flexible linker comprises the sequence (S)-(lxGGGGS)-G-(S) (SEQ ID NO:6), (S)-(2xGGGGS)-G-(S) (SEQ ID NO:7), (S)- (3xGGGGS)-G-(S) (SEQ ID NO:8), (S)-(4xGGGGS)-G-(S) (SEQ ID NO:9), (S)-(5xGGGGS)-G-(S) (SEQ ID NQ:10), or (S)-(6xGGGGS)-G-(S) (SEQ ID NO:22). In certain embodiments, all protein-based components of the multifunctional molecule according to the invention, i.e., the TCR fragment, the CD3 agonist, the CD2 agonist, optionally, the antibody Fc region, are linked with a flexible linker. In certain embodiments, at least two, preferably all of the TCR fragment, the CD3 agonist, and the CD2 agonist comprised in the multifunctional molecule according to the invention are linked with a flexible linker; preferably with a flexible linker consisting of or comprising the sequence (S)-(NxGGGGS)-G-(S) where N = 1,2, 3,4,5 or 6; preferably with a linker comprising or consisting of SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:22.

[0334] In certain embodiments, the linker is an alpha-helical linker. Due to their rigid structure, alphahelical linkers can be used to establish a defined spatial distance between the two components of the multifunctional molecule that are linked with the alpha helical linker. Alpha-helical linkers are known in the art and the skilled person is capable of identifying alpha-helical linkers. Non-limiting examples of alpha-helical linkers include (S)-A(3xEAAAK)A-G-(S) (SEQ ID NO:11), (S)-A(6xEAAAK)A-G-(S) (SEQ ID NO:12) and (S)-(A-4xEAAAK-A)-LE-(A-4xEAAAK-A)-G- (S) (SEQ ID NO:13).

[0335] Alternatively, linkers from naturally occurring proteins may be used to connect two components of the multifunctional molecule of the invention. Non limiting examples are the naturally occurring linker derived from the EPO receptor (S-(G-INEVVLLDAP)-G-(S) (SEQ ID NO:14)) and the natural occurring linker derived from T LYMPHOCYTE ACTIVATION ANTIGEN (CD80) (S-(G-LSVKADF)-G-(S) / SEQ ID NO:15)).

[0336] Multifunctional molecules comprising three or more components may comprise two or more identical or different peptide linkers to connect the individual components. That is, the multifunctional molecule of the invention may comprise different types of linkers, including flexible, alpha-helical and / or naturally occurring linkers. Preferably, all components of the multifunctional molecule are linked with a peptide linker.

[0337] 7. Nucleic acids and cells encoding the multifunctional molecule of the invention

[0338] In a particular embodiment, the invention relates to a nucleic acid molecule encoding the multifunctional molecule of the invention.

[0339] That is, the present invention further relates to a nucleic acid molecule encoding the multifunctional molecule provided and described herein. As used herein, unless specifically defined otherwise, the term "nucleic acid" or "nucleic acid molecule" is used synonymously with "oligonucleotide", "nucleic acid strand", "polynucleotide", or the like, and means a polymer comprising one, two, or more nucleotides. The term "nucleic acid molecule" relates to the sequence of bases comprising purine- and pyrimidine bases which are comprised by polynucleotides, whereby said bases represent the primary structure of a nucleic acid molecule. Herein, the term "nucleic acid molecule" includes all kinds of nucleic acid, including DNA, cDNA, genomic DNA, RNA, synthetic forms of DNA and mixed polymers comprising two or more of these molecules, and preferably relates to DNA and cDNA. As readily understood by those of skill in the art, the nucleic acid sequences provided herein represent sequences of DNA and also comprise corresponding RNA sequences where T is replaced by U. The term "nucleic acid molecule" generally comprises sense and antisense strands. "Nucleic acid molecule" mayfurther comprise non-natural or derivatized nucleotide bases as well as natural or artificial nucleotide analogues, e.g., in order to protect the nucleic acid molecule against endo- and / or exonucleases as will be readily appreciated by those skilled in the art.

[0340] In certain embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule, such an mRNA molecule, encoding one or more polypeptide chains of the multifunctional molecule according to the invention. Encompassed herein are also compositions comprising two or more nucleic acid molecules encoding different polypeptide chains of the multifunctional molecule according to the invention. Preferably, the two or more nucleic acid molecules encode all polypeptide chains of the multifunctional molecule according to the invention.

[0341] The present invention further relates to a vector comprising the nucleic acid molecule described and provided herein.

[0342] The term "vector" as used herein generally comprises all kinds of linear or circular nucleic acid molecules which can replicate autonomously is a suitable host cell. Such vectors comprise, but are not limited to, plasmids, cosmids, phages, virus (e.g., adeno-, adeno-associated-, lenti-, or preferably retroviral vectors), and other vectors or shuttles known in the art which are suitable to carry and transfer genes into host cells in order to allow stable or transient translation and constitutive or conditional expression of the inventive fusion protein in the host cell. The vector is usually not integrated into the cell genome, but may also be integrated. Vectors according to the present invention which comprise nucleic acid molecules as described and provided herein preferably allow stable expression of the fusion protein of the present invention in the host cell (expression vector). Vectors of the present invention may further comprise marker genes, promoter and / or enhancer sequences (operably linked to the nucleic acid molecule of the present invention), replication origin suitable for the respective host cell, restriction sited, multiple cloning sites, labels and further functional units as known in the art. The vectors may inter alia be transferred into host cells via a shuttle such as a virus (which may itself be considered a vector), or be nakedly transformed or transduced into host cells. The vector is preferably adapted to suit to the respective host cell where it is to be transformed or transduced into. The skilled person will readily understand that different host cells will require different kinds of vectors. For example, plasmids are suitable vectors for transformation of bacterial cells, while retroviral vectors are suitable for transduction into eukaryotic cells (e.g., T cells).

[0343] In one embodiment of the present invention, the vector of the present invention is a viral vector, e.g., a retroviral or lentiviral vector or an AAV vector.

[0344] In a particular embodiment, the invention relates to a cell comprising the nucleic acid molecule or the vector according to the invention.

[0345] The present invention further relates to a host cell comprising the nucleic acid molecule or the vector as described and provided herein. In one embodiment, the host cell of the present invention is transduced or transformed with the nucleic acid molecule or the vector as described and provided herein.

[0346] Generally, as used herein unless specifically defined otherwise, the terms "transduced" or "transformed" (as well as "transduction" or "transformation") or the like may be used interchangeably and generally mean any kind of transfer of a nucleic acid molecule and / or vector into a host cell, regardless of the kind of host cell and regardless of the way of transfer (e.g., (chemical) transformation, (viral) transduction, electroporation, transfection, etc.). The nucleic acid molecule and / or the vector may be stably integrated into the genome of the host cell, or be extrachromosomal (i.e. transient expression). Examples for suitable methods for achieving transient expression in a host cell are known in the art and comprise mRNA transfection. In one embodiment, the nucleic acid molecule and / or the vector is stably integrated into the genome. The host cell described and provided in context with the present invention comprising the nucleic acid molecule or the vector as described and provided herein is preferably able to stably or transiently (e.g., stably) express (either constitutively or conditionally) the fusion protein of the present invention. The host cell may generally be transduced or transformed by any method with any suitable nucleic acid molecule or vector.

[0347] Examples of suitable host cells according to the present invention include, but are not limited to, T cells, e.g. CD8+ T cells, CD4+ T cells, NK (natural killer) cells, y6 T cells, macrophages, dendritic cells, as well as cells suitable store and / or reproduce the nucleic acid molecule or vector of the present invention, including bacterial cells (e.g., E. coli) and further eukaryotes. The cells may be autologous or non-autologous, but are preferably autologous. Also, the cells may be allogeneic or non-allogeneic as readily clear for the skilled person.

[0348] The multifunctional molecule of the invention can be produced using various mammalian cell lines as host cells, which are well-suited for the expression of heterologous proteins. Examples of suitable mammalian cell lines include Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, NSO cells, Baby Hamster Kidney (BHK) cells, PerC6 cells, Sp2 / 0 cells, and COS cells (COS-1 and COS-7). These cell lines may provide a robust and versatile platform for the efficient production of the multifunctional molecule of the invention, ensuring proper folding, stability, and functional activity through appropriate post- translational modifications.

[0349] The present invention also relates to a method of preparing a host cell of the present invention as described and provided herein, said method comprising

[0350] (1) transducing or transforming a host cell as described above with a nucleic acid molecule or a vector as described and provided herein;

[0351] (2) cultivating the transduced host cell of step (1) in a suitable medium allowing growth of the cell and expression of the fusion protein encoded by said nucleic acid molecule or said vector; and

[0352] (3) collecting the host cells from the medium.

[0353] In a preferred embodiment of the present invention, the host cell is transduced or transformed outside the human body. Methods for obtaining, isolating and culturing cells (e.g., T cells such as CD8+ T cells, CD4+ T cells) from donors (e.g., human donors) are known in the art and comprise inter alia blood draw or bone marrow removal.

[0354] 8. Methods of producing the molecule of the invention In a particular embodiment, the invention relates to a method of producing the multifunctional molecule according to the invention, the method comprising a step of culturing the cell according to the invention.

[0355] That is, the present invention also relates to a method of preparing the multifunctional molecule of the present invention as described and provided herein, said method comprising

[0356] (1) transducing or transforming a host cell as described above with a nucleic acid molecule or a vector as described and provided herein;

[0357] (2) cultivating the transduced host cell of step (1) in a suitable medium allowing growth of the cell and expression of the fusion protein encoded by said nucleic acid molecule or said vector; and

[0358] (3) retrieving the multifunctional molecule of the invention from the culture supernatant and / or from the host cells of step (2).

[0359] A suitable medium and growth conditions for the production of the multifunctional molecule according to the invention in HEK293 cells is provided in the appended examples.

[0360] 9. Pharmaceutical compositions and therapeutic applications

[0361] In a particular embodiment, the invention relates to a pharmaceutical composition comprising the multifunctional molecule according to the invention, the nucleic acid molecule according to the invention and / orthe cell according to the invention, and a pharmaceutically acceptable carrier.

[0362] That is, the present invention further relates to a pharmaceutical composition comprising a multifunctional molecule, a nucleic acid molecule, a vector, and / or a host cell as described and provided by the present invention. Such pharmaceutical composition is suitable to be administered to a patient (preferably, a human patient).

[0363] Medicaments in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

[0364] The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0365] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0366] The pharmaceutical compositions may contain preserving agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colourants, odorants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

[0367] Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. The dosage may be repeated as often as appropriate. If side effects develop the amount and / or frequency of the dosage can be reduced, in accordance with normal clinical practice.

[0368] As is common in anti-cancer the multifunctional molecule of this invention may be used in combination with other agents for the treatment of cancer, and other related conditions found in similar patient groups.

[0369] Accordingly, the present invention also relates to methods for treating a disease or disorder by administering comprising a pharmaceutical composition comprising a multifunctional molecule, a nucleic acid molecule, a vector, and / or a host cell as described and provided by the present invention. Thus, in a particular embodiment, the invention relates to the multifunctional molecule according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in medicine, in particular for use in the treatment of cancer.

[0370] The multifunctional molecule of the invention may be used in the treatment of various diseases, including cancer, viral infections and autoimmune diseases.

[0371] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating cancer. Various cancer antigens, including neoantigens, have been described in the art. The skilled person would thus have no difficulties generating a multifunctional molecule of the invention comprising a soluble TCR fragment that binds to a specific cancer antigen.

[0372] The term "cancer" as used herein refers to any malignant neoplasm. The malignant neoplasm refers to diseases resulting from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism. The cells giving rise to cancer are genetically impaired and have usually lost their ability to control cell division, cell migration behaviour, differentiation status and / or cell death machinery. Most cancers form a tumour but some hematopoietic cancers, such as leukaemia, do not. Symptoms and staging systems for the different cancers are well known in the art and described in standard textbooks of pathology. Cancer as used herein encompasses any stage, grade, morphological feature, invasiveness, aggressiveness or malignancy of the cancer or the tissue or organ affected thereby.

[0373] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating viral infections.

[0374] The skilled person is aware of soluble TCR fragments that may be used in the context of the present invention for treating viral infections. In particular, the skilled person is aware of viral antigens that may be presented by virus-infected cells in an MHC-dependent manner and may be targeted with a soluble TCR fragment.

[0375] In certain embodiments, the invention relates to the multifunctional molecule according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating autoimmune diseases.

[0376] In autoimmune diseases, B cells play a crucial role in producing autoantibodies that erroneously target and attack the body's own tissues. The multifunctional molecule according to the invention may facilitate the formation of an immune synapse between autoreactive B cells and T cells, resulting in the depletion of these autoreactive B cells (Perico et al., Front Immunol. 2024 Feb 26;15:1335998). The skilled person is capable of identifying antigens presented on the surface of autoreactive B cells in an MHC-dependent manner, which can be targeted with a soluble TCR fragment.

[0377] In certain embodiments, the autoimmune disease may be, without limitation, lupus or multiple sclerosis.

[0378] In certain embodiments, the invention relates to a method of treating cancer in a subject in need, wherein the method comprises the administration of a multifunctional molecule according to the invention to said subject.

[0379] In certain embodiments, the invention relates to a method of treating a viral infection in a subject in need, wherein the method comprises the administration of a multifunctional molecule according to the invention to said subject.

[0380] In certain embodiments, the invention relates to a method of treating an autoimmune disease in a subject in need, wherein the method comprises the administration of a multifunctional molecule according to the invention to said subject.

[0381] The term "subject," as used herein, generally refers to any animal, e.g., a mammal. A subject may be a patient. A subject may be symptomatic or asymptomatic with respect to a disease or ailment. A subject may be primate (e.g., a human), non-human primate (e.g., rhesus or other types of macaques), dog, cat, mouse, pig, horse, donkey, cow, sheep, rat, and fowl. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Preferably, the multifunctional molecule of the invention is used for the treatment of humans. To reduce the risk of immunogenic reactions, it is thus preferred that all components of the multifunctional molecule of the invention, i.e., the soluble TCR fragment, the CD2 agonist, the CD3 agonist and, optionally, the antibody Fc region are of human origin.

[0382] In a particularly preferred embodiment, the multifunctional molecule of the invention is for use in the treatment of cancer in a subject, wherein the subject's cancer is characterized by the presence of exhausted T-cells. T-cell exhaustion is a state of T-cell dysfunction that arises during many chronic infections and cancers. It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T-cells. Overcoming T-cell exhaustion is a central goal of modern immunotherapy.

[0383] The inventors have surprisingly and unexpectedly found that the multifunctional molecules of the present invention are not only potent activators of fresh T-cells but are also exceptionally effective at reactivating exhausted T-cells. As demonstrated in FIG. 11 and FIG. 19, when cocultured with target-positive cancer cells, the trispecific molecules of the invention (e.g., format 376) induced a dramatic and potent IFN-y response from T-cells that had been driven into an exhausted state through repeated prior stimulation. This effect was substantially greater than that observed with the corresponding bispecific molecules, which lack the CD2 agonist. For example, the gplOO-targeting trispecific molecule showed a 15-fold increase in potency compared to its bispecific counterpart in activating exhausted T-cells (FIG. 11). This demonstrated ability to reverse the functional inertia of exhausted T-cells represents a significant and non-obvious therapeutic advantage, rendering the molecules of the invention particularly suitable for treating cancers where the tumour microenvironment is dominated by exhausted T-cells.

[0384] In the context of the present invention, a cancer "characterized by the presence of exhausted T-cells" is understood to mean that a clinically significant population of T-cells, particularly within the tumour microenvironment, exhibits an exhausted phenotype. This phenotype is identified by the co-expression of multiple canonical exhaustion markers, such as Programmed cell death protein 1 (PD-1), Lymphocyte-activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). A skilled person, such as a clinical immunologist, would consider such a population to be present when it constitutes a substantial fraction of the tumour-infiltrating lymphocytes (TILs), distinguishing it from the baseline levels of exhausted cells typically found in healthy tissue or peripheral blood.

[0385] More specifically, a clinically significant population of exhausted T-cells may be defined by quantitative thresholds as measured by standard methods such as multi-colour flow cytometry. This analysis is typically performed on the CD8+ or CD4+ T-cell compartment of tumour-infiltrating lymphocytes (TILs). For example, a subject's cancer may be considered to be characterized by the presence of exhausted T-cells if: at least 10%, preferably at least 20%, and more preferably at least 30% of the CD8+ TILs are positive for the inhibitory receptor PD-1; or at least 5%, preferably at least 10%, of the CD8+ TILs co-express two or more exhaustion markers, such as both PD-1 and TIM-3; or at least 5%, preferably at least 10%, of the CD8+ TILs co-express both PD-1 and LAG-3.

[0386] Such percentages may be considered significant as they represent a notable deviation from the levels typically observed in healthy peripheral blood, where such deeply exhausted phenotypes are rare. The precise thresholds and marker panels may be adapted by the skilled artisan based on the specific cancer type and validated assays, but the principle of identifying a substantial, non-trivial population of T-cells with a multi-marker exhausted phenotype remains the guiding factor.

[0387] Accordingly, a subject who "has been identified as having exhausted T-cells" is a subject from whom a biological sample, such as a tumour biopsy or blood sample, has been obtained and analysed in accordance with the methods described herein. The subject is thereby determined to have a cancer characterized by a clinically significant population of exhausted T-cells, for example by meeting one or more of the quantitative thresholds defined above. Such a subject is therefore selected as a suitable candidate for treatment with the multifunctional molecule of the invention due to the particular effectiveness of the molecule in reactivating said exhausted T-cells.

[0388] The person skilled in the art, such as a clinical immunologist or oncologist, is readily capable of determining whether a subject's cancer is characterized by the presence of exhausted T- cells. Such a determination can be made using standard and well-established techniques. For instance, T-cells, particularly tumour-infiltrating lymphocytes (TILs) obtained from a tumour biopsy or peripheral blood mononuclear cells (PBMCs) from a blood sample, can be analysed for the expression of a characteristic panel of biomarkers. This typically involves quantifying the surface expression of multiple co-inhibitory receptors, such as Programmed cell death protein 1 (PD-1), Lymphocyte-activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), often using methods like multi-colour flow cytometry or immunohistochemistry. Furthermore, the exhausted state can be functionally confirmed by assessing the T-cells' impaired effector functions, such as reduced proliferative capacity or diminished production of cytokines like interferon-gamma (IFN-y) upon ex vivo stimulation. These methods are routine in the field and allow for the clear identification of patients having cancers characterized by the presence of exhausted T-cells.

[0389] In another particularly preferred embodiment, the multifunctional molecule of the invention is for use in the treatment of cancer in a subject who is refractory to, or has relapsed after, a prior immunotherapy. In a specific embodiment, the prior immunotherapy is a checkpoint inhibitor therapy, such as an anti-PD-1, anti-PD-Ll, or anti-CTLA-4 antibody. A large population of cancer patients fails to respond to or relapses after treatment with existing immunotherapies, including checkpoint inhibitors. A primary mechanism underlying this treatment failure is the development of T-cell exhaustion, which renders the T-cells unresponsive to checkpoint blockade. The data presented herein provides a direct mechanistic rationale for using the multifunctional molecules of the invention in this high- unmet-need patient population. As shown in FIG. 11 and FIG. 19, the molecules of the invention potently reactivate T-cells that are in a state of exhaustion analogous to that found in patients who have failed prior immunotherapy. By providing both a primary activation signal (via the CD3 agonist) and a potent co-stimulatory signal (via the CD2 agonist) in a singleagent format, the invention can overcome the functional blockade in these exhausted cells. This provides a novel and inventive therapeutic strategy for treating patients who have been failed by current standards of care.

[0390] In a particularly preferred embodiment, the invention relates to a multifunctional molecule for use in the treatment of melanoma, particularly gplOO-positive melanomas such as metastatic uveal melanoma (mUM) and cutaneous melanoma. The current standard of care for HLA-A*02:01-positive mUM is tebentafusp (Kimmtrak), a bispecific molecule comprising a gplOO-specific TCR fragment fused to an anti-CD3 scFv. While tebentafusp has shown a survival benefit, its objective response rates are modest, indicating a clear need for more potent therapies.

[0391] The multifunctional molecules of the present invention, when configured to target gplOO, represent a direct and significant improvement over the tebentafusp bispecific format. The inventors have demonstrated across multiple assays that the addition of a CD58 ectodomain to a gpl00-TCR / anti-CD3 bispecific backbone results in markedly superior activity. As shown in FIG. 6 and FIG. 7, the trispecific molecules of the invention induce significantly higher levels of IFN-y secretion and more potent target cell cytolysis compared to the bispecific control, which is analogous to the tebentafusp architecture. This enhanced potency is particularly pronounced in the context of exhausted T-cells, as shown in FIG. 11, where the trispecific molecule was 15-fold more potent. Therefore, the multifunctional molecule of the invention provides a more effective means of treating gplOO-positive melanomas, including metastatic uveal melanoma and cutaneous melanoma by inducing a more powerful and durable antitumour T-cell response.

[0392] The terms "treatment" and "treating," as used herein, refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0393] It is to be understood that any and all definitions, limitations, features, variants, and embodiments described herein for the multifunctional molecule per se are intended to apply mutatis mutandis to the methods of treatment, medical uses, and Swiss-type claims of the invention. This includes, without limitation, any specific structural formats (such as format 376), amino acid or nucleic acid sequences, complementarity-determining regions (CDRs), linker types, or Fc region modifications.

[0394] 10. Ex vivo methods for reactivating T-cells

[0395] In another aspect, the present invention relates to an ex vivo or in vitro method for reactivating a population of exhausted T-cells. Such methods may be particularly useful for the preparation of cells for adoptive cell therapy.

[0396] The method comprises providing a population of T-cells which contains a subpopulation of exhausted T-cells. This population of cells may be obtained from a subject, such as a cancer patient (thereby providing autologous cells) or from a healthy donor (providing allogeneic cells). The cells can be isolated from various biological samples, including but not limited to peripheral blood mononuclear cells (PBMCs) obtained from a blood draw, or from tumor tissue as tumor-infiltrating lymphocytes (TILs).

[0397] The method further comprises the step of contacting said population of T-cells with a multifunctional molecule according to the invention. This contacting step is typically performed by culturing the T-cells in a suitable cell culture medium in the presence of the multifunctional molecule, under conditions that promote T-cell survival and activation (e.g., at approximately 37°C in a humidified atmosphere).

[0398] As surprisingly demonstrated by the inventors and shown in FIG. 11 and FIG. 19, contacting exhausted T-cells with a trispecific molecule of the invention leads to their potent reactivation. This reactivation is characterized by a restoration of key effector functions, including a substantial increase in cytokine production, such as interferon-gamma (IFN-y) and Granzyme B, and an enhanced capacity to kill target cancer cells. This effect is significantly and unexpectedly greater than that achieved with a corresponding bispecific molecule lacking the CD2 agonist, highlighting the unique therapeutic benefit of the inventive molecules in this context. Accordingly, the invention also provides a population of reactivated T-cells obtainable by the ex vivo or in vitro methods described herein. Such a population of cells is characterized by its restored effector functions and enhanced anti-tumor potency compared to the original, untreated population of exhausted T-cells. These reactivated cells represent a novel and improved cellular therapeutic agent.

[0399] The reactivated T-cells produced by this method are particularly useful for adoptive cell therapy. The method may therefore further comprise the steps of expanding the population of reactivated T-cells ex vivo and formulating them into a pharmaceutical composition suitable for administration to a subject. When administered to a patient, this potent population of reactivated T-cells can mediate a powerful and durable anti-cancer immune response. Any of the multifunctional molecules described herein, including those defined herein, may be used in these ex vivo methods for the preparation of a reactivated T-cell population for therapeutic use.

[0400] It is to be understood that any and all definitions, limitations, features, variants, and embodiments described herein for the multifunctional molecule per se are intended to apply mutatis mutandis to the methods of treatment, medical uses, and Swiss-type claims of the invention. All such combinations are considered to be explicitly disclosed herein.

[0401] Unless otherwise defined, the terms "comprising," "comprises," and "comprised of" are construed as being inclusive and open-ended, and not exclusive. Specifically, when used in this specification including the claims, the terms "comprising," "comprises," and "comprised of" and variations thereof mean that the specified features, steps, or components are included. These terms are not to be interpreted as excluding the presence of other features, steps, or components.

[0402] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0403] Where a numerical range is stated, it is to be understood that all values and sub-ranges within that range are also specifically disclosed, as if each and every value and sub-range had been explicitly written out. For example, a range of from 1 to 5 includes the individual values 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, and any sub-range, such as from 1 to 3, from 2 to 4, from 3 to 5, etc.

[0404] The term "about" or "approximately" when used in conjunction with a numerical value is intended to have its ordinary and customary meaning to a person of ordinary skill in the art for the particular context. It is to be understood as encompassing variations normal in the art, for example, within experimental error or measurement accuracy for a given technique, or referring to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined.

[0405] All documents, patents, patent applications, and publications cited or referred to in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of a conflict in terminology, the present specification is to control.

[0406] The headings and sub-headings used herein are for convenience only and are not to be construed as limiting the invention in any way.

[0407] BRIEF DESCRIPTION OF THE DRAWINGS

[0408] FIG.l shows a schematic overview of trispecific format compositions. TCRs linked via flexible or rigid protein linkers to either an anti-CD3 binding domain (here scFv or Fab) at the N terminus of the beta chain (bright grey, TCR-scFv) or at the C terminus of the alpha chain (dark grey, TCR-Fab) as well as an anti-CD2 binding domain (denoted as X), such as a CD58 ectodomain, or an anti-CD2 binding antibody (scFv, VHH or Fab), fused via flexible or rigid protein linkers either directly to the TCR or to the anti-CD3 domain.

[0409] FIG.2 shows an SDS-PAGE analysis of purified trispecific soluble TC engagers in Fab-Fab format under reducing and non-reducing conditions on stain-free gels. Molecules run higher than predicted molecular weights due to glycosylation. Trispecific molecules run ~25 kDa higher than the bispecific, corresponding to the size of CD58. Under reducing conditions the expected amount of chains is visible. The TCR alpha - VH chain runs at a similar size as the TCR beta - CD58 chain. Two bands are visible around 30 kDa, which both correspond to the TCR beta chain in different glycosylation states.

[0410] FIG.3 shows an SDS-PAGE analysis of purified trispecific soluble TCR engagers in Fab-scFv format under reducing and non-reducing conditions on stain-free gels. Molecules run higher than predicted molecular weights due to glycosylation. Trispecific molecules run ~25 kDa higher than the bispecific, corresponding to the size of the CD58 ectodomain. Under reducing conditions the expected amount of chains is visible. The TCR beta - scFv and the TCR alpha - CD58 chains run at similar sizes. The alpha chain alone is barely visible on the gel, due to the low amount of tryptophans. Construct 378 expressed at low yields, therefore less protein was loaded on the gel resulting in a weak band.

[0411] FIG.4 shows affinity measurements of selected trispecific soluble TCR engagers against pMHC by means of biolayer interferometry (BLI). Bispecific and trispecific soluble TCR engagers were used as analytes at 3-fold dilutions starting from 30 nM. Trispecific molecules show slightly lower affinities (up to 2.5-fold reduction in KD). Solid lines show measured curves. Dashed lines show curve fits generated using a 1:1 Langmuir kinetic model.

[0412] FIG.5 shows a schematic representation of a natural immune synapse (left) and an artificial immune synapse redirected by a trispecific soluble TCR engager co-ligating CD3 and CD2 on effector T cells.

[0413] FIG.6 shows the assessment of trispecific soluble TCR engager activity by means of interferongamma secretion. NCI-H2452 target cells were pulsed with either 50 pg / mL (bottom panels) or 100 ng / mL (upper panels) of the gplOO-YLE target peptide or with vehicle (DMSO). Soluble TCR engagers in Fab-scFv format (left panels) or Fab-Fab format (right panels) were tested at indicated concentrations in the presence of primary human T cells and target cells mixed at a 10:1 effector-to-target cell ratio. The levels of IFN-y in culture supernatants was detected after 111 hours of co-culture by means of chemiluminescent ELISA.

[0414] FIG.7 shows the assessment of trispecific soluble TCR engager cytotoxic activity by real-time fluorescence microscopy. NCI-H2452 target cells were pulsed with either 50 pg / mL (bottom panels) or 100 ng / mL (upper panels) of the gplOO-YLE target peptide or with vehicle (DMSO). Soluble TCR engagers in Fab-scFv format (left panels) or Fab-Fab format (right panels) were tested at indicated concentrations in the presence of primary human T cells and target cells mixed at a 10:1 effector-to-target cell ratio. Target cell cytotoxicity was assessed using red fluorescent microscope images acquired every 3 hours for quantification of target cell proliferation and death for 111 h. Specific target cell lysis [Cytolysis (%)] was calculated according to the formula: Cytolysis (%) = 100% - [data point - Positive control (TritonX-100)] / [co-culture without engager - Positive control (TritonX-100)].

[0415] FIG.8 shows the assessment of trispecific soluble TCR engager format activity in a T cell redirection assay. The activity of the selected trispecific soluble TCR engager format 376 and its bispecific Fab-scFv counterpart was assessed by means of interferon-gamma ELISA (left panel) and real-time fluorescence microscopy (right panel). NCI-H2452 cells were pulsed with either 100 ng / mL of the gplOO-YLE target peptide or vehicle (DMSO). Soluble TCR engagers were tested at indicated concentrations in the presence of primary human T cells and target cells mixed at a 10:1 effector-to-target cell ratio. The levels of IFN-y in cell culture supernatants and target cell killing were assessed after 111 hours of co-culture.

[0416] Fig.9 shows format 376 of Fig.l linked to an antibody Fc region.

[0417] Fig.10 shows antibody Fc fusions of the formats 368, 369, 370, 371, 372 and 373 of Fig.l.

[0418] FIG. 11 shows IFN-y secretion from co-cultures of exhausted human T-cells and two different gplOO-positive target cell lines: MP-41 (top panel) and A375-PMEL (bottom panel). The activity of the trispecific gplOO-376 TCR engager is compared to its bispecific counterpart. The inset tables show the calculated EC50 values, demonstrating significantly higher potency for the trispecific molecule in reactivating exhausted T-cells.

[0419] FIG. 12 demonstrates the target specificity of the TCR engagers, showing a lack of non-specific T-cell activation in co-cultures of exhausted human T-cells with two different gplOO-negative target cell lines: NCIH2452 (top panel) and A375-WT (bottom panel). The data confirms that IFN-y secretion is not induced by either the bispecific or trispecific gplOO TCR engagers in the absence of the target antigen.

[0420] FIG. 13 shows IFN-y secretion from co-cultures of fresh human T-cells with either gplOO- positive A375-PMEL target cells (top panel) or gplOO-negative A375-WT target cells (bottom panel). The activity of the trispecific gplOO-376 TCR engager is compared to its bispecific counterpart. The inset table shows the calculated EC50 values for the gplOO-positive setting.

[0421] FIG. 14 shows Granzyme B secretion from co-cultures of fresh human T-cells with either gplOO-positive A375-PMEL target cells (top panel) or gplOO-negative A375-WT target cells (bottom panel). The activity of the trispecific gplOO-376 TCR engager is compared to its bispecific counterpart. The inset table shows the calculated EC50 values.

[0422] FIG. 15 shows target cell cytolysis, measured by luminescence, in co-cultures of fresh human T-cells with either gplOO-positive A375-PMEL target cells (top panel) or gplOO-negative A375- WT target cells (bottom panel). The activity of the trispecific gplOO-376 TCR engager is compared to its bispecific counterpart and a Digitonin lysis control. The inset table shows the calculated EC50 values.

[0423] FIG. 16 shows biolayer interferometry (BLI) sensorgrams for the binding of gplOO bispecific, gplOO-376 trispecific (L7 linker), and gplOO-376 trispecific (Lil linker) molecules to immobilized gplOO pMHC (left panels) and hCD3e6 (right panels). Calculated equilibrium dissociation constants (KD) are indicated for each interaction. FIG. 17 shows biolayer interferometry (BLI) sensorgrams for the binding of Glyptodon-4 bispecific, Glyptodon-4-376 trispecific (L3 linker), and Glyptodon-4-376 trispecific (L7 linker) molecules to immobilized MAGE-A3 pMHC (left panels) and CD3E6 (right panels). Calculated equilibrium dissociation constants (KD) are indicated for each interaction.

[0424] FIG. 18 shows IFN-y secretion from co-cultures of fresh human T-cells with either MAGE-A3- positive A375 target cells (top panel) or MAGE-A3-negative HEK-A1 target cells (bottom panel). The activity of the trispecific Glyptodon-4 TCR engagers (L3 and L7 linkers) is compared to the bispecific counterpart. The inset table shows the calculated EC50 values.

[0425] FIG. 19 shows IFN-y secretion from co-cultures of exhausted human T-cells with either MAGE- A3-positive A375 target cells (top panel) or MAGE-A3-negative HEK-A1 target cells (bottom panel). The activity of the trispecific Glyptodon-4 TCR engagers (L3 and L7 linkers) is compared to the bispecific counterpart. The inset table shows the calculated EC50 values.

[0426] FIG. 20 shows biolayer interferometry (BLI) sensorgrams for the binding of the bispecific Squid-1 (top panel) and trispecific Squid-1-376 (bottom panel) molecules to immobilized GPC3 pMHC. Calculated equilibrium dissociation constants (KD) are indicated.

[0427] FIG. 21 shows IFN-y secretion from co-cultures of fresh human T-cells with either GPC3- positive ACDC-A2-GPC3 target cells (top panel) or GPC3-negative ACDC-A2 target cells (bottom panel). The activity of the trispecific Squid-1-376 TCR engager is compared to its bispecific counterpart. The inset table shows the calculated EC50 values.

[0428] EXAMPLES

[0429] The present invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention in any way. The examples provided herein describe specific embodiments of the invention but are not intended to limit the invention to the specific embodiments described. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the spirit and scope of this disclosure. The examples are not intended to limit the scope of the invention as defined in the appended claims.

[0430] Example 1: General methods

[0431] Construct design and cloning

[0432] Gene cassettes encoding desired soluble TCR engager building blocks were manufactured by gene synthesis (Twist Biosciences) and deposited into storage vectors by restriction cloning. PCR amplification of building blocks was performed for Golden-gate assembly of constructs encoding desired soluble TCR engager chains into the expression vector pTwist_CMV_WPRE (Twist Biosciences). The resulting assemblies were transformed into E. coli cells and the resulting clones were sequence verified prior to small-scale plasmid preparation by means of spin-column purification (Qiagen, #27106). Combinations of purified plasmids encoding individual soluble TCR engager designs were then co-transfected into Expi293 cells for transient expression.

[0433] Recombinant expression

[0434] Polypeptide chains were expressed from separate plasmids or from one plasmid separated by a self-processing 2A peptide. All soluble TCR engagers contain a 6xHis tag for purification at the N-terminus of the TCR beta chain, the CD58 ectodomain or the UCHT1 scFv CD3 agonist depending on the construct. To increase stability and expression yields all TCRs contain stabilising mutations in their constant domains (priority filing EP24171724) and an additional interchain disulphide bond in the constant domain (i.e., T166C in TRAC and S173C in TRBC) (Boulter et al. Protein Engineering, 2003, 16 (9): 707-11.). Suspension-adapted HEK293 cells (Expi293, Thermo Fisher) were transfected with the single plasmids or co-transfected with separate plasmids in a 1:1 ratio. Secreted soluble TCRs were purified from culture supernatants using a HisTrap excel column (5 mL, Cytiva, #17371206 on an Akta pure system (Cytiva) with a 20 mM sodium phosphate, 0.5 M NaCI and 25 mM imidazole wash buffer and a single step elution with 20 mM sodium phosphate, 0.5 M NaCI, 500 mM imidazole elution buffer. Purified proteins were buffer exchanged and concentrated into PBS using 30 MWCO concentrators (PierceTM). To evaluate the correct size of all Spectrs, 2 pg of each protein was analysed on SDS-PAGE under reducing and non-reducing conditions using Mini-PROTEAN TGX Stain-Free Precast Gels (Biorad).

[0435] Affinity measurements

[0436] Biolayer interferometry (BLI) measurements were performed using the Octet instrument (ForteBio). Biotinylated peptide-MHC class I monomers (Biolegend, #280013) were immobilised on streptavidin biosensors at 5 pg mL-1 for 300 s. Binding curves were obtained using soluble TC engagers diluted serially in a 3-fold fashion, starting from 30 nM with 1200 s association and 1200 s dissociation times. Kinetic curves were fitted with a 1:1 binding model using BLI Discovery 14.0 software and re-plotted using the Prism GraphPad software.

[0437] Cell culture

[0438] NCI-H2452 target cells expressing the red fluorescent protein mKate2 were cultured in RPMI 1640 Medium (ATCC modification) (Gibco, # A1049101) supplemented with 10 % FBS (Gibco, #16000-044) and 1 % Penicillin-Streptomycin (Gibco, #15140-122). For prolonged storage, cells were frozen in Serum-free cryopreservation media Bambanker (Nippon Genetics, #BB02) and stored in liquid nitrogen. A375-PMEL and A375-WT cells were cultured in Complete DMEM (Gibco, # 61965026) supplemented with 10 % FBS (Gibco, #16000-044), 1 % Penicillin- Streptomycin (Gibco, #15140-122) and ImM sodium pyruvate (Gibco, #11360070). MP41 cells were cultured in RPMI 1640 Medium (Gibco, # 11875093) supplemented with 10 % FBS (Gibco, #16000-044) and 1 % Penicillin-Streptomycin (Gibco, #15140-122). HEK293-derived cells (i.e., ACDC-A2-GPC3, ACDC-A2 and HEK293-A1) were cultured in Complete DMEM (Gibco, # 61965026) supplemented with 10 % FBS (Gibco, #16000-044), 1 % Penicillin-Streptomycin (Gibco, #15140-122).

[0439] Blood samples from healthy donors were obtained from the Blutspendezentrum SRK beider Basel in buffy coat format. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples through Ficoll (Sigma Aldrich, #10771) gradient centrifugation as per standard protocol and subsequently used for primary T cell isolation using the EasySep Human T Cell Isolation kit (Stemcell Technologies, #17951) following the manufacturer's instructions. Isolated human T cells were cryopreserved by resuspension in CS10 media (StemCell Technologies, # 07930) and aliquoted into cryovials which were placed in a freezing container for overnight storage at -80C and transfer into liquid nitrogen vapour phase storage the following day. One day before the co-culture assay, human T cells were thawed and rested overnight in complete T cell medium consisting of X-VIVO 15 Medium (Lonza, #BE02-060F) supplemented with 5% FBS, 50 pM |3-mercaptoethanol (Gibco, #31350-010), 100 pg mL-1 Primocin (Invivogen, #ant-pm-2) and, when indicated, freshly added 100 IU mL-1 recombinant human IL-2 (Peprotech, #200-02). HLA typing was performed by the Hematology Diagnostics unit at University Hospital Basel. Sequential T cell stimulation

[0440] Primary human T cells were subjected to six rounds of stimulation using CD3 / CD28 T cell simulation beads (Biolegend Cat. No. 422604) to induce exhaustion. Purified human T cells were seeded at a density of lxlO6cells / mL in complete T cell medium supplemented with 30 U / mL IL-2 and CD3 / CD28 T cell stimulation beads added at a 1:1 bead:cell ratio. Every second or third day of culture (37°C, 5% CO2 in humidified atmosphere), beads were removed by magnetic separation and T cells were washed twice in T cell medium lacking IL-2. After washing, T cells were counted and seeded lxlO6cells / mL in complete T cell medium supplemented with fresh 30 U / mL IL-2 and fresh CD3 / CD28 T cell stimulation beads added at a 1:1 bead:cell ratio. Following a total of six cycles of stimulation, expanded T cells were analysed by flow cytometry to confirm elevated markers of T cell exhaustion (Live dead staining, CD8, PD-1, Tim-3 and LAG-3) for subsequent use in co-culture assays. Alternatively, exhausted T cells were cryopreserved at -150°C according to standard protocols for prolonged storage.

[0441] Assessment of T cell killing and activation

[0442] One day before co-culture, human T cells were thawed and rested in complete T cell medium with 100 lU / mL recombinant human IL-2 (Peprotech, #200-02). Target NCI-H2452 cells were seeded in 384-well microtiter plates at 2.5xl0A3 cells per well in 50 pL one day prior to the experiment setup. Primary T cells were washed twice and re-suspended in complete T cell medium in the absence IL-2. Prior to T cell addition and soluble TCR addition, 37.5 pL of target cell supernatants were removed without disrupting seeded cells.

[0443] When required, target cells were pulsed with the target peptide gplOO (at 100 ng / mL or 50 pg / mL final concentrations in serum-free RPMI-1640) or DMSO, and incubated at 37°C for 1.5 hours. Next, pulsed cells were washed 50 pL of pre-warmed T cell medium three times. At wash three, 62.5 pL of the media was removed with 12.5 pL of the culture volume remaining. 7.5 uL of the media was added to yield the starting volume of the pulsed cells of 20 pL. Next, primary T cells were added (2.5xlOA4 cells / well) into target cell wells. Recombinant soluble TCRs were filtered through a 0.1 pm filter (Merck, UFC30VV), their concentrations measured via absorbance at 280 nm using a Nanodrop OneC instrument (Thermofisher) and diluted to specific concentrations in complete T cell medium in the absence of IL-2 prior to addition into co-culture wells. Co-culture plates were placed in an Incucyte S3 instrument for monitoring by means of real-time fluorescent microscopy. Images were acquired every 3 hours for quantification of target cell proliferation and death for up to 111 h. Image analysis was performed with the Incucyte S3 Software Module.

[0444] Following co-culture, supernatants were collected and used for IFN-y, Granzyme B ELISA and quantification of cell death using the CytoTox-Glo™ reagent. For IFN-y ELISA, standard 96-well ELISA plates (Thermo Scientific, #442404) or luminescencecompatible 96-well ELISA plates (Thermo Scientific, #436110) were coated with 2.5 pg mL-1 purified anti-human IFN-y capture antibody (clone MD-1, BioLegend, #507502) in PBS for 1-3 days at 4°C. On the day of supernatant harvest, ELISA plates were washed three times with wash buffer (0.05% Tween 20 in PBS) and blocked with 1% Sure Block in PBS (Lubio Science, #SB232010). After 90 min blocking at RT, ELISA plates were washed three times with wash buffer, followed by addition of 50 pL / well co-culture supernatant. Recombinant human IFN-y (Peprotech, #300-02-250UG) was diluted freshly with T cell medium in the absence of IL-2 and serially diluted to generate a standard curve. After 2 h incubation at RT, ELISA plates were washed 5 times with a wash buffer. Detection antibody was then added at 1.25 pg mL-1 in 1% Sure Block for 1 h at RT (clone 4S.B3, BioLegend, #502504). After incubation, wells were washed five times with wash buffer, followed by addition of 1:2000 diluted HRP Streptavidin (BioLegend, #504210) for 20 min at RT in the dark. After incubation, wells were washed five times with a wash buffer. For colorimetric ELISA development, 1-Step™ TMB ELISA Substrate Solutions (Thermo Scientific, #34028) was added into each well. After 2 min incubation in the dark, development was stopped by adding 2N H2SO4 (Fluka, #35276). Plate reading and data analysis were performed using an Infinite 200 PRO Plate Reader (Tecan). For Luminescent ELISA development, ELISA Pico Chemiluminescent Substrate (Thermo Scientific, #37070) was added into every well for 3-20 min prior to reading (2-19 min in the dark with 1 min shaking at 200 rpm) using an LU Ml star-0 mega Microplate Reader (BMG Labtech). Granzyme B ELISA was performed using the ELISA MAX Deluxe Set Human Granzyme B kit according to manufacturer's instructions (BioLegend 439204). For quantification of cell death using the CytoTox-Glo™ reagent (Promega, Cat. No. G9292), 25 pL of co-culture supernatant was used for mixture with 12.5 pL of freshly prepared CytoTox-Glo Cytotoxicity Assay Reagent, followed by incubation at RT for 15 min and quantification of luminescence using a LUMIstar-Omega Microplate Reader (BMG Labtech).

[0445] Example 2: Recombinant expression and affinity measurements of trispecific TCR molecules

[0446] To test the feasibility of expressing trispecific soluble TCR engagers recombinantly, gene cassettes encoding several trispecific molecular designs based on the Fab-Fab scaffold (formats 368, 369, 370, 371, 372 and 373) or the Fab-scFv scaffold (formats 374, 375, 376 and 378) were designed. In this experiment, sequences encoding tebentafusp as a model TCR targeting the gplOO antigen (See Tables 2 and 3 for sequences), the humanised UCHT1 antibody in Fab or scFv fragment format as a CD3 agonist (comprising SEQ ID NO:16 and 17) and the human CD58 ectodomain (SEQ ID NO:3) as a CD2 agonist were used. For each scaffold family, a no-CD58 control bispecific molecule was also included (Fig. 1). Designed gene cassettes were cloned into mammalian expression vectors that were co-transfected into suspension-adapted HEK293 cells for transient expression of desired molecular configurations.

[0447] Following an expression period of 5 days, cell culture supernatants were collected, filtered and subjected to immobilised metal affinity chromatography to purify recombinant proteins. SDS-PAGE analysis of the resulting preparations revealed that all proteins displayed the molecular weights expected from their original design, with a migration profile slightly above 100 kDa for the control bispecific TCR engager in the Fab-Fab format and an additional ~25 kDa for its corresponding trispecifics (Fig. 2, left), and a migration profile slightly above 75 kDa for the control bispecific TCR engager in the Fab-scFv format and an additional ~25 kDa for its corresponding trispecifics (Fig. 3, left). Similarly, SDS-PAGE analysis of p-mercaptoethanol- reduced preparations confirmed the molecular weights expected from the polypeptide chains forming the designed soluble TCR engager configurations (Fig. 2, right and Fig. 3, right).

[0448] Purified trispecific soluble TCR engagers were next assessed for their binding affinity to the target gplOO28o-288 YLEPGPVTA peptide (gplOO-YLE; SEQ. ID NO:23). To this end, gplOO-YLE- loaded HLA-A*02:01 biotinylated monomers were immobilised onto streptavidin-coated biosensors in preparation for biolayer interferometry measurements (BLI). These experiments revealed that the binding affinity of trispecific soluble TCR engagers to their gplOO target in both Fab-Fab (Fig. 4, top) and Fab-svFv (Fig. 4, bottom) formats was similar to that observed for their corresponding bispecific control molecules (i.e., dissociation constants KD within 2.5- fold of control).

[0449] Overall, the results show that trispecific soluble TCR engagers incorporating the human CD58 ectodomain are expressible from mammalian cells, display the molecular weights expected from their design and show similar levels of target binding affinity as their bispecific parental formats.

[0450] Example 3: Functional assessment of trispecific soluble TCR engagers in T cell redirection assays

[0451] The activity of a panel of trispecific soluble TCR engagers targeting the tumour antigen gplOO was assessed in T cell re-direction assays in the presence of target cells. The goal of these experiments was to assess the ability of designed molecules to form an artificial immune synapse between T cells and target cells (Fig. 5), and to evaluate the contributions of molecular format and addition of the CD58 ectodomain towards the potency of target-specific T cell activation and target cell cytotoxicity (i.e., redirected killing). Titrations of trispecific soluble TCR engagers alongside their corresponding bispecific controls were added into microtiter plate wells containing primary human T cells and fluorescent NCI- H2452 target cells pulsed with 100 ng / mL or 50 pg / mL gplOO-YLE peptide. Co-cultures were performed for a duration of 5 days to assess T cell activation by means of IFN-y ELISA (Fig. 6) and to monitor target cell survival (a proxy for redirected cytotoxicity) by real-time fluorescence microscopy (Fig. 7).

[0452] T cell activation assessment revealed that all tested molecules displayed increased activity in the context of high levels of pulsed target peptide (Fig. 6 lower panels) compared to low levels of pulsed target peptide (Fig. 6 upper panels). In addition, activity was higher for soluble TCR engagers in the Fab-scFv format (Fig. 6 left panels) compared to those in the Fab-Fab format (Fig. 6 right panels). In the context of soluble TCR engagers in the Fab-scFv format, the trispecific format 376 was found to have enhanced activity over the bispecific soluble TCR control, while the other trispecific molecules (i.e., 374 and 378) were found to have similar or lower activity than the bispecific control (Fig. 6 left panels). In the context of soluble TCR engagers in the Fab-Fab format, every trispecific molecule showed enhanced activity over the bispecific soluble TCR control (Fig. 6 right panels).

[0453] Assessment of target cell killing by means of real-time fluorescence microscopy in the same experiment revealed a similar pattern of activity to that revealed by IFN-y ELISA. This is, higher overall activity in the presence of high levels of target, higher overall activity of Fab-scFv formats over Fab-Fab formats, enhanced activity of the Fab-scFv trispecific format 376 relative to bispecific Fab-scFv control, and enhanced activity for all trispecific molecules in the Fab-Fab format relative to bispecific Fab-Fab control (Fig. 7).

[0454] Assessment of the Fab-scFv trispecific format 376 in a second experiment confirmed its enhanced capacity to activate and redirect T cell killing relative to the Fab-scFv bispecific control, as measured by IFN-y ELISA (Fig. 8 left panel) and real-time fluorescence microscopy (Fig. 8 right panel), respectively. In this experiment, the trispecific format 376 showed potent T cell activation in the context of low levels of pulsed target. This activity was approximately 10-fold higher than control, while maintaining similarly low levels of background IFN-y release (Fig. 8 left panel). In terms of redirected killing, the trispecific format 376 displayed a 10-fold broader activity window (i.e., killing of gplOO-pulsed versus DMSO-pulsed target cells) over the activity window observed for the bispecific Fab-scFv control (Fig. 8 right panel).

[0455] The experiments show that the smaller Fab-scFv format, either in the bispecific or trispecific context, has higher potency than the Fab-Fab format in redirecting T cell activity. These findings highlight that if using Fab-Fab formats for therapeutic purposes, a higher dose will likely be required to achieve the same efficacy as their Fab-scFv counterparts. It should be noted that, due to a larger size differential to the renal filtration cutoff (approximately 50 kDa), the 100 kDa Fab-Fab format is expected to have a longer serum half-life than the smaller 75 kDa Fab-scFv format, which may compensate for its reduced levels of in vitro efficacy.

[0456] Importantly, the experiments demonstrate that addition of the CD58 ectodomain as a second immunoligand in addition to the CD3 agonistic UCHT1 antibody fragment results in more potent target-specific T cell killing and activation. This increase in potency was observed for a discrete configuration in the smaller Fab-scFv format (i.e., format 376) and for all configurations in the larger Fab-Fab format, indicating that positioning to the CD58 arm in the context of more compact immune synapses is crucial for potentiation of T cell activity.

[0457] We next evaluated the activity of the gplOO-targeting soluble TCR engager in trispecific format 376 (SEQ ID NO:24 and 25; see Table 1) using exhausted T cells as effectors. To this end, primary human T cells were subjected to repeated stimulation using CD3 / CD28 beads over a period of 15-16 days (a total of 6 sequential stimulations) prior to evaluation in T cell redirection assays. Co-cultures between exhausted T cells and gplOO-positive (MP41 and A375-PMEL) or gplOO-negative cells (NCIH2452 and A375-WT) were performed in the presence of gplOO-targeting soluble TCR engagers in trispecific (i.e., format 376, co-ligating CD3 and CD58) or its bispecific counterpart (i.e., ligating CD3 only). In co-cultures with gplOO- positive cell lines, the trispecific molecule displayed a substantial increase in activity relative to its bispecific counterpart, with 15-fold increased potency and 2-3-fold increase in the magnitude of IFN-y secretion (FIG. 11). In co-cultures with gplOO-negative cells, IFN-y secretion was observed for the trispecific molecule only, however, this was only the case at high concentrations of engager (starting at 3 nM, compared to 40 pM for gplOO-positive cells) and IFN-y secretion levels that were substantially lower than those observed in co-cultures with gplOO-positive cells (FIG. 12).

[0458] In order to evaluate the activity of trispecific engager across additional parameters, we performed co-cultures with A375-PMEL (gplOO-positive) and A375-WT cells (gplOO-negative) and measured granzyme B secretion and target cell cytolysis, in addition to IFN-y secretion after 48 hours incubation with trispecific or bispecific molecules. We identified a substantial increase of IFN-y secretion (FIG. 13), granzyme B secretion (FIG. 14) and target cell cytolysis (i.e., killing) (FIG. 15) for the trispecific engager in format 376 relative to its bispecific counterpart. Notably, the trispecific molecule displayed low or negligible levels of activity on gplOO-negative A375-WT cells across all readouts, highlighting a safe activity window (FIG. 13, FIG. 14 and FIG.15). Of note, addition of the CD58 ectodomain to the trispecific molecule did not have an impact on affinity to either gplOO-YLE pMHC complex or to CD3E6 heterodimer that its enhanced activity is the direct result of harbouring a third arm engaging CD2 (FIG. 16). This was the case for trispecific molecules incorporating two different linkers (i.e., L7 (SGGGGSGGGGSGGGGSGGGGSGS; SEQ ID NO:9) and Lil (SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGS; SEQ ID NO:22)) connecting the CD58 ectodomain and the TCR alpha chain.

[0459] Overall, this data demonstrates that the combination of CD3 and CD2 co-ligation in trispecific soluble TCR engagers is an effective approach to increase the in vitro potency of this class of therapeutics, both when using fresh and exhausted T cells as effectors, providing translational potential for enhanced clinical activity.

[0460] Example 4: Enhanced activity of soluble TCR trispecific T cell engagers targeting additional antigens

[0461] Having demonstrated increased activity of a soluble TCR engager targeting gplOO, we sought to apply this concept to soluble TCR engagers targeting different antigens. First, we generated trispecific soluble TCR engagers (in format 376) incorporating an affinity-enhanced TCR-Fab arm binding the MAGE-A3i68-i76 peptide (MAGE-EVD) restricted on HLA-A*01:01 (i.e., Glyptodon-4-376 or Glyptodon-4 bispecific). Of note, addition of the CD58 ectodomain to this soluble TCR engager reduced its affinity to MAGE-EVD pMHC 4-5-fold but had no impact on its affinity to the CD3E6 heterodimer (FIG. 17). Despite this reduction in target affinity, coculture assays using fresh T cell effectors and MAGE-A3-positive cells (A375-WT) revealed a modest increase in activity of the trispecific molecule relative to its bispecific equivalent (FIG. 18). This was the case for trispecific molecules incorporating two different linkers (i.e., L3 (SGGGGSGGGGSGS; SEQ ID NO:7) and L7 (SGGGGSGGGGSGGGGSGGGGSGS; SEQ ID NO:9)) connecting the CD58 ectodomain and the TCR alpha chain.

[0462] Notably, co-cultures using exhausted T cell effectors and A375 cells showed a substantial increase in both the potency and magnitude of IFN-y secretion by Glyptodon-376 trispecific molecule compared to bispecific equivalents (FIG. 19). In both sets of experiments, T cell activation in the presence of MAGE-A3 negative cells (HEK-A0101) was low or negligible (FIG. 18 and FIG. 19).

[0463] We next generated a soluble TCR engager trispecific (format 376) using an affinity-enhanced TCR fragment targeting a A*02:01-restricted peptide derived from the oncofetal antigen glypican-3 (GPC3) (i.e., Squid-1-376 or Squid-1 bispecific). For this soluble TCR engager, addition of the CD58 ectodomain had a negligible impact on its affinity to GPC3-A2-MLL pMHC (FIG. 20). Co-cultures of fresh T cells with GPC3-positive cells (ACDC-A2-GPC3) in the presence of trispecific or bispecific versions of this soluble TCR engager demonstrated a higher potency for the trispecific molecule, as measured by a 7-fold improvement in IFN- secretion EC50 values. Similar to previous experiments, control co-cultures with GPC3-negative cells (ACDC- A2) resulted in negligible levels of T cell activation in the presence of trispecific soluble TCR engagers (FIG. 21).

[0464] Overall, this work demonstrates a generalisable approach to enhance the potency of soluble TCR engagers through incorporation of CD3 and CD2 co-ligation in a specific molecular configuration, namely in trispecific format 376.

[0465] Table 1. Sequences of gplOO-376 trispecific polypeptide chains.

[0466] Table 2. Sequences of gplOO-376 trispecific variable TCR domains. Table 3. CDR sequences of gplOO-376 trispecific molecule according to IMGT definition.

Claims

New PCT-Patent ApplicationEngimmune Therapeutics AG c / o Switzerland Innovation Park Basel Area AGVossius Ref.: AJ3536 PCT BSCLAIMS1. A multifunctional molecule comprising i) a soluble T cell receptor (TCR) fragment; ii) a CD3 agonist; and iii) a CD2 agonist.

2. The multifunctional molecule according to claim 1, wherein the soluble TCR fragment is a heterodimeric TCR fragment comprising a first and a second polypeptide chain, in particular wherein the soluble TCR fragment is an a|3-heterodimeric TCR fragment comprising extracellular fragments of a TCR a-chain and a TCR p-chain.

3. The multifunctional molecule according to claim 1 or 2, wherein the soluble TCR fragment has been engineered for increased affinity, specificity and / or stability.

4. The multifunctional molecule according to any one of claims 1 to 3, wherein the soluble TCR fragment has been engineered to comprise one or more artificial disulphide bonds.

5. The multifunctional molecule according to any one of claims 1 to 4, wherein the soluble TCR fragment specifically binds to MAGE-A3, EBNA-1, GPC3, KRAS proto-oncogene neoantigens, TCF-1, AFP or PSA.

6. The multifunctional molecule according to any one of claims 1 to 5, wherein the CD3 agonist is an anti-CD3 antibody, or an antigen-binding fragment thereof.

7. The multifunctional molecule according to claim 6, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv fragment or a nanobody, or wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment.

8. The multifunctional molecule according to any one of claims 1 to 7, wherein the CD2 agonist is a CD2 ligand, an anti-CD2 antibody, an antigen-binding fragment thereof, or a non-antibody binding scaffold.

9. The multifunctional molecule according to any one of claims 1 to 8, wherein the CD2 agonist is a CD58 ectodomain.

10. The multifunctional molecule according to claim 9, wherein the CD58 ectodomain comprises or consists of (i) an amino acids sequence as set forth in any one of SEQ ID NOs:l-4, or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:l- 4, wherein the CD58 domain remains the ability to engage CD2 on the surface of a T cell.

11. The multifunctional molecule according to any one of claims 1 to 10, wherein the soluble TCR fragment is linked via a first polypeptide chain comprised in the soluble TCR fragment to a polypeptide chain comprised in the CD3 agonist.

12. The multifunctional molecule according to any one of claims 1 to 11, wherein the CD3 agonist is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

13. The multifunctional molecule according to claim 12, wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment, or a single chain CD3 agonist, in particular an scFv; preferably wherein the CD3 agonist is an scFv.

14. The multifunctional molecule according to claim 12 or 13, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv, and wherein the C-terminal end of the single chain CD3 agonist is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

15. The multifunctional molecule according to claim 14, wherein the CD2 agonist is linked: a) to the N-terminal end of the single chain CD3 agonist; b) to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment; or c) to a C-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment.

16. The multifunctional molecule according to claim 12 or 13, wherein the CD3 agonist is a heterodimeric CD3 agonist, in particular a Fab fragment, and wherein an N-terminal end of a first polypeptide chain comprised in the heterodimeric CD3 agonist is linked to a C- terminal end of a first polypeptide chain comprised in the soluble TCR fragment.

17. The multifunctional molecule according to claim 16, wherein the CD2 agonist is linked: a) to an N-terminal end of a second polypeptide chain comprised in the heterodimeric CD3 agonist;b) to a C-terminal end of a first or second polypeptide chain comprised in the heterodimeric CD3 agonist; c) to an N-terminal end of a first or second polypeptide chain comprised in the soluble TCR fragment; or d) to a C-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

18. The multifunctional molecule according to any one of claims 1 to 17, wherein the molecule further comprises an antibody Fc region.

19. The multifunctional molecule according to claim 18, wherein the antibody Fc region is derived from a human IgG antibody heavy chain.

20. The multifunctional molecule according to claim 18 or 19, wherein the antibody Fc region is a heterodimeric antibody Fc region.

21. The multifunctional molecule according to any one of claims 18 to 20, wherein the antibody Fc region comprises one or more mutations that reduce immune effector functions.

22. The multifunctional molecule according to any one of claims 18 to 21, wherein a first polypeptide chain comprised in the antibody Fc region is linked to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment or the CD3 agonist.

23. The multifunctional molecule according to any one of claims 18 to 22, wherein the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

24. The multifunctional molecule according to claim 23, wherein the CD3 agonist is linked to an N-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

25. The multifunctional molecule according to claim 24, wherein the CD3 agonist is a single chain CD3 agonist, in particular an scFv.

26. The multifunctional molecule according to claim 24 or 25, wherein the CD2 agonist is linked: a) to an N-terminal end of a polypeptide chain comprised in the single chain CD3 agonist, in particular to the N-terminal end of the scFv;b) to an available N-terminal end of a polypeptide chain comprised in the soluble TCR fragment; or c) to an available C-terminal end of a polypeptide chain comprised in the soluble TCR fragment.

27. The multifunctional molecule according to any one of claims 18 to 26, wherein: a) the antibody Fc region is linked via an N-terminal end of a polypeptide chain comprised in the antibody Fc region to a C-terminal end of a polypeptide chain comprised in the soluble TCR fragment; b) the CD3 agonist, in particular the scFv, is linked to an N-terminal end of a first polypeptide chain comprised in the soluble TCR fragment; and c) the CD2 agonist, in particular the CD58 ectodomain, is linked to an N-terminal end of a second polypeptide chain comprised in the soluble TCR fragment.

28. A nucleic acid encoding the multifunctional molecule of the preceding embodiments.

29. A cell comprising the nucleic acid according to claim 28.

30. A method of producing the multifunctional molecule according to any one of claims 1 to 27, the method comprising a step of culturing the cell according to claim 29.

31. A pharmaceutical composition comprising the multifunctional molecule according to any one of claims 1 to 27, the nucleic acid molecule according to claim 28 and / or the cell according to claim 29, and a pharmaceutically acceptable carrier.

32. The multifunctional molecule according to any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31 for use in medicine, in particularfor use in the treatment of cancer, viral infections or autoimmune diseases.

33. The multifunctional molecule according to any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31 for use in the treatment of cancer in a subject, wherein the subject's cancer is characterized by the presence of exhausted T-cells.

34. The multifunctional molecule according to any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31 for use in the treatment of cancer in a subject, wherein the subject is refractory to, or has relapsed after, a prior immunotherapy.

35. The multifunctional molecule for use according to claim 34, wherein the prior immunotherapy is a checkpoint inhibitor therapy.

36. An ex vivo or in vitro method for reactivating a population of exhausted T-cells, the method comprising the step of contacting a population of T-cells comprising exhausted T-cells with the multifunctional molecule according to any one of claims 1 to 27.

37. A population of reactivated T-cells obtainable by the method of claim 36.

38. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the multifunctional molecule according to any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31, wherein the subject has been identified as having exhausted T-cells prior to administration of the multifunctional molecule.

39. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the multifunctional molecule according to any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31, wherein the subject has previously been treated with and failed to respond to a checkpoint inhibitor therapy.

40. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of the multifunctional molecule of any one of claims 1 to 27, the nucleic acid according to claim 28, the cell according to claim 29 or the pharmaceutical composition according to claim 31, wherein the administration of the molecule reactivates exhausted T-cells in the subject.

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