Engineered allogeneic immune cells

Abstract

The present disclosure relates to engineered immune cells, in particular engineered T cells which are useful in allogeneic cancer therapy.

Description

[0001] ENGINEERED ALLOGENEIC IMMUNE CELLS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to engineered immune cells for cell therapy, in particular cancer cell therapy.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancer remains one of the leading causes of mortality worldwide, with solid tumors presenting significant challenges for effective treatment. Traditional therapies, such as chemotherapy and radiation, often result in severe side effects and limited efficacy. In recent years, cell therapy has emerged as a promising approach to cancer treatment, offering targeted and potentially curative options. Among these, allogeneic T cell therapy has gained attention due to its unique advantages over autologous approaches.

[0006] Allogeneic T cell therapy involves the use of T cells derived from healthy donors rather than the patient's own cells. This approach offers several benefits, including immediate availability, standardized quality, and the potential for large-scale production. Unlike autologous therapies, which require the extraction and processing of the patient's own cells, allogeneic therapies can be prepared in advance and stored for off-the-shelf use.

[0007] However, there is still the need for improved allogeneic cell therapies with optimized genetic engineering for maximal safety and efficacy.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention provides a novel and effective knock-out / knock-in gene engineering strategy for the production of off-the-shelf (allogeneic) cell products, particularly gamma delta (y5 or gd) T cells or alpha beta (a|3 or ab) T cells, which are genetically modified to express multiple heterologous open reading frames (ORFs) to achieve optimized targeting, cloaking, armouring and / or purity. In this context, targeting refers to the introduction of highly efficient T cell receptors (TCRs) or chimeric antigen receptors (CARs) that enhance the cells' ability to recognize and bind to specific targets, such as cancer cells. Cloaking encompasses genetic modifications aimed at minimizing immune recognition and rejection of the transplanted cells by the recipient’s immune system. Armouring entails genetic enhancements designed to improve the cells' functionality, persistence, and resistance to immunosuppressive signals within the tumor microenvironment (TME).

[0010] The gene engineering strategy disclosed herein enables the simultaneous expression of multiple ORFs in immune cells, specifically T cells, to enhance their therapeutic efficacy and overcome challenges typically associated with adoptive celltherapies. Notably, the strategies disclosed herein are, in combination, unprecedented in their application to y5 and a|3 T cells, offering a novel approach with potential advantages in terms of safety, efficacy, and immune evasion.

[0011] Specific advancements in the design of the off-the-shelf (allogeneic) T cell products disclosed herein include the use of carefully optimized nucleic acid constructs, wherein the arrangement of gene cassettes and the selection of promoters are configured to achieve maximal and controlled expression of the encoded sequences. This design enables high-level expression of the transgenic TCR and, in certain embodiments, co-expression of CD8. Further improvements relate to an optimized cloaking strategy, which employs a B2M knockout and the knock-in of a single-chain HLA-E fusion protein engineered to confer enhanced cell-surface expression and effective protection from host-versus-graft immune responses, thereby improving the persistence of the administered cells.

[0012] Additional improvements were implemented for the design of specific T cell products encompassed by the present disclosure. For allogeneic a|3 T cell products, such improvements include knock-out of the endogenous TCR by targeted disruption of the TRAC, TRB1, and TRB2 loci, thereby efficiently preventing mispairing between transgenic and endogenous TCR chains. Furthermore, the transgenic TCR contains a defined mutation in the constant domain that enables selective depletion of residual endogenous TCR chains using an antibody-based enrichment or sorting procedure. Collectively, these genetic modifications yield an efficient, functional, and highly standardized allogeneic a|3 T cell product.

[0013] For allogeneic y5 T cell products, additional design improvements include knock-out of endogenous TGFBR2, thereby rendering the engineered y5 T cells resistant to inhibitory TGF-[3 signaling and enhancing their functional stability and cytotoxic activity within the immunosuppressive tumor microenvironment. Optional cytokine support via membrane-bound interleukins can further enhance their proliferative capacity, persistence, and anti-tumor functionality.

[0014] Taken together, the present disclosure provides improved, genome-engineered allogeneic T cell products with enhanced safety, functionality, manufacturability, and clinical applicability.

[0015] In a first aspect, the present disclosure relates to an immune cell comprising a) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

[0016] Preferably, the immune cell further expresses a peptide that is presented by the fusion protein on the surface of said cell. Said peptide can be covalently linked to atleast a portion of said HLA-I protein, such that the fusion protein does not present any other peptides on the cell surface.

[0017] In the context of the present disclosure, the “portion” of the HLA-I protein preferably comprises or consists of an HLA class I a chain or a variant or derivative thereof. Said HLA class I a chain may be selected from HLA-E, HLA-F and HLA-G.

[0018] In view of the above, embodiments of the present disclosure include a fusion protein comprising or consisting of a HLA class I a chain covalently linked, either directly or via a linker sequence, to a B2M protein and a peptide. The fusion protein may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 or 2 or as shown in Table 2 below. It is envisaged herein that the immune cell comprises a genetically engineered inactivation of all copies of the B2M gene. Preferably, this leads to a B2M knockout, which may refer to minimal or no expression of the endogenous B2M protein.

[0019] The immune cell of the present disclosure may preferably be a primate cell, more preferably a human cell. It is particularly envisaged that the immune cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably an a|3 or y5 T cell.

[0020] The genetically engineered inactivation of the at least one B2M gene in the immune cell may preferably be achieved through insertion of the at least one heterologous nucleic acid sequence(s) into the B2M gene locus. That is, the ORF encoding the fusion protein, and / or the ORF encoding the antigen binding protein, and / or any other ORF described herein, and optionally further heterologous nucleic acid sequences such as regulatory sequences, may be knocked into the B2M gene locus, such that the B2M gene is knocked out.

[0021] The immune cell may further comprise at least one heterologous nucleic acid sequence(s) encoding a CD8 a and / or CD8 [3chain.

[0022] In embodiments of the present disclosure, the at least one nucleic acid sequence(s) encoding said fusion protein and / or the at least one nucleic acid sequence(s) encoding said antigen binding protein and / or the at least one nucleic acid sequence(s) encoding said CD8 a and / or CD8 [3 chain is / are inserted into the B2M gene locus. In this way, said heterologous nucleic acid sequence(s) is / are knocked in and the B2M gene is knocked out.

[0023] The immune cell may further comprise a genetically engineered inactivation of a gene associated with any one of NK cell inhibition, T cell exhaustion, and / or graft-versus-host disease, and / or a gene selected from a TGFBR2 gene, a TRAC gene, a TRBC1 gene, a TRBC2 gene, a CIITA gene, a CISH gene, a PD-1 gene, a CTLA-4 gene, a NKG2A gene, a CBLB gene, a REG1 gene, a PTPN2 gene, a LAG3 gene, a TIM3 gene and / or a FAS gene.In such embodiments, the immune cell preferably comprises a genetically engineered inactivation of all copies of the TGFBR2, TRAC, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3, TRBC1, TRBC2 and / or FAS genes, respectively, depending on the engineering approach. Preferably, said genetically engineered inactivation causes a knockout of the affected genes, i.e. TGFBR2, TRAC, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3, TRBC1, TRBC2 and / or FAS, respectively.

[0024] The immune cell may further comprise at least one heterologous nucleic acid sequence(s) encoding a cytokine or a cytokine fusion protein, said cytokine optionally being selected from interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21. Said cytokines or cytokine fusion proteins may be expressed in soluble or membrane-bound form. In embodiments of the present disclosure, the immune cell comprises a heterologous nucleic acid encoding a membrane-bound interleukin-15 fusion protein. In other words, said immune cell expresses a membrane-bound interleukin-15 fusion protein.

[0025] In view of the above, the present disclosure provides embodiments of the immune cell, wherein the at least one nucleic acid sequence(s) encoding the fusion protein and optionally the at least one nucleic acid(s) encoding the CD8 a and / or [3 chain is / are inserted into the B2M gene locus; and the at least one nucleic acid sequence(s) encoding the antigen binding protein is / are inserted into the TRAC locus. Preferably, in such embodiments the immune cells are a|3 T cells. Said a|3 T cells may further comprise a genetically engineered inactivation, i.e. knockout, of the TRBC1 and TRBC2 loci.

[0026] In view of the above, the present disclosure further provides embodiments of the immune cell, wherein the at least one nucleic acid sequence(s) encoding the fusion protein and optionally the at least one nucleic acid(s) encoding the CD8 a and / or [3 chain is / are inserted into the B2M gene locus; and the at least one nucleic acid sequence(s) encoding the antigen binding protein is / are inserted into the TGFBR2 locus. Preferably, in such embodiments the immune cells are y5 T cells.

[0027] Any of the heterologous protein-encoding nucleic acid sequences described herein may be operably linked to at least one suitable regulatory element, such as a heterologous or endogenous promoter. As such, the at least one nucleic acid sequence(s) encoding the fusion protein, the at least one nucleic acid sequence(s) encoding the antigen binding protein and / or optionally the at least one nucleic acid sequence(s) encoding the CD8 a and / or CD8 [3 chain may be operably linked to a heterologous or endogenous promoter, preferably a B2M, EF1 alpha, EF1alpha-HTLV, MSCV, MND or MND-HTLV promoter.

[0028] In embodiments of the present disclosure, the at least one nucleic acid sequence(s) inserted into the B2M gene locus is / are operably linked to the endogenous B2M promoter. In further embodiments of the present disclosure, the atleast one nucleic acid sequence(s) inserted into the TRAC gene locus is / are operably linked to a heterologous MND or B2M promoter. In further embodiments the at least one nucleic acid sequence(s) inserted into the TGFBR2 locus is / are operably linked to a heterologous MND-HTLV promoter.

[0029] The antigen binding protein preferably comprises six complementarity determining regions (CDR) CDRal, CDRa2, and CDRa3. The antigen binding protein may comprise a first polypeptide and a second polypeptide. The first polypeptide preferably comprises three complementarity determining regions (CDR) CDRal, CDRa2 and CDRa3. The second polypeptide preferably comprises three CDRs CDRbl, CDRb2, and CDRb3. Preferably, said antigen binding construct is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), or a derivative or fragment of a TCR or CAR.

[0030] Accordingly, the first polypeptide preferably comprises or consists of a TCR alpha-chain variable domain (Va) or a derivative or fragment thereof, and / or the second polypeptide preferably comprises or consists of a TCR beta-chain variable domain (V|3) or a derivative or fragment thereof. The first and / or said second polypeptide may further comprise a TCR constant domain.

[0031] It is envisaged that the antigen binding protein is preferably capable of specifically binding to a target peptide presented by an MHC-I molecule. Said target peptide may be a CT45, PRAME, COL6A3, NY-ESO-1, MAGE-A3, KLK3, KLK4, KK-LC-1, MART-1, CEA, HA-1, KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide.

[0032] The heterologous nucleic acid sequence(s) is / are preferably introduced into the immune cell through adeno-associated vectors (AAVs) and a CRISPR / Cas system. Preferably, the CRISPR / Cas system comprises a Cas protein selected from Cas9 or a variant or derivative thereof, or Cas12 or a variant or derivative thereof, more preferably Cas12a (Cpf1) or Cas12i.

[0033] In a further aspect, the present disclosure provides a (pharmaceutical) composition comprising the immune cell, and optionally a pharmaceutically acceptable carrier or excipient.

[0034] In a further aspect, the present disclosure provides a kit comprising the immune cell or (pharmaceutical) composition, and optionally at least one further therapeutic or co-therapeutic agent.

[0035] In a further aspect, the present disclosure provides the immune cell or (pharmaceutical) composition or kit for use in medicine.Specifically, the present disclosure provides the immune cell or (pharmaceutical) composition or kit for use in treating cancer, an autoimmune disease, or an infection.

[0036] In a further aspect, the present disclosure accordingly also provides a method of treating cancer, an autoimmune disease, or an infection, said method comprising administering the immune cell or (pharmaceutical) composition or kit to a patient in need thereof.

[0037] The uses and methods of the present disclosure may further comprise administering said immune cell, (pharmaceutical) composition or kit and, additionally, at least one further therapeutic or co-therapeutic agent or treatment.BRIEF DESCRIPTION OF THE FIGURES

[0038] FIG. 1 shows the successful knock-out of B2M and knock-in of heterologous TCR, CD8 and HLA-E fusion protein (“HLA-E”). To generate engineered y5 T cells, electroporation was used to introduce Cas12a ribonucleoproteins (RNPs), followed by transduction with adeno-associated viral vectors (AAVs) encoding the HLA-E fusion protein, TCR and CD8. (A) Flow cytometric validation of successful gene KO by either direct surface staining of B2M or intracellular staining of phosphorylated SMAD proteins after TGF-[3 stimulation as readout for TGF[3R2 KO. (B) Surface staining of endogenous V52 TCR as well as transgenic a|3 TCR, CD8 and HLA-E on expanded y5 T cell product.

[0039] FIG. 2 demonstrates double knock-in strategies in which either CD8 (“CD8”) or HLA-E fusion protein and CD8 (“HLA-E_CD8”) were successfully expressed from the B2M locus, while transgenic T cell receptors (TCRs) were successfully expressed from either the TRAC locus (FIGS. 2A and 2B) or the TGFBR2 locus (FIGS. 2C and 2D), each under the control of different promoters. Engineered T cells were generated using the protocol described in connection with FIG. 1, involving electroporation and transduction with adeno-associated viral vectors (AAVs), along with Cas12a ribonucleoproteins (RNPs). Flow cytometry (FACS) analysis confirmed successful integration and expression of the knock-in constructs.

[0040] FIG. 3 shows the successful cloaking of y5 T cells against allogeneic rejection.

[0041] (A) Schematic representation of two co-culture setups used to assess successful cloaking against T and NK cells, respectively. B2M KO y5 T cells were generated according to the methods described previously and subsequently co-cultured with both HLA-matched and H LA-mismatched peripheral blood mononuclear cells (PBMCs). HLA negative K562 cells served as a control to confirm NK cell-mediated killing. (B) Fraction of dividing, CD25+ T cells after 4d coculture with expanded y5 T cells of indicated genotype. Values normalized to the HLA-matched reference sample are plotted for CD8+ and CD4+ responders. B2M knock-out cells. Knock-out of B2M reduces activation of CD8+ T cells in an HLA mismatched setting, but had no detectable effect on CD4+ T cell activation, demonstrating selective modulation of immune responses. (C) Exemplary flow cytometry plots after 24h coculture of NK cells (CD3- CD56+) and y5 T cells (CD3+) of indicated genotype. The gate shown was used to count surviving T cells and calculate % killing plotted in (D). Overall, knock-out of B2M reduces activation of CD8+ T cells in an HLA mismatched setting, but induces y5 T cell killing by NK cells in a „missing self" reaction. Knock-in of a HLA-E fusion protein (“HLA-E”) effectively inhibits NK cell recognition and cloaks engineered T cells against NK cell mediated cytotoxicity.

[0042] FIG. 4 shows the effects of TGF-[3 on T cell killing for various engineered y5 T cells. Target cells (SNU475) presenting the TCR target antigen were provided. Engineered T cells without (“WT”) or with TGFBR2 knock-out (“KO”), heterologousTCR, CD8 and HLA-E fusion protein (“HLA-E”) were added at an effector: target (E:T) ratio of 1:1 to the target cells in a serial re-challenge assay: after each round, all effectors were added to a fresh target cell culture in the presence of 5U / ml IL2 and 5ng / ml IL15. Effectors were added either in the presence (“+”) or the absence (“-“)10 ng / ml TGF-[3.. T cell cytotoxicity was assessed using Incucyte (FIG 4A), ATP assay, IFNy ELISA and flow cytometry. FIG. 4B shows that added exogenous TGF-[3 can reduce yd T cell function (light grey curve), the effect can be rescued by TGFBR2 knockout. In addition to cytotoxicity, interferon gamma secretion (FIG. 4C) and y5 T cell proliferation (FIG 4D) were reduced in samples treated with TGF-[3 during coculture, no inhibitory effect is observed on TGFBR2 KO cells.

[0043] FIG. 5 illustrates the cytokine dependence of some engineered y5 T cells in culture and their rescue through membrane-bound IL-15. Engineered y5 T cells, incorporating a transgenic TCR at the TRAC locus and CD8 at the B2M locus, were generated as described previously. Non-transduced (“NTD”) yd T cells served as a control. In the presence of IL-2 and IL-15, the engineered cells showed enhanced proliferation and survival up to day 23 compared to cultures without cytokines (FIG 5A). Annexin V and 7-AAD staining demonstrated that double knock-in (KI) cells underwent apoptosis in the absence of IL-2 and IL-15 (FIG. 5B, C). However, these cells could be rescued by the inclusion of a transgenic membrane-bound IL-15 in one of the knock-in constructs, even under cytokine-free conditions (FIG. 5D). Flow cytometry analysis of a coculture experiment with T2 cells loaded with the TCR target peptide further revealed that mblL15-engineered cells (TCR in TRAC locus, CD8+mblL15 in B2M locus) exhibited comparable cytotoxic activity after prolonged culture with or without cytokine supplementation.

[0044] FIG. 6 illustrates the successful integration and expression of two different constructs encoding transgenic TCR and CD8 in the B2M locus. y5 T cells were engineered as described previously and analysed by flow cytometry.

[0045] FIG. 7 illustrates the successful integration and expression a construct encoding an HLA-E fusion protein (“HLA-E”) and both chains of a transgenic TCR in the B2M locus. y5 T cells were engineered as described previously and analysed by flow cytometry.

[0046] FIG. 8 illustrates the successful integration and expression two different constructs encoding an HLA-E fusion protein (“HLA-E”), CD8a and membrane-bound IL-15 in the B2M locus. y5 T cells were engineered as described previously and analysed by flow cytometry.

[0047] FIG. 9 demonstrates the repeated in vitro tumor cell killing and cytotoxic potential of engineered y5 T cells in target expressing tumor cells. Time-lapse monitoring of RFP-expressing Hs695T cells (A) or SNU-475 cells (B) in coculture with y5 T cells according to the legend below the Figure. Dotted lines indicate transfer of the effector cells onto a fresh plate of pre-seeded target cells. Dots indicate valuesmeasured for individual culture wells. Target cells with low peptide expression (FIG 11 A) and higher target peptide expression (FIG 11 B) are efficiently and repeatedly killed over 10 days of coculture even with low target expression and at low effector: target (E:T) ratios.

[0048] FIG. 10 shows that a single dose of engineered yd T cells can clear a solid tumor in an Hs695T xenograft model. (A) Schematic overview of the experimental protocol. (B) Tumor size measurements of mice treated with 20*106non-engineered yd T cells (grey) or cells from three donors expressing a transgenic TOR, CD8 and HLA-E (dark grey). Values are averages of four mice per experimental group. (C) Comparison of mice treated with the same cell product (engineered yd T cells, donor 1) supported by 3x weekly injections of IL-2 and IL-15 (dark grey) or IL-2 only (light grey). (D) Tumor size curves of mice treated with yd T cells with and without TGFbR2 KO. As the Hs695T tumors do not secrete substantial levels of TGF-[3 the equal performance of both products is expected. Overall, the Figure shows that Engineered yd T cell products from multiple donors can induce complete remission in a solid tumor xenograft model. The engineered cells were independent on IL-15 availability for in vivo functionality, similar results (with a slightly slower kinetic) are achieved with IL-2 injections. Different exemplary knock-in strategies of the TCR show comparable efficacy, demonstrating the flexibility of the platform.

[0049] FIG. 11 illustrates an engineering strategy and corresponding protocol for the generation of allogeneic a|3 T cells. FIG. 11A depicts the overall genetic engineering scheme, which includes both armouring (via TCR, CD8) and cloaking (via HLA-E fusion protein) modifications. In this approach, the endogenous T-cell receptor (TCR) is disrupted, and a transgenic TCR is inserted, optionally together with CD8, preferably by targeted integration into the TRAC locus and additional disruption of the TRBC1 and TBC2 loci. Additionally, the endogenous HLA class I expression is ablated, and an HLA-E fusion protein is introduced, preferably by targeted knock-in at the B2M locus. FIG. 11 B outlines an exemplary workflow for engineering allogeneic a|3 T cells. On day 0, peripheral blood mononuclear cells (PBMCs) are thawed and activated using antibodies against CD3 and CD28. On day 2 / 3, Cas ribonucleoprotein (RNP) complexes are delivered into the activated cells by electroporation, and homology-directed repair templates are supplied in the form of adeno-associated virus (AAV) vectors to enable multiplex genome editing. Following editing, the cells are expanded in cytokine supplemented media and subsequently harvested and cryopreserved on day 10.

[0050] FIG. 12 illustrates the efficient multiplex engineering of allogeneic a|3 T cells, including the targeted knock-in of five open reading frames (ORFs): an a|3 TCR comprising two ORFs, a CD8 heterodimer comprising two ORFs, and an HLA-E fusion protein encoded by a single ORF, all five ORFs inserted into two knockout sites. These genetic modifications are coupled with robust cellular expansion (>100-fold post-editing). FIG. 12A presents the knockout efficiencies achieved at the TRAC locus(upper panels) and the B2M locus (lower panels). Cells are stained for CD3 to detect TCR surface expression and B2M to detect HLA-I surface expression after harvest at day 10. FIG. 12B shows the knock-in efficiencies for the heterologous TCR integrated into the TRAC locus, and for the HLA-E fusion protein together with the heterologous CD8 integrated into the B2M locus. FIG. 12C demonstrates that a strong fold-expansion of the engineered T cells was obtained following multiplex genome editing.

[0051] FIGs. 13 to 19 illustrate an improved engineering concept for allogeneic a|3 T cells, including B2M / TRAC / TRBC1 / 2 triple knockouts and point mutations in the transgenic TCR constant region to increase the quality of the resulting allogeneic a|3 T cell product.

[0052] FIG. 13 illustrates engineered mutations introduced into the transgenic TCR [3-chain constant region (TRBC1 / 2) to enable selective depletion of endogenous T-cell receptors. Sequence diagrams compare the wild-type TRBC epitope with three mutant variants carrying defined amino-acid substitutions within the antibody-binding region of an anti a[3TCR antibody (clone BW242 / 412) that recognizes endogenous TCRs and is used to detect and deplete any residual donor T cells expressing the endogenous TCR repertoire.

[0053] FIG. 14 compares the expression and mispairing of mutated transgenic and endogenous TCR chains. Cells with only mutated transgenic TCR chains are in the left upper quadrant (Q1 ) of each plot. Cells with mutated transgenic, endogenous and mixed / mispaired (transgenic+endogenous) TCR chains are shown in the right upper quadrant (Q2). The right lower panel (Q3) shows cells with endogenous TCRs and mixed / mispaired (endogenous+mutated transgenic) TCR chains. Finally, cells with an endogenous TCR knockout (but no transgenic TCR knock-in) are in the lower left quadrant Q4). Mutant 2 (EWKAK) lead to better expression and less mispairing (mid panel).

[0054] FIG. 14A shows that while >98% of cells in the population express an endogenous TCR (Q2&3), this number drops to 0.44% as the TCRA, TRBC1 / 2 loci are disrupted (Q3). FIG. 14B shows that all three mutated TCRs are expressed from the TRAC locus, but indicates residual mispairing (Q2, Q3).

[0055] FIG. 15 shows that a knockout of both TRAC and TRBC1 / 2 significantly reduces TCR chain mispairing. The knocking out of TRAC and TRBC1 / 2 efficiently disrupts the expression of the endogenous TCR (second panel, Q3). Even the expression of a transgenic (mutant) TCR from the TRAC locus results only in rare mispairing events (third panel, Q2, Q3), which are further reduced by depleting residual, endogenous TCR-expressing cells as shown in FIG. 16.

[0056] FIG. 16 demonstrates the integration of the depletion step into the manufacturing workflow. After engineering and a first expansion phase, the depletionis performed at day 7. The cells are then cultured for three additional days until harvest at day 10. Even rare residual T cells bearing mispaired endogenous+transgenic (mutant) TCRs can be removed from the final T cell product by using the anti a[3TCR antibody (clone BW242 / 412). MACS depletion with the BW242 / 412 antibody clone efficiently removes mispaired TCR-Ts from the repertoire (Q2, Q3), while purely transgenic TCR-Ts are enriched due to their mutated BW242 / 412 binding epitope (Q1 ). The effect is retained even after three additional days of expansion (right panels).

[0057] FIG. 17 depicts recovery of viable cells after depletion (A) and fold-expansion of the edited cell product (B). The data indicate that products that are low in endogenous TCR expression (<0.1% residual endogenous TCR) are obtainable without loss of viability or proliferative capacity using the presented in-process depletion strategy.

[0058] FIG. 18 compares allogeneic ct|3 T-cell products generated with B2M / TRAC Knockout (A) and B2M / TRACTRBC1 / 2 triple knockouts (B) before (upper panels) and after (lower panels) depletion of residual endogenous a[3-TCR-positive cells using BW242 / 412 antibody coupled to magnetic beads. The mutated epitope in the transgenic TCR enables selective identification and depletion of residual donor cells expressing the endogenous TCR or mispaired TCRs comprising the endogenous TCR [3 chain. Optimal yield and purity are achieved with a combination of the triple knockout and point mutations. The resulting T cell product is essentially free of residual donor T cells expressing the endogenous TCR (FIG. 18B, Q2, Q3).

[0059] FIG. 19 illustrates the effect of HLA-E fusion protein expression on the rescue of engineered T cells from natural killer (NK)-cell-mediated cytotoxicity following [32-microglobulin (B2M) knockout. A co-culture assay is shown in which expanded and activated NK cells are incubated with a|3 T cells carrying combinations of B2M knockout, TRAC knockout, TCR knock-in, CD8 knock-in, and HLA-E knock-in modifications (A). The bar graph (B) presents NK-cell-mediated target-cell killing for non-engineered cells, B2M / TRAC-deficient cells, and B2M / TRAC-deficient cells further modified with transgenic TCR and HLA-E fusion protein variant knock-ins. Representative flow-cytometric plots (C) depict proportions of NK and T cells under the coculture assay conditions. The results demonstrate that loss of B2M markedly increases NK-cell killing, whereas introduction of HLA-E fusion protein substantially restores resistance to NK-cell cytotoxicity.

[0060] FIG. 20 shows an in-vitro cytotoxicity assay in which engineered allogeneic a|3 T cells are repeatedly challenged with freshly seeded Hs695T (A) and SNU475 (B) tumor cells expressing a target antigen at high and low copy numbers per cell, respectively. Allogeneic a|3 T cells were engineered to express a transgenic TCR against the target antigen (knocked into the TRAC locus) and a single chain HLA-E fusion protein and CD8a0 heterodimer (knocked into the B2M locus). Further modifications include TRBC1 / 2 knockouts and point mutations in the TCR constantregion as described above. Kinetics are plotted over time for target cells alone, and coculture with engineered allogeneic a|3 T cells from two independent donors at an effector to target ratio of 1 : 1. The curves demonstrate TCR-dependent killing, with both donor-derived allogeneic a|3 T-cell products inducing sustained tumor-cell clearance across multiple challenge rounds, whereas control conditions show continued tumor expansion. The results indicate potent and reproducible cytotoxic activity of the allogeneic a|3 T cells against Hs695T and SNU475 cells.

[0061] FIG. 21 illustrates interferon-y (IFN-y) release by engineered allogeneic a|3 T cells in response to target antigen-expressing tumor cells under repeated challenge conditions. IFN-y concentrations are shown for co-cultures with Hs695T cells and SNU475 cells, each carrying defined levels of target antigen. Bar graphs show responses from allogeneic a|3 T cells across multiple rounds of re-challenge (rounds 1 , 3, 5, and 7), with negative-control conditions included. The data demonstrate robust and TCR-dependent IFN-y secretion by the allogeneic a|3 T-cell products.

[0062] FIG. 22 depicts the dose-dependent, response of engineered allogeneic a|3 T cells to target antigen. Interferon-y (IFN-y) release (A), target cell killing (B) of engineered allogeneic a|3 T cells upon coculture with target cells loaded with increasing concentration of antigenic peptide are quantified. T cells from multiple donors show highly similar, robust response to the target peptide after 24 hours. (C) shows the polyfunctionality of the response of allogenic a|3 T cells to antigen. Upon coculture with target peptide loaded cells for 30 h, a large fraction of effector T cells stains positive for intracellular cytokines as response to antigen mediated activation. (D) shows the corresponding cumulative percentage of cells producing individual cytokines (IL-2, TNFa, IFN-y). The results demonstrate that allogeneic a|3 T cells from multiple donors exhibit multi-cytokine effector responses indicative of polyfunctionality.

[0063] FIG. 23 illustrates the in-vivo antitumor activity of allogeneic a|3 T cells in a mouse tumor model. Tumor volume over time is shown for non-engineered control cells and for allogeneic a|3 T-cell products engineered to express a TCR, CD8ab heterodimer and scHLA-E. The T cells were administered at three dose levels (6x106, 3x106, and 1.5X106cells per mouse). The growth curves demonstrate that all dose levels achieved tumor control compared with the non-engineered control group.

[0064] DEFINITIONS

[0065] Where an indefinite or definite article is used when referring to a singular noun, e.g., “a”, “an” or “the”, does not exclude a plurality, unless something else is specifically stated. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Accordingly, the articles “a”, “an” or “the” preceding an element or component are intended to be non-restrictive regarding the number of instances (i.e. , occurrences) of the element. Therefore, “a”, “an” or “the” is to be read to include oneor at least one, and the singular word form of the element also includes the plural unless the number is obviously meant to be singular.

[0066] The terms “about” or “approximately” mean 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, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, ±15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, for instance within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. The terms “about” and “approximately” also include the concrete value, e.g., "about 50" includes the value "50".

[0067] The term “adoptive cell therapy” (“ACT”') is a type of immunotherapy in which T cells are administered to a patient to treat a disease, such as cancer. In autologous ACT, T cells that have been extracted from a patient are cultured in vitro and are typically genetically modified and are then returned to the same patient for therapy. Comparatively, allogeneic ACT involves T cells isolated and expanded from a donor or donors different from the patient receiving the T cells for therapy.

[0068] The term “administration” of an agent (e.g., host cells or polynucleotides of the disclosure or compositions or formulations comprising such host cells or polynucleotides) to a subject (e.g., a subject in need thereof) includes any suitable route of introducing or delivering the agent to a subject to perform its intended function. Administration of a therapeutic agent and compositions containing same can be performed in one dose or in several doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective routes and means of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, and the disease and subject being treated.

[0069] The term “administered in combination” or “combined administration” or “combination therapy” means that two or more therapeutic agents are administered to a patient at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient.

[0070] “Affinity” is defined, in the context of the present disclosure by the equilibrium binding between the antigen binding protein and its antigen, e.g. the PRAME-004 TCR and the PRAME-004 peptide in a complex with a MHC protein. Affinity is usually expressed as equilibrium dissociation constant (KD).The term “amino acid” refers to one of the 20 naturally occurring amino acids or any non-natural analogues. Preferably, the term “amino acid” refers to one of the 20 naturally occurring amino acids. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0071] “Amino acid mutations” may be deletions, insertions or substitutions.

[0072] The term “amino acid substitution” refers to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. An amino acid can be substituted in a parent or reference sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. In the context of the present disclosure, substitutions (even when they referred to as amino acid substitution) are typically conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.

[0073] Accordingly, a reference to a “substitution at position X” refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position X, and Y and Z are alternative substituting amino acid residue.

[0074] Amino acid substitutions may be conservative or non-conservative. For instance, and specifically in the context of antigen binding proteins, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. An amino acid substitution may also be a post-translational modification of the antigen binding protein and is herein also encompassed.

[0075] A conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class, for example, (non-polar amino acids substituted by other non-polar amino acids.

[0076] A conservative amino acid substitution may be made in accordance with Table 1. Methods for predicting tolerance to protein modification may be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.Conservative Amino Acid substitutions

[0077] Amino acid Substitutions (others are known in the art)

[0078] Ala Ser, Gly, Cys

[0079] Arg Lys, Gin, His

[0080] Asn Gin, His, Glu, Asp

[0081] Asp Glu, Asn, Gin

[0082] Cys Ser, Met, Thr

[0083] Gin Asn, Lys, Glu, Asp, Arg

[0084] Glu Asp, Asn, Gin

[0085] Gly Pro, Ala, Ser

[0086] His Asn, Gin, Lys

[0087] lie Leu, Vai, Met, Ala

[0088] Leu lie, Vai, Met, Ala

[0089] Lys Arg, Gin, His

[0090] Met Leu, lie, Vai, Ala, Phe

[0091] Phe Met, Leu, Tyr, Trp, His

[0092] Ser Thr, Cys, Ala

[0093] Thr Ser, Vai, Ala

[0094] Trp Tyr, Phe

[0095] Tyr Trp, Phe, His

[0096] Vai lie, Leu, Met, Ala, Thr

[0097]

[0098] Table 1

[0099] An antigen binding protein of the present disclosure can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and may include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, [3-phenylserine [3-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a,y-diaminobutyric acid, a,[3-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0100] An antigen binding protein or nucleic acid(s) encoding the antigen binding protein of the present disclosure can be recombinant, isolated, engineered and / or purified.

[0101] The term “analogue” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0102] The term “and / or,” as used herein should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. In other words, the term “and / or” is to be interpreted as encompassing that one or more of the cases it connects may occur. Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0103] The term “antibody” as used herein is meant to include native and engineered antibodies. The term “engineered antibody includes functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

[0104] A “native antibody” comprises two heavy and two light chains, wherein the heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct domains (also referred to as regions). The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four or five domains depending on the antibody isotype; a variable domain (VH) and three or four constant domains (CHi, C 2 and CH3, and optionally CH4, collectively referred to as C ). The variable domains of both light (VL) and heavy(VH) chains determine binding recognition and specificity to the antigen. The constant domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0105] The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are made up of residues that are primarily from the “antibody complementarity determining regions” (CDRs) or hypervariable regions. Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the binding site. CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native antibody binding site. The light and heavy chains of an antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. An antibody antigen binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. “Antibody framework regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of antibody light and heavy chain variable regions that are relatively conserved among different antibodies in a single species. The light and heavy chains of an antibody each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain may be described as (FR1 -L)-(CDR1 -L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain may be described as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody. In the context of the disclosure, CDR / FR in a TCR is determined based on IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Accordingly, amino acid sequences of the CDR1, CDR2 and CDR3 of a given variable chain and the amino acid sequences of the framework regions (e.g. FR1, FR2, FR3, and FR4) are indicated according to said IMGT definition in the herein provided disclosure.

[0106] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule or complex that is capable of being bound by an antigen binding site, wherein said antigen binding site is present in an antigen binding protein, preferably an antigen binding protein of the present disclosure. A target antigen may generally be a protein or antigenic peptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.

[0107] The term “antigen binding protein” (occasionally abbreviated to “ABP”) herein refers to a polypeptide or a complex of two or more polypeptides comprising an antigen binding site that specifically binds to an antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein, and that polypeptide or the two or more polypeptides comprise(s) the CDRs as herein provided, such as CDRal,CDRa3, and optionally CDRa2, and CDRbl, CDRb3, and optionally CDRb2. The two or more polypeptides of the antigen binding protein may be covalently or non-covalently linked together. As used in the context of the present specification, the term antigen binding protein includes antigen binding proteins that comprise fragment(s) of the herein provided TCRs. The herein provided antigen binding proteins may be used in different formats as also described below, such as membrane bound antigen binding proteins, fusion proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins.

[0108] The term includes antigen binding proteins having the overall structure of a TCR, an antibody and / or a chimeric antigen receptor (CAR). The antigen binding protein can comprise TCR-derived CDRs, in particular a variable domain VAcomprising TCR-derived CDRal, CDRa3, and optionally CDRa2, and a variable domain VBcomprising TCR-derived CDRbl, CDRb3, and optionally CDRb2. Antigen binding proteins can comprise a variable domain VAcomprising complementarity determining regions (CDRs) CDRal, CDRa2, and CDRa3, e.g. on a first polypeptide, and a variable domain VBcomprising CDRbl, CDRb2, and CDRb3, e.g. on a second polypeptide, wherein CDRal, CDRa2, CDRa3, CDRbl, CDRb2 and CDRb3 form an “antigen binding domain A”. “Antigen binding domain A” denotes a binding domain that binds to the antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein. For instance, the entire VAdomain and / or the entire VBdomain may be TCR-derived and thus be TCR alpha, beta, gamma or delta variable domains (Va, V|3, Vy or Vb). Preferably, the antigen binding protein may be a TCR or functional fragment(s) thereof, e.g. the variable domains VAand VBof the TCR. In some instances, the antigen binding protein can comprise CDRs and optionally the VAand VBas herein provided, and can comprise further (an) additional domain(s) fused directly or indirectly to VAor VB. The further domains may form (an) additional binding domain(s) or (a) binding site(s). For example and in particular instances, the additional binding domains may form antigen binding domain B. Further binding domains may be comprised that, e.g., form further antigen binding domains, e.g. antigen binding domain C, etc. The additional / further domains comprised in the antigen binding protein may also be a further protein.

[0109] The antigen binding protein thus also includes fusion proteins wherein fragment(s) of the herein provided TCRs further comprise other binding domains. Examples of additional domains comprised in an antigen binding protein of the disclosure that is a fusion protein are listed below.

[0110] The term “antigenic peptide in a complex with an MHC protein”, herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. In particular, the antigenic peptide is located to a “peptide-binding groove” formed by the MHC molecule. A complex of an MHC molecule and an antigenic peptide is herein also referred to as “peptide-MHC complex” or “pMHC complex”. For instance, in the caseof the PRAME antigenic peptide, the complex is also referred to as “PRAME antigenic peptide-MHC complex” or “PRAME:MHC complex”.

[0111] An “antigen-expressing” cancer” (also referred to as an antigen “positive” cancer), is characterized by the over-presentation of the antigenic peptide (e.g., PRAME-004) in cancer cells. Some cancer indications are known to express a certain antigenic peptide. Others reguire testing of the patient. In such cases, a cancer biopsy can be used and the antigenic peptide can be identified using the XPresident® and related methods (according to WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, US 7,811,828, US 9,791,444, and US 2016 / 0187351, the contents of each are hereby incorporated by reference in their entirety.

[0112] An “antigenic peptide presenting cell” or “antigenic peptide:MHC complex presenting cell” refers to a cell that presents on its surface the a certain antigenic peptide (e.g., PRAME-004) in a complex with an MHC molecule. Specifically, the antigenic peptide:MHC complex presenting cell may be a tumor cell, wherein the tumor is preferably a cancer as defined herein. In the context of the present disclosure, an antigenic peptide:MHC complex is “over-presented” on the cell surface of a an antigenic peptide:MHC complex presenting cell, compared to levels of said complex on the surface of cells in normal (healthy) tissue (also referred to as “healthy cells”) or on the surface of control cells loaded with a different antigen presenting peptide or no peptide. By "over-presented" is meant that the antigenic peptide:MHC complex is present at a level at least 2-fold, preferably between 5-fold to 10-fold of the level present in healthy tissue or control cells.

[0113] The term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain instances, the term “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0114] As used herein with respect to a disease, the term “associated with” means that the symptom, measurement, characteristic, or status in guestion is linked to the diagnosis, development, presence, or progression of that disease. As association can, but need not, be causatively linked to the disease. For example, symptoms, seguelae, or any effects causing a decrease in the guality of life of a patient having cancer are considered associated with the cancer and in some instances of the present disclosure can be treated, ameliorated, or prevented by administering the polynucleotides of the present disclosure to a subject in need thereof.

[0115] The term “at least one” herein refers to one or more of the specified elements such as 1, 2, 3, 4, 5 or 6 or more of the specified elements. Thus, as a non-limitingexample, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to 1 , 2, 3, 4, 5 or 6 or more A, with no B present (and optionally including elements other than B); in another embodiment, to 1 , 2, 3, 4, 5 or 6 or more B, with no A present (and optionally including elements other than A); in yet another embodiment, to 1, 2, 3, 4, 5 or 6 or more A, and 1 , 2, 3, 4, 5 or 6 or more B (and optionally including other elements); etc.

[0116] The term “binding affinity” refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay).

[0117] The term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.

[0118] The term “biodegradable” means capable of being broken down into innocuous products by the action of living things.

[0119] The phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and / or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. For instance, a polynucleotide (e.g. mRNA) or antigen binding protein can be considered biologically active if even a only a portion of the polynucleotide or antigen binding protein retains or mimics the desired biological effect.

[0120] The term “(pharmaceutically acceptable) carrier” refers to a type of excipient used to deliver a therapeutic agent to a specific target site in the patient’s body. It acts as a vehicle or medium to transport the therapeutic agent, and it can help to enhance the solubility, stability, and bioavailability of the therapeutic agent. Carriers are usually inert substances that do not interfere with the therapeutic action of the therapeutic agent.The carrier typically serves as a vehicle or medium to transport the therapeutic agent to the target site and ensure its proper distribution and absorption in the body. Examples of pharmaceutically acceptable carriers include solvents, diluents, binders, and lubricants, among others. These carriers are typically selected based on their compatibility with the therapeutic agent and other components of the composition or formulation, as well as their safety and effectiveness in delivering the therapeutic agent to the patient. The use of a pharmaceutically acceptable carrier can help to ensure the stability, efficacy, and safety of the final product. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. In the context of polynucleotides, e.g. mRNAs, pharmaceutically acceptable carriers include nanoparticles, such as an polymeric nanoparticle carrier or an lipid nanoparticle (LNP).

[0121] The terms “coding region” and “region encoding” and grammatical variants thereof, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.

[0122] The terms “codon substitution” or “codon replacement” in the context of sequence optimization refer to replacing a codon present in a reference nucleic acid sequence with another codon. A codon can be substituted in a reference nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a "substitution" or "replacement" at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon.

[0123] The term “complementary” with respect to sequences refers to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some instances, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0124] The term “comprising” is to be interpreted as encompassing all specifically mentioned elements as well optional, additional, unspecified elements. In other words, the term “comprising” does not exclude other elements. For the purposes of the present disclosure, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

[0125] The term “consisting of” refers to the inclusion of exactly one element of a number or list of elements, and preferably excluding more than trace elements of other ingredients other than the element or list of elements.

[0126] The term “consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like.

[0127] The term “contacting” means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a compound means that the mammalian cell and compound are made to share a physical connection. Methods of contacting cells with entities both in vivo and ex vivo are known in the biological arts. For example, contacting a compound and a mammalian cell disposed within a mammal can be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve varied amounts of compound. Moreover, more than one mammalian cell can be contacted by a compound.The term “copy number” refers to the number of antigenic peptide / MHC complexes as defined in the context of the present disclosure that are present on the cell surface of a cell, such as a antigenic peptide / MHC presenting cell, for example a cancer cell, or a healthy cell. Copy numbers of a protein can be determined by a variety of art known methods including FACS analysis of diseased cells with fluorescently labelled antigen binding proteins.

[0128] The term "cytotoxic agent" refers to a compound, molecule, or biological entity that induces cell death or inhibits cell proliferation by disrupting essential cellular processes. Cytotoxic agents may function through various mechanisms, including but not limited to, DNA damage, inhibition of mitosis, induction of apoptosis, disruption of protein synthesis, or generation of reactive oxygen species. Examples include chemotherapeutic drugs (e.g., doxorubicin, paclitaxel), immunotoxins, oncolytic viruses, radionuclides, and targeted payloads in antibody-drug conjugates (ADCs). Cytotoxic agents may be used in therapeutic, diagnostic, or research applications, particularly in the treatment of cancer, autoimmune diseases, or other pathological conditions involving aberrant cell growth.

[0129] The term “delivering” means providing an entity to a destination. For example, delivering a polynucleotide or host cell to a subject can involve administering a composition including the polynucleotide or host cell to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route).

[0130] The term "disease" or "disorder" refers to any condition that would benefit from treatment with the therapeutic agent(s) of the disclosure. The term may include chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question.

[0131] The term “diagnostic agent” as used herein refers to a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other labels known in the art that provide (either directly or indirectly) a signal.

[0132] A “domain” may be any region of a protein or nucleic acid sequence, generally defined on the basis of sequence homologies and often having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions). The terms “domain” and “region” may be used interchangeably herein.

[0133] The term "dosage" or "unit dose" or refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e. , the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinicalstate of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0134] The term “(therapeutically / pharmaceutically) effective amount” refers to a quantity of a therapeutic agent, either alone or on combination with one or more other therapeutic agents, sufficient to treat a disease as defined elsewhere herein. In the context cancer treatment, an effective amount of the therapeutic agent preferably produces at least one desired response, which includes inhibiting the progression of the cancer, either by slowing its progression temporarily, or by stopping its progression permanently. The effective amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the polynucleotide(s) administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease, the time of administration, route of administration, and rate of excretion, exhaustion or degradation of the therapeutic agent; the duration of the treatment; drugs used in combination or coincidental with the specific therapeutic agent employed; and other factors. The skilled artisan will be able to determine the effective amount depending on these and other factors. An effective amount can be administered to a subject in one or more doses. The terms “(therapeutically / pharmaceutically) effective amount” can be used interchangeably with “(therapeutically / pharmaceutically) effective dose”, respectively.

[0135] The term “(therapeutic) efficacy” refers to as a parameter that describes the capability of a therapeutic agent to exert its desired therapeutic effect, e.g. for a host cell expressing an antigen binding protein to kill cancer cells. The efficacy can be determined in a functional assay, for example a live-cell monitoring cytotoxicity assay as described below.

[0136] The term “E:T ratio” refers to the ratio of effector cells (i.e. immune cells, in particular T cells, expressing the antigen binding protein, in particular the TCR) to target cells. In some instances, the E:T ratio corresponds to the seeding ratio, i.e. the ratio of the total number of immune cells, in particular T cells, to target cells. In some instances, the E:T ratio is lower than the seeding ratio. This applies to cases where not all immune cells express the antigen binding protein, i.e. not all immune cells are effector cells, for example due to a low electroporation efficiency. In some instances, the seeding ratio is used as approximation of the E:T ratio. In some instances, the E:T ratio is determined by adjusting the seeding ratio taking into account the electroporation efficiency.In an example of a lactate dehydrogenase (LDH)-release assay, the effector cells are immune cells. These effector cells are co-cultured with tumor cells endogenously expressing and presenting the antigenic peptide and optionally additionally loaded with the antigenic peptide. In some instances, the tumor cells are SKMEL-5 cells, RPMI7951 cells or SCC25 cells. In some instances of the LDH-release assay, the seeding ratio of total immune cells and target cells is 10:1. The efficacy of an effector cell or its antigen binding protein is considered high if in a LDH-release assay as defined above, killing of tumor cells (as determined LDH release) is observed at an E:T ratio of 10: 1. Alternatively, the efficacy of an effector cell or its antigen binding protein is considered high if in a cytotoxicity assay, preferably a LDH-release assay as defined above, the cytotoxic activity of the effector cells against the target cells at the highest concentration of the antigen binding protein tested is at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of the cytotoxic activity of a control toxic reagent. The skilled in the art is aware that the cytotoxic activity can be higher than 100%. This is due to the fact that 100% cytotoxic activity is defined by a “maximum lysis control”, which refers to incubation of the target cells with the toxic reagent. In some instances, the toxic reagent is a detergent, e.g. Triton-Xi 00, Tween-20, Tween-80 or NP-40, that effects lysis of the target cells. In some specific examples, the maximum lysis control comprises adding a 0.9% Triton-X100 solution to the target cell culture. The cytotoxic activity of the toxic reagent, i.e. the number of target cells killed by the toxic reagent is defined as 100%. Since the target cells can still proliferate during the co-culture, the effector cells may eventually kill an even higher number of target cells during the cytotoxicity assay than the toxic reagent killed during the maximum lysis control. In such instances, the calculated cytotoxic activity will be higher than 100%.

[0137] In an example of a cytokine production assay and cytokine release assay, the effector cells are immune cells expressing the antigen binding protein as host cells. These effector cells are co-cultured with cells (either target cells, e.g. tumor / cancer cells, or peptide loaded cells, e.g. T2 cells) with different expression of the antigenic peptide in complex with MHC, such as an HLA protein, for instance HLA-A*02. Preferably, the effector cells and the target cells are seeded, e.g. at a ratio between 10:1 and 1:1. For the cytokine release assay, after a defined time of co-culture, e.g.

[0138] 24-48 hours, preferably about 48 hours, the supernatants of the co-culture (effector cells + target cells) are collected and subjected to a cytokine release ELISA assay, for example IFN-gamma, TNF a, IL-2 and Granzyme B, to determine the amount of cytokine released by the effector cells. In order to determine the efficacy, a cytokine production assay can be applied using target cells, e.g. tumor or cancer cells. Alternatively, the killing of a target cell / cancer cell / tumor cell can be determined in e.g. an LDH assay or a live cell imaging assay. The efficacy of an antigen binding protein may be considered high if the antigen is capable of activating effector cells in a cytokine production assay, in particular if the amount of produced cytokines upon coculture with target cells is at least about 100 pg / ml, at least about 300 pg / ml, preferably at least about 500 pg / ml, more preferably at least about 1000 pg / ml.The term “engineered” or “genetically engineered” in the context of a nucleic acid, protein, cell or organism, means that the nucleic acid, protein, cell or organism has been modified using biotechnological methods. Such genetically modified nucleic acids, proteins, cells or organisms typically do not occur in nature. For instance, genetically engineered nucleic acids, proteins, cells or organisms can be obtained by introducing a DNA vector or an RNA encoding a heterologous gene.

[0139] The term “epitope”, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system. As used herein, the term epitope comprises the terms “structural epitope” and “functional epitope”. The term “structural epitope” refers those amino acids of the antigen, e.g. peptide-MHC complex, that are covered by the antigen binding protein when bound to the antigen. Typically, all amino acids of the antigen are considered covered that are within 5 A of any atom of an amino acid of the antigen binding protein. The structural epitope of an antigen may be determined by art known methods including X-ray crystallography or NMR analysis. The structural epitope of an antibody typically comprises 20 to 30 amino acids. The structural epitope of a TCR typically comprises 20 to 30 amino acids. A “functional epitope” as herein defined is a subset of those amino acids forming the structural epitope and comprises the amino acids of the antigen that are critical for formation of the interface with the antigen binding protein, either by directly forming non-covalent interactions such as H-bonds, salt bridges, aromatic stacking or hydrophobic interactions or by indirectly stabilizing the binding conformation of the antigen and is, for instance, determined by mutational scanning. In the context of the present disclosure, the functional epitope is also referred to as “binding motif”. Typically, the functional epitope of an antigen bound by an antibody comprises between 4 and 6 amino acids. Typically, the functional epitope of a peptide-MHC complex comprises between 2 to 6 or 7 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since MHC I presented peptides typically have a length between 8 to 10 amino acids only a subset of amino acids of each given peptide is part of the functional epitope of a peptide-MHC complex. The epitope, in particular the functional epitope bound by the antigen binding protein comprises or consists of the amino acids of the antigen that are required for formation of the binding interface.

[0140] Term “excipient” refers to a substance that is added to a therapeutic agent to facilitate its manufacturing, administration, stability or therapeutic efficacy. Excipients are typically inactive substances that are used, inter alia, as carriers, solvents, fillers, binders, disintegrants, lubricants, or flavoring agents.

[0141] The term "expression system" means a host cell and compatible expression vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[0142] An “expression vector” refers to vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capableof effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0143] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.

[0144] “Fluorescent molecules” are known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or uv laser (e.g. Pacific blue, pacific orange).

[0145] A “format” in relation to an antigen binding protein relates to a defined spatial arrangement of domains, in particular of variable and optionally constant domains. Characteristics of such antigen binding protein formats are the number of polypeptide chains (single chain, double chain or multiple chains), the type and length of linkers connecting different domains, the number of variable domains (and thus the number of valences), the number of different variable domains (and thus the number of specificities for different antigens, e.g. bispecific, or multispecific), and the order and orientation of variable domains (e.g. cross-over, parallel).

[0146] The term "formulation" refers to a specific therapeutic product that has been developed with a particular set of ingredients, dosage form, and delivery method to meet specific therapeutic needs. It is a final product that has typically undergone several steps of formulation development, such as selecting the appropriate ingredients, optimizing the drug delivery system, and ensuring its safety and efficacy. Different formulations of the same therapeutic agents may have different pharmacokinetic and pharmacodynamic properties, and may be used to treat different conditions or patient populations.

[0147] A "fragment," as used herein, refers to a portion of a nucleic acid or protein. For example, fragments of proteins can comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. A fragment may be a subsequence of a full-length protein wherein N-terminal, and / or C-terminal, and / or internal subsequences have been deleted. Preferably, fragments of a protein are functional fragments (i.e.,exhibit or mimic the protein’s desired biological activity), or exhibit a novel desired biological activity.

[0148] The term “free of”, as used herein, means not comprising the referenced component. For example, when a composition, solution, or formulation is described as being “free of X”, the composition, solution, or formulation does not comprise X.

[0149] In a “functional assay”, an antigen binding protein is, for example, expressed in an “effector cell (E)”, and the effector cell is co-cultured with “target cells (T)”, i.e. with antigen presenting cells presenting a peptide:MHC complex. Functional assays can thus also be described as “co-culture assays”. For all cell culture assays described herein, the cell culture temperature preferably is at about 37°C. Preferably, the effector cell is a T cell. The target cells may be cells that are artificially loaded with the antigenic peptide (e.g. T2 cells) or may be cells that endogenously present the target antigenic peptide on their surface (e.g. cancer cells expressing PRAME). Binding of the antigen binding protein to the peptide:MHC complex leads to activation of the effector cell. Depending of the type of functional assay, there are different readouts for measuring the degree of activation. In a cytokine production assay or cytokine release assay, such as an ELISA, the production of cytokines (e.g. TNF-a, IFN-y, CD107a+, IL-2 and / or Granzyme B) by the effector cells is determined. In a cytotoxicity assay, the killing of target cells by the effector cells is determined, e.g. by measuring a decline in proliferation of target cells, in particular cancer cells or by measuring the release of intracellular proteins from the target cells. Suitable intracellular proteins to be measured in a cytotoxicity assay can be endogenous proteins, e.g. LDH release assay.

[0150] “Functional avidity” is defined, in the context of the present disclosure, as a parameter that describes the capability of an antigen binding protein to activate an effector cell, preferably a T cell, upon binding to its target antigenic peptide in a complex with MHC. The activation of the effector cell, preferably T cell, can be measured in a functional assay, e.g., a cytokine production assay or a cytotoxicity assay as described below. In some embodiments, the functional avidity of an antigen binding protein is considered high if the EC50determined in a functional assay is low, such as less than about 50 nM, less than about 20 nM, or less than about 5 nM in a cytotoxicity assay as described below, and / or the activity determined in a functional assay is high, such as at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of a maximal activity defined in the respective functional assay. Depending on the functional assay, the maximal activity may be the activity of a reference protein with known high functional avidity or the activity of a “maximum lysis control” as described below.

[0151] The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such aspromoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0152] “Half maximal effective concentration” also called “ECso”, typically refers to the concentration of a molecule, which induces a response halfway between the baseline and maximum after a specified exposure time. EC50and affinity are inversely related, the lower the EC50value the higher the affinity of the molecule.

[0153] The term “heterologous” refers to an element, such as a nucleic acid sequence, a gene or a protein that is foreign to its genomic location and / or host in that it originates from a different cell, genomic location, organism or species. The term is, inter alia, used in the context of “heterologous” gene expression, which is typically achieved through recombinant DNA technology. Essentially, a gene from one cell or organism is optionally engineered and inserted into a different cell or organism, which his then also referred to as a “recombinant” cell or organism. A “heterologous” protein is produced in a system (e.g., a cell) different from their native host and is also referred to as a “transgenic” protein herein.

[0154] The term “HLA-A*02” signifies a specific HLA allele, wherein the letter A signifies the allele and “*02” indicates the A2 serotype.

[0155] The term "homology" refers to the overall relatedness between polymeric molecules, e.g., between polynucleotides (e.g., DNA molecules and / or RNA molecules) and / or between (poly-)peptides. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity. As used herein, polymeric molecules may be considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0156] A “host cell” is a cell that comprises a heterologous nucleic acid, gene or protein. Specifically, a host cell can express a heterologous nucleic acid. The host cell may be transfected, infected or transduced or transformed, in particular with nucleic acid(s) and / or a vector(s) as described herein. Host cells include immune cells and other types of cells.The term "human," when referring to an antigen binding protein, such as a TCR, or any component of a TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, alpha chain, and / or beta chain), means a TCR (or component thereof), which is derived from a human unrearranged corresponding gene locus, such as a TCR locus, respectively. Similarly, the term “murine” refers to an antigen binding protein (or a component thereof) which is derived from a corresponding murine unrearranged gene locus, respectively.

[0157] The term “humanized” in the context of an antigen binding protein, such as a TCR or antibody refers to an antigen binding protein which is completely or partially of non-human origin and which has been modified by replacing certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antigen binding protein are mainly human CHand CLdomains. Numerous methods for humanization of an antibody sequence are known in the art. For example, a “humanized” antigen binding protein can be made by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations, see, e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and the review by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR grafting, which, for example, involves grafting of the CDR sequences of a donor antibody, generally a mouse antibody, into the framework scaffold of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues. Another alternative technique is known as “guided selection” (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive from for example a murine or rat antibody a fully human antibody conserving the epitope and binding characteristics of the parental antibody. A further method of humanization is the so-called 4D humanization. The 4D humanization protocol is described in the patent application US20110027266 A1 (the content of which is incorporated by reference in its entirety) (W02009032661A1) and is exemplified in the following applying the 4D humanization to humanize the rat antibody variable light (VL) and heavy (VH) domains. Such techniques are equally applicable to other antigen binding proteins, such as TCRs, mutatis mutandis.

[0158] For chimeric antigen binding proteins, such as antibodies or TCRs, humanization typically involves modification of the framework regions of the variable region sequences. Knowing the amino acid sequence of the CDRs an antigen bindingprotein of the disclosure, e.g., a TCR, one skilled in the art can easily determine the framework regions, such as the TCR framework regions. In cases where the CDRs are not indicated, the skilled in the art can first determine the CDR amino acid sequences based on the IMGT definition for TCRs and then determine the amino acid sequences of the framework regions.

[0159] The term “immunoglobulin (Io) domain” refers to a protein domain that consists of a 2-layer sandwich of 7-9 antiparallel [3-strands arranged in two [3-sheets with a Greek key topology. Proteins containing Ig domains are subsumed into the immunoglobulin superfamily, including e.g. antibodies, T cell receptors (TCRs) and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

[0160] The term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0161] The term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0162] The term “isolated” refers to the separation of a specific molecule or entity from its natural environment or context. For nucleic acids, isolation can refer to the separation of a specific DNA or RNA sequence from the rest of the naturally occurring genomic material. This can be done through techniques such as PCR, gel electrophoresis, or hybridization. An “isolated” nucleic acid can be present in solution, or in a host cell. For polypeptides, isolation can refer to the purification of a specific protein from a complex mixture of proteins or cell culture components. This can be done using techniques such as chromatography, electrophoresis, or immunoaffinity purification. For host cells, isolation can refer to the removal of a specific cell type from a mixed population of cells. This can be done using techniques such as fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or microfluidic cell sorting.

[0163] “Kp” is the equilibrium dissociation constant, a ratio of kOff / kOn, between an antigen binding protein and its antigen. KDand affinity are inversely related. The KDvalue relates to the concentration of an antigen binding protein and the lower the KDvalue, the higher the affinity of an antigen binding protein. The KDvalue can be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates with surface plasmon resonance (SPR) or bio-layer interferometry (BLI). For example, an antigen binding protein can be produced as soluble molecule, for instance, by removing the transmembrane domain and introducing an artificial disulfide bridge (Boulter et al. 2003; Stable, soluble T-cell receptor molecules for crystallization and therapeutics; Protein Engineering vol. 16 no.

[0164] 9 pp. 707-711) or dimerization via a leucin zipper domain. (Willcox et al. 1999; TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface; Immunity, Vol. 10,357-365). Binding interactions can be measured at a broad range of settings, including, but not limited to, a temperature range of 25°C to 37°C and a shake speed range of 500 rpm to 1500 rpm using a suitable buffer that minimizes nonspecific binding and maintains protein stability. Examples of such buffers are phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS), or other physiological buffers, with or without additives such as Tween, BSA, DMSO, or EDTA. The analyte can be immobilized on various sensors at a concentration range, including, but not limited to, 1 pg / ml to 100 pg / ml for a duration range of 30 s to 300 s. KDdetermination can be measured at various molarities of the analyte sample for detecting potential off-target reactivities with high sensitivity. Exemplary determination of the KD is herein provided in the Examples. For example the KD may be determined by the following bio-layer interferometry (BLI) method: using a 384-well tilted bottom microplate, black and loading with 50 pg / ml pMHC (100 pl / well). The antigen binding protein can then be added, e.g. as soluble TCR in 7 concentrations as well as a reference well (only HEPES / Tween): 50 pM; 25 pM; 12,5 pM; 6,25 pM; 3,1 pM; 1,6 pM; 0,8 pM with 60 pl / well. The association of off-target TCRs can be against NYES01-001 at 25pM TCR concentration. Then, the dissociation of soluble TCR can be 100 pl / well HEPES / Tween. The measurements can be conducted by 16 streptavidin sensors: duplicates measured with different sensors (sensor offset: 3).

[0165] A “linker”, as long as it is not specified otherwise in the respective context, can be from at least 1 to 30 amino acids in length. For instance, a linker can be 2-25, 2- 20, or 3-18 amino acids long. In some instances, a linker can be a peptide of a length of no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In other instances, a linker can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids long. In other instances, a linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In a particular instance, a linker can be less than 24, less than 20, less than 16, is less than 12, less than 10, for example from 5 to 24, 10 to 24 or 5-10 amino acid residues in length. In some instances, said linker is equal to 1 or more amino acid residues in length, such as more than 1 , more than 2, more than 5, more than 10, more than 20 amino acid residues in length, more than 22 amino acid residues in length. In preferred instances, the linker is an alanine linker, i.e. a linker consisting of or essentially consisting of one or more alanine residues. In some instances, the linker is a single alanine linker. In some instances, a linker comprises or consists of 2 alanine residues. In some instances, a linker comprises or consists of 3 alanine residues.

[0166] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of nonlimiting example, B cells, T cells, NKT cells, and NK cells.

[0167] The “Major Histocompatibility Complex” (MHC) is a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules invertebrates, which in turn determines histocompatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or just HLA). Thus, the terms “MHC” and “HLA” can be used interchangeably. The MHC gene family is divided into three subgroups: class I, class II, and class III. Complexes of peptide and MHC class I molecules (MHC I) are usually recognized by CD8-positive T cells (CD8+ T cells) bearing the appropriate T cell receptor (TCR), whereas complexes of peptide and MHC class II molecules (MHC II) are usually recognized by CD4-positive helper-T cells (CD4+ T cells) bearing the appropriate TCR. CD4 and CD8 usually function as coreceptors of a TCR in binding to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptide and MHC I are recognized by CD8-negative (in particular CD8-negative, CD4-positive) T cells (Soto et al., 2013, Cancer Immunol Immunother.

[0168] 2013 Feb; 62(2): 359-369). Since the responses of CD8-positive and CD4-positive T cells contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens and corresponding T cell receptors is important in the development of cancer immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger, a-chain, constituent of HLA-A. Variation of HLA-A a-chain is key to HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of certain structures, greater variety of HLAs means greater variety of antigens to be 'presented' on the cell surface. The MHC class I HLA protein in the context of the present disclosure may be an HLA-A, HLA-B or HLA-C protein, suitably HLA-A protein, for example HLA-A*02. In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T cells bearing specific T cell receptors (TCR).

[0169] The term “messenger RNA” or “mRNA” refers to any polynucleotide (a ribonucleic acid) which encodes a (poly-)peptide of interest and which is capable of being translated to produce the encoded (poly-)peptide of interest in vitro, in vivo, in situ, or ex vivo. Typically, the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail.

[0170] The term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Nucleotides are referred to by their commonly accepted singleletter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.The term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a (poly-)peptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0171] As used herein, “operably linked” with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a (poly-)peptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0172] Herein a phrase of the form "optionally substituted X" is intended to be equivalent to "X, wherein X is optionally substituted". It is not intended to mean that the feature "X" per se is optional.

[0173] As used herein, a "part" or "region" of a polynucleotide or polypeptide is defined as any portion of the polynucleotide or polypeptide that is less than the entire length of the polynucleotide or polypeptide, respectively.

[0174] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0175] The terms "pharmaceutical composition" as used herein refers to a mixture or formulation of one or more therapeutic agent(s), typically along with one or more excipients, which are added to the formulation to aid in its manufacture, stability, or administration. The pharmaceutical composition is typically designed to deliver the therapeutic agent(s) in a safe, effective, and convenient manner for the treatment or prevention of a particular disease or medical condition. Pharmaceutical compositions can take various forms, such as tablets, capsules, injections, creams, ointments, or inhalers, and can be administered by various routes, including oral, topical, intravenous, intramuscular, or inhalation. The composition may need to meet regulatory requirements for safety, efficacy, and quality before it can be approved for marketing and distribution to patients."Pharmaceutically acceptable" refers to the suitability of a therapeutic agent for use in a pharmaceutical formulation, which typically means that the substance is safe, effective, and compatible with the other ingredients in the formulation. A therapeutic agent that is considered "pharmaceutically acceptable" may need to meet certain criteria, including purity, stability, and absence of harmful impurities or contaminants, and it must not cause any adverse effects on the patient's health. In addition, the substance may need to be able to perform its intended function within the formulation and be compatible with the manufacturing process, packaging, and storage conditions.

[0176] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides encoding antigen binding proteins are typically provided as part of vectors. In some instances, nucleic acids may be isolated nucleic acids. In some instances, nucleic acids may be a recombinant or heterologous nucleic acids. In some instances, nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form.

[0177] As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some instances, the polymeric lipid is a lipid comprising a polymer component. In some instances, the polymeric lipid is a PEG lipid. In some instances, the polymeric lipid is not a PEG lipid. In some instances, the polymeric lipid is Brij or OH-PEG-stearate.The term "prevent" refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0178] The terms "prophylactic" or “prophylaxis” refer to a therapeutic agent or measure used to prevent the spread of disease.

[0179] The term “proliferative diseases refers to a type of disease characterized by an uncontrolled or abnormal growth of cells, which can lead to the formation of tumors or other forms of abnormal tissue growth. This growth can occur in various parts of the body. Examples of proliferative diseases include cancer, benign tumors, and conditions such as hyperplasia and dysplasia. The causes of proliferative diseases can vary, but they may be related to genetic mutations, exposure to toxins or radiation, or other environmental factors.

[0180] The terms "protein" and “polypeptide” are used interchangeably herein to refer to polymers of amino acids of any length. A protein can be a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it can be produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The protein can be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, proteins containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.The term "purified" in the context of nucleic acids, polypeptides, and host cells, refers to the separation of a specific molecule or entity from its (natural) environment or context. Purification is a process of removing impurities, contaminants, or unwanted molecules to obtain a more homogeneous or highly concentrated form of the molecule or entity of interest. “Purified” thus denotes a degree of separation that is higher than isolation. Isolation can be the first step in the purification process, but it does not necessarily imply that the molecule or entity has been completely purified. A “purified” or “biologically pure” nucleic acid, polypeptide, or host cell is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the nucleic acid, polypeptide, or host cell. That is, a nucleic acid, polypeptide or host cell is purified if it is substantially free of cellular material, viral material, culture medium or other supplements and additives.

[0181] For nucleic acids, purification can involve removing proteins, lipids, and other cellular debris to obtain a highly concentrated and pure DNA or RNA sample. This can be done using techniques such as column chromatography, gel electrophoresis, or spin column purification.

[0182] For polypeptides, purification can involve removing other proteins, nucleic acids, or contaminants to obtain a highly purified form of the polypeptide of interest. This can be done using techniques such as affinity chromatography, size exclusion chromatography, or ion exchange chromatography.

[0183] For host cells, purification can involve removing dead cells, debris, and other unwanted materials to obtain a highly purified population of cells. This can be done using techniques such as density gradient centrifugation, magnetic separation, or fluorescence-activated cell sorting (FACS).

[0184] Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0185] The term “radioactive isotope” or “radioactive molecule” is intended to include radioactive isotopes suitable for treating cancer, such as At211, Bi212, Er169, I131, I125, Y90, In111, P32, Re186, Re188, Sm153, Sr89, and radioactive isotopes of Lu. Such radioisotopes generally emit mainly beta-radiation. For instance, the radioactive isotope can be an alpha-emitter isotope, more precisely Thorium 227 which emits alpha-radiation. The term also includes spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.The term "recombinant" refers to any nucleic acid, protein, cell or organism that has been genetically engineered using molecular biology techniques, such as through the artificial modification, insertion, or alteration of genetic material. A “recombinant” nucleic acid, protein, cell or organism is not naturally occurring. A “recombinant” cell can be modified to express a heterologous protein.

[0186] The term "reference sequence" refers to a starting nucleic acid or amino acid sequence that can be sequence optimized. A reference nucleic acid sequence may be a wild-type nucleic acid sequence, a fragment or a variant thereof, or a previously sequence optimized nucleic acid sequence.

[0187] As used herein, “regulatory sequence” of a nucleic acid molecule means a cisacting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0188] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EF1 -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. For instance, the promoter can be a constitutive promoter. The term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus 40 (SV40) promoter, a polyubiquitin C (UBC) promoter, an EF1 -alpha promoter, a PGK promoter and a CAG promoter. Alternatively, the promoter can be a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. The conditional promoter may be an immune cell specific promoter, which allows for continual transcription of the codingsequence or gene in an immune cell. Non-limiting examples of the immune cell specific promoters include a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-|3 gene promoter, a WASP gene promoter, a T-cell receptor [3-chain gene promoter, a V9 y (TRGV9) gene promoter, a V25 (TRDV2) gene promoter, and the like.

[0189] Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0190] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0191] The term “safety profile” herein refers to the capacity to distinguish tumor cells from healthy tissue cells and this is often determined by determining the safety window.

[0192] The term “safety window” or “therapeutic window” herein refers to a factor that compares the half maximal concentration of a compound that is required for inducing 100% cytotoxicity in a tumor cell line in comparison to the half maximal concentration of a compound that is required for inducing 100% cytotoxicity healthy tissue cells. If for an antigen binding protein of interest the EC50determined for a tumor cell line is 1 pM and the EC50value determined for, for instance, primary cells is 1000 pM then the safety window is 1000 since the EC50 for the tumor cell line is 1000 times smaller than the EC50for the primary cells.

[0193] The term "sample" or "biological sample" refers to a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further can include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which can contain cellular components, such as proteins or nucleic acid molecule.

[0194] The term "stable" when used in the contex of compounds or compositions refers to a compound that is sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and in some cases capable of formulation into an efficient therapeutic agent.

[0195] The term "stabilize," "stabilized," "stabilized region" means to make or become stable.

[0196] The term “sequence optimization” refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties, e.g., improved protein expression or decreased immunogenicity of the nucleic acid itself.

[0197] In general, the goal in sequence optimization is to produce a synonymous nucleotide sequence than encodes the same polypeptide sequence encoded by the reference nucleotide sequence. Thus, there are no amino acid substitutions (as a result of codon optimization) in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the reference nucleotide sequence. In some instances, codon optimization is achieved by modification of a coding sequence according to at least one of the following; (i) replacing the naturally occurring codon sequence with an alternative codon that retains the amino acid sequence encoding the protein but alters the composition and / or structure of the encoding RNA; (ii) adjusting the guanosine cytosine content of the coding sequence relative to the naturally occurring guanosine cytosine content of the coding sequence; (iii) adjusting the number of CpG sites of the coding sequence relative to the number of CpG sites in the naturally occurring coding sequence; (iv) substituting the naturally occurring codon sequence with an alternative codon relative to (ii) guanosine cytosine content and / or (iii) number of CpG sites. Codon optimization may include adjusting codons in the context of tRNA expression in a particular tissue and / or may include methods for evading the effects of natural, tissue-specific shRNAs or miRNAs.

[0198] The term “sequential” therapeutic use refers to administration of at least two therapeutic agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the therapeutic agents before administration of the other or others commences. It is thus possible to administer one of the therapeutic agents over several minutes, hours, or days before administering the other therapeutic agent or ingredients. There is no simultaneous treatment in this case.

[0199] As used herein, the phrases "signal sequence," "signal peptide," and "transit peptide" are used interchangeably and refer to a sequence that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence polypeptide and the nucleic acid sequence encoding the signal sequence. Thus, references to a signal sequence in the context of a nucleic acid refer in fact to the nucleic acid sequence encoding the signal sequence polypeptide.The term “sequence identity” refers to a measure of the similarity between two biological sequences, such as polynucleotide or polypeptide sequences. It is expressed as a percentage of the number of identical positions in the two sequences divided by the total number of positions. In other words, sequence identity indicates how much two sequences match each other in terms of the order and composition of their building blocks, such as nucleotides or amino acids. If the two sequences to be compared are not of equal length, they can be aligned to give the best possible fit, allowing the insertion of gaps or alternatively, truncation at the ends of the nucleic acid sequences or amino acid sequences. The skilled person will acknowledge that various means for comparing sequence identity are available (see below). A higher sequence identity may indicate a closer evolutionary relationship between the two sequences, or a higher degree of functional similarity or homology. Sequence identity is commonly used in bioinformatics and molecular biology to compare and analyze the structure and function of biological molecules, such as proteins, and to infer their evolutionary history and relationships.

[0200] For example, in the context of the present disclosure, a sequence that is “at least 85% identical to a reference sequence” may be a sequence having, over its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of a reference sequence (e.g., a variable domain disclosed herein). Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% Identical” to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence.

[0201] In the context of the present disclosure, the sequence identity can be calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle” program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol.

[0202] 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides or polynucleotides, in accordance with the disclosure, can be calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

[0203] In the context of the present disclosure “similar peptides” herein refers to potential off-target peptides, i.e. peptides that may potentially be bound by the antigen binding proteins of the disclosure based on their biochemical / biophysical characteristics, including but not limited to a homologous sequence or a similar motif.Similar peptides comprise typically 8 to 12 amino acids in length. The similar peptides in the context of the present disclosure are typically MHC, in particular MHC I, presented. Furthermore, similar peptides in the context of the present disclosure include peptides that comprise or consist of an amino acid sequence that is similar to the amino acid sequence of the PRAME antigenic peptide, more particular, peptides that, in comparison to the epitope of the PRAME antigenic peptide, comprise an epitope wherein some or all amino acids have identical and / or similar biochemical / biophysical characteristics as the amino acids that constitute the epitope of the PRAME antigenic peptide. In some examples, similar peptides investigated in the context of the present disclosure were selected from a database of tumor and normal tissue-presented HLA-A*02 bound peptides (XPRESIDENT® database) using a similarity scoring within the binding-relevant positions of PRAME and the requirement of at least one detection on normal tissues. Binding of an antigen binding protein to a similar peptide presented by an MHC protein may lead to adverse reactions. Such adverse reactions may be “off-tumor” side effects, such as crossreactivity of a specific TCR with a similar peptide in healthy tissues as reported in Lowdell etal., Cytotherapy, published on December 4, 2018).

[0204] In particular, the “similar” peptides disclosed in WO2018172533 A1 are similar peptides in the context of the present disclosure.

[0205] The skilled person is aware that among the similar peptides, there are some that are not bound by the antigen binding proteins of the disclosure to a detectable degree, e.g. peptides for which no binding signal during affinity determination or no response in a functional assay beyond the background level is detectable. “Background level” in this context refers to a response in a functional assay observed for the co-culture of target cells and effector cells at the respective E:T ratio without the addition of bispecific TCR-antibody fusion protein.

[0206] For other similar peptides, a low, but non-significant binding may be detectable. These latter similar peptides may also be described as "potentially relevant" similar peptides. An antigen binding protein is considered to not significantly bind to a similar peptide and to be specific for its target antigenic peptide if at least one of the following applies when binding to the similar peptide and the target antigenic peptide is compared under similar, preferably identical experimental conditions:

[0207] The functional avidity in response to the similar peptide, determined in a functional assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the functional avidity in response to the target antigenic peptide.

[0208] The cytotoxic activity in response to the similar peptide, determined in a cytotoxicity assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the cytotoxic activity in response to the target antigenic peptide.The EC50of the similar peptide, determined in a functional assay, preferably a cytotoxicity assay, as described above, is increased by a factor of at least 50, at least 100, at least 200 or at least 500, compared to the EC50of the target antigenic peptide.

[0209] The KDfor the similar peptide is increased by a factor at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100, compared to the KDfor the target antigenic peptide.

[0210] The relative response signal for the similar peptide is not higher than 30%, not higher than 25%, not higher than 20%, or not higher than 15%, compared to the response signal to the target antigenic peptide.

[0211] The term “simultaneous” therapeutic use refers to the administration of at least two therapeutic agents by the same or different routes and at the same time or at substantially the same time.

[0212] A "single unit dose" is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0213] The term “specificity” or “specific binding” or “specifically binds” or “specifically targets” refers to the ability of an antigen binding molecule such as a TCR to recognize and bind to a single target (antigen) while avoiding interactions with unrelated targets. A specific TCR binds only to its intended target and does not or not substantially crossreact with other proteins or similar epitopes. For instance, the term "specificity" or "antigen specificity" or "specific for" a PRAME-004 peptide in the context of TCRs means that the TCR can specifically bind to said PRAME-004 peptide, more preferably with high avidity, when said PRAME peptide is presented by HLA, preferably by HLA A2. Typically, an antigen binding protein is considered “specific” for a target peptide, if binding to the target peptide presented in an MHC molecule occurs with a significantly higher affinity and / or higher functional avidity than the binding to similar peptides. The specificity of the antigen binding protein is determined by the amino acid seguences CDRal, CDRa3, CDRbl and CDRb3. The amino acid seguences of CDRa2 and CDRb2 contact the MHC molecule and may in some instances not be reguired for antigen specificity. Therefore, the term “specifically binds” or “specifically binds to” or “specifically target” can be exhibited, for example, by a molecule having a Kd for the molecule to which it binds to of about 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, IO’9M, IO’10M, IO’11M, or 10’12M.

[0214] The term “specific delivery,” “specifically deliver,” or “specifically delivering”, in particular in the context of polynucleotides (e.g., mRNAs) means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen). The level of delivery of a nanoparticle to a particular tissuecan be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue. For example, for renovascular targeting, a polynucleotide is specifically provided to a mammalian kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more polynucleotide per 1 g of tissue is delivered to a kidney compared to that delivered to the liver or spleen following systemic administration of the polynucleotide. It will be understood that the ability of a nanoparticle to specifically deliver to a target tissue need not be determined in a subject being treated, it can be determined in a surrogate such as an animal model (e.g., a rat model).

[0215] The term “stable expression” or “stably expressed refers to the sustained and reproducible production of a nucleic acid, RNA, or protein in a host cell, organism, or system over multiple generations, passages, or extended periods without the need for continuous transduction. Stable expression may result from genomic integration of the nucleic acid sequence, the use of self-replicating vectors, or other mechanisms that allow for long-term maintenance and inheritance of the expressed sequence. The term includes expression in prokaryotic or eukaryotic systems, whether natural, engineered, or recombinant.

[0216] The term “start codon”, used interchangeably with the term “initiation codon”, refers to the first codon of an open reading frame that is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases. The initiation codon is depicted by the first letter codes of adenine (A), uracil (U), and guanine (G) and is often written simply as “AUG”. Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as “alternative initiation codons”, the initiation codons of polynucleotides described herein use the AUG codon. During the process of translation initiation, the sequence comprising the initiation codon is recognized via complementary base-pairing to the anticodon of an initiator tRNA (Met-tRNAiMet) bound by the ribosome. Open reading frames may contain more than one AUG initiation codon, which are referred to herein as “alternate initiation codons”.

[0217] As used herein, by "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, prophylaxis or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such asdeer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferably, the subject is a human subject.

[0218] The term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness in many biological and chemical characteristics.

[0219] The term “substantially equal” as it relates to time differences between doses, the term means plus / minus 2%.

[0220] The term “sub-therapeutic dose” refers to a dose of an agent that does not achieve a particular therapeutic effect (e.g., wherein the particular therapeutic effect is achieved using a therapeutically effective amount). Typically, a sub-therapeutic dose of an agent is an amount of a therapeutic dose that is less than a therapeutically effective amount of the agent.

[0221] The term “suffering from” refers to a subject who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0222] A subject who is "susceptible to" a disease, disorder, and / or condition has not been diagnosed with and / or cannot exhibit symptoms of the disease, disorder, and / or condition but harbors a propensity to develop a disease or its symptoms. In some instances, a subject who is susceptible to a disease, disorder, and / or condition (for example, cancer) can be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microbe associated with development of the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0223] In the context of the present disclosure, the term “T2 cell” refers to a cell that expresses an MHCI molecule (HLA-A2) that lacks TAP function. T2 cells can be easily artificially loaded with different concentrations of exogenous antigenic peptides. T2 cell are described e.g. in (Hosken and Bevan, Science 1990 Apr 20;248(4953):367-70). T2 cells are commercially available, e.g. from ATCC (American Type CultureCollection). Loading of T2 cells can be achieved under standard cell culture conditions known to the skilled in the art by incubating the T2 cells for about 2 hours with a desired concentration of antigenic peptide. In the context of the present disclosure, T2 cells that are incubated with a certain concentration of antigenic peptide, such as 1 pM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, are referred to as T2 cells loaded with said concentration of antigenic peptide, e.g. T2 cells incubated with 10 pM of antigenic peptide are referred to as T2 cells loaded with 10 pM of antigenic peptide.

[0224] The term “TCR” as used herein includes both native and engineered TCRs. A “native” TCR refers to a wildtype TCR that can be isolated from nature, whereas an “engineered” TCR may be a protein resembling a native TCR, but comprising further modifications e.g. in the variable and / or constant domains compared to the naturally occurring sequence, e.g. a humanized TCR or a TCR with altered characteristics (e.g. altered binding, heterodimerization or expression level).

[0225] Native TCRs are heterodimeric cell surface proteins of the immunoglobulin super-family, which are associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Native heterodimeric TCRs exist in a|3 and yb forms, which are structurally similar but have distinct locations and probably functions. The terms “a / p TCR” or a ”y / b TCR” thus refer to a TCR comprising an a-chain and a p-chain as described above, or a y-chain and a b-chain, respectively. Such TCRs may also be described as “full length TCRs” or “conventional TCRs”. An a / p TCR or a y / b TCR may be a native TCR or may be an engineered TCR that retains the structure of a native TCR, i.e. an engineered TCR comprising minor modifications in the variable and / or constant domains as described above, such as a humanized TCR.

[0226] Native, full-length ap heterodimeric TCRs consist of an a-chain and a p-chain. The a-chain comprises a variable region (V region) encoded by a TRAV gene, a joining region (J region) encoded by a TRAJ gene, and a constant region (C region) encoded by a TRAC gene. The p-chain comprises a variable region (V region) encoded by a TRBV gene, a joining region (J region) encoded by a TRBJ gene and a constant region (C region) encoded by a TRBC gene, and usually a short diversity region (D region) encoded by a TRBD gene between the V and J regions, although this D region is often considered as part of the J region (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10). The genes encoding different a-chain and p-chain variable, joining and constant regions are referred to in IMGT nomenclature by unique numbers (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Further information on TCR genes can be found in the international ImMunoGeneTics information system®, Lefranc M-P et al., (Nucleic Acids Res. 2015 Jan;43(Database issue): D413-22; and http: / / www.imgt.org / ).

[0227] The alpha chain TRAC constant domain sequence and the beta chain TRBC1 or TRBC2 constant domain are in the following, also referred to as TCR constantdomain sequences. The TCR constant domain sequences may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human. The TCR constant domain sequences may be modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. Also, further stabilizing mutations as known from the state of the art (e.g. WO 2018 / 104407, PCT / EP2018 / 069151, WO 2011 / 044186, WO 2014 / 018863) may be introduced, such as replacement of unfavorable amino acids in the variable regions and / or the introduction of a disulfide bridge between the TCR C domains and the removal of unpaired cysteine. Thus, the disclosure also relates to proteins that may comprise the antigen binding domains (e.g. the CDRs as provided herein) and further comprise domains / amino acid sequences that are not found in the naturally occurring TCR.

[0228] On the protein level, TCR a-, (3-, y- and 5-chains comprise two immunoglobulin domains, the variable domain and the constant domain. The variable domain corresponds to the V(D)J region. The constant domain corresponds to the C region. The constant domain is the membrane-proximal domain and in the context of the present disclosure also includes the transmembrane (TM) domain and a short cytoplasmic tail. Each of the constant and variable domains include an intra-chain disulfide bond. The variable domains (Vaand Vpin a|3 TCRs and VYand V5in y5 TCRs) contain highly polymorphic loops comprising the complementarity determining regions (CDRs).

[0229] Each TCR variable domain comprises three “TCR complementarity determining regions (CDRs)” embedded in a framework sequence, one being the hypervariable region named CDR3. In the context of the present disclosure, CDRal , CDRa2 and CDRa3 denote a-chain CDRs, and CDRbl, CDRb2 and CDRb3 denote [3-chain CDRs. The sequences encoding CDRal and CDRa2 are comprised in TRAV, the sequences encoding CDRa3 are comprised in TRAV and TRAJ, the sequences encoding CDRbl and CDRb2 are comprised in TRBV, and the sequences encoding CDRb3 are comprised in TRBV, TRBD and TRBJ. In TCRs, the CDR1 and CDR3 amino acid residues make contact with the antigenic peptide, while the CDR2 amino acid residues mainly contact the HLA molecule (Stadinski et al., J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et al., J Biol Chem. 2014 Jan 10;289(2):628-38). The antigen specificity of a TCR is thus defined by the CDR3 and CDR1 sequences. The CDR2 sequences are not required for the determination of antigen specificity, but may play a role in the overall affinity of a TCR towards a peptide:MHC complex.

[0230] “TCR framework regions” (FRs) refer to amino acid sequences interposed between the CDRs, i.e. to those portions of the variable domains that are to some extent conserved among different TCRs. The a-, (3-, y- and 5-chain variable domains each have four FRs, herein designated FR1-a, FR2-a, FR3-a, FR4-a (for an a- or y-chain), and FR1-b, FR2-b, FR3-b, FR4-b (for a [3- or 5-chain), respectively. Accordingly, an a-chain or y-chain variable domain may be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a) and a [3- or 5-chain variable domain may be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In the context of the present disclosure, the CDR / FR sequences in an a-, (3, y- or b-chain variable domain is determined based on IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Accordingly, CDR / FR amino acid positions when related to TCR or TCR-derived domains are indicated according to said IMGT definition. Preferably, the IMGT position of the CDR / FR amino acid positions of the variable domain Va is given in analogy to the IMGT numbering of TRAV24*01 and / or the IMGT position of the CDR / FR amino acid positions of the variable domain V|3 is given in analogy to the IMGT numbering of TRBV12-3*01.

[0231] A “fragment” of a TCR” refers to a fragment of a TCR that retains or substantially retains the affinity, functional avidity and / or specificity of the parental TCR from which it is derived for a target antigen. In other words, a “fragment” of a TCR is preferably a “functional” fragment of the TCR. The term “parental TCR” in this context refers to a full length TCR from which a functional fragment may be derived.

[0232] As binding to the target antigenic peptide is defined by the CDR1 and CDR3 sequences, and binding to the target antigenic peptide MHC complex is defined by CDR1, CDR2 and CDR3, antigen binding proteins comprising the CDR1 and CDR3 and optionally CDR2 sequences of a TCR retain the affinity, functional avidity and / or specificity of the parental TCR for a target antigen. The person skilled in the art is aware that the CDRs have to be interspersed with framework regions (FRs), however the specific amino acid sequences of the framework regions are not directly involved in target antigen specificity. Examples of functional TCR fragments include single variable domains, such as TCR alpha, beta, gamma or delta variable domains, or fragments of the a, (3, 5 or y chain, such as an a, (3, 5 or y chain without transmembrane domain and short cytoplasmic tail. The term “fragment” as used herein refers to naturally occurring fragments (e.g. splice variants or peptide fragments) as well as artificially constructed fragments, in particular to those obtained by gene-technological means.

[0233] A functional fragment of a TCR is considered to have retained or substantially retained the affinity for a target antigen, if, for example, the KDfor binding to the target antigen measured as outlined below is identical to the KDof the TCR or is increased or reduced, preferably reduced, no more than 10x, 5x, 3x, or 2x.

[0234] A functional fragment of a TCR may have retained or substantially retained the functional avidity for a target antigen, if, for example, the functional avidity for the target antigen is identical to that of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1%. In particular, a functional fragment of a TCR is considered to have retained or substantially retained the functional avidity for a target antigen, if, for example, its cytotoxic activity in response to the target of the parent protein measured in a cytotoxicity assay is identicalto the cytotoxic activity of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1%, preferably 10%, 8%, 5%, 3%, 2% or 1%.

[0235] A functional fragment of a TCR is considered to have retained or substantially retained the specificity for a target antigen (i.e. the ability to specifically bind to a target antigen), if it does not significantly bind to peptides other than the target antigenic peptide of the TCR.

[0236] “Does not significantly bind” in the context of antigenic peptide variants and in the context of antigen binding proteins of the disclosure, denotes, typically, a functional avidity was determined for the antigen binding protein binding to an antigenic peptide variant that is not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, preferably not higher than 20% of the functional avidity obtained for binding to the target antigenic peptide consisting of the amino acid sequence of SEQ ID NO: 24, preferably in the same experimental conditions. For instance, the functional avidity obtained for the antigen binding protein binding to a similar peptide is not higher than 30% of the signal obtained in the same experimental conditions for the antigen binding protein binding to the target antigenic peptide consisting of the amino acid sequence of SEQ ID NO: 24 (see WO2018172533 A1 ). The skilled person knows how to determine whether an antigen binding protein does not significantly bind to an antigenic peptide. An exemplary method is herein disclosed below and exemplified in the appended examples.

[0237] “Single chain TCR (scTCR)” as used herein denotes a TCR in which the variable domains of the TCR are located on a single polypeptide. Typically, the variable domains in scTCRs are separated by a linker, wherein said linker typically comprises 10 to 30 amino acids, such as 25 amino acids.

[0238] The term "therapeutic agent" or “active agent” refers to an agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. For example, a host cell expressing an antigen binding protein, or an mRNA encoding an antigenic peptide can be a therapeutic agent.

[0239] The term "therapeutically effective outcome" means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0240] The term "transcription" refers to methods to produce mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence).

[0241] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or heterologous) nucleic acid such as a gene, DNA or RNA to a host cell, so that the hostcell will express the introduced nucleic acid to produce a desired substance, e.g. the PRAME antigenic peptide or the antigen-binding protein described herein. A host cell that receives and expresses introduced DNA or RNA bas been "transformed". The nucleic acid can be introduced into the host cell by means of non-viral methods (e.g., via plasmids, electroporation, chemical transfection, heat shock, calcium phosphate, microinjection) or viral methods (i.e., infection with genetically engineered viruses). Non-virally introduced DNA can remain episomal (plasmid-based), typically leading to transient (short-term) expression, or be inserted into the genome for stable expression. Similarly, virally introduced DNA can integrate into the genome (retroviruses) or remain episomal (adenoviruses), but is typically stably expressed in either case. Transformation can combine viral and non-viral methods (e.g., electroporation for viral infection). The introduction of foreign nucleic acids into host cells via viral methods is also referred to as “transduction” and is often preferred for mammalian cells. Throughout the specification, the terms “transformation” and “transformed” include the terms “transduction” and “transduced”, respectively.

[0242] The term "treating" or "treatment" or "therapy" refers to the application or administration of a composition including one or more therapeutic agents to a subject with the aim of curing, healing, preventing, reducing, delaying, slowing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting a condition, disorder or disease such as cancer and / or its symptoms. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. For instance, cancer treatment may include, without limitation, inhibiting the recurrence of cancer, alleviating its symptoms, diminishing any direct or indirect pathological consequences, preventing or reducing metastases, decreasing the rate of cancer progression, ameliorating or palliation of the disease state, and remission or improved prognosis.

[0243] The term “treatment regimen” refers to a protocol or plan for administering a therapeutic agent to a patient. A treatment regimen may encompass parameters such as the dosage amount, frequency of administration, duration of treatment, and the method or route by which the therapeutic agent is delivered.

[0244] As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.

[0245] The term “variant” in relation to a nucleic acid or amino acid sequence refers to both natural variants (e.g., polymorphisms, isoforms, etc.) and artificial variants in which at least one nucleic or amino acid residue in a native or starting sequence (e.g.,a wild type sequence) has been removed and a different nucleic or amino acid inserted in its place at the same position, respectively. These variants can be described as “substitutional variants.” The substitutions can be single, where only one nucleic or amino acid in the molecule has been substituted, or they can be multiple, where two or more nucleic or amino acids have been substituted in the same molecule. If nucleic or amino acids are inserted or deleted, the resulting variant would be an “insertional variant” or a “deletional variant” respectively.

[0246] “Va” in the context of the present disclosure refers to a variable domain of a TCR a-chain.

[0247] “VA” or Vain the context of the present disclosure refers to a TCR variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (Va), [3-chain (Vp), y-chain (VY) or b-chain (V5), preferably from a Va. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, [3-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR a-chain.

[0248] The CDR and framework sequences of the VAdomain may not necessarily be derived from the same TCR chain. For example, the CDRs derived from one TCR variable domain (of the donor TCR) could also be grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VAencoded by TRAV5 and TRAJ17, and the acceptor TCR may comprise a VAencoded by TRAV14 and TRAJ33.

[0249] ”VP” in the context of the present disclosure refers to a variable domain of a TCR [3-chain.

[0250] “VB” or Vbin the context of the present disclosure refers to a variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (Va), [3-chain (Vp), y-chain (VY) or b-chain (V5), preferably from a Vp. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, [3-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR [3-chain. In the examples, various framework and CDR mutations / substitutions are shown.

[0251] The CDR and framework sequences of the VBdomain in context of the present disclosure may not necessarily be derived from the same TCR. For example, the CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR maycomprise a VBencoded by TRBV2 and TRBJ2-1 , and the acceptor TCR may comprise a Vg encoded by TRBV27 and TRBJ1-5.

[0252] CDRs may not only be exchanged / grafted between different alpha variable domains or different beta variable domains, but also may be grafted from a TCR alpha to a TCR beta, gamma or delta variable domain, or from a TCR beta to a TCR alpha, gamma or delta variable domain.

[0253] ”VY” in the context of the present disclosure refers to a variable domain of a TCR y-chain.

[0254] ”Vs” in the context of the present disclosure refers to a variable domain of a TCR b-chain.

[0255] ”V ” in the context of the present disclosure refers to a variable domain of an antibody light chain.

[0256] “VH” in the context of the present disclosure refers to a variable domain of an antibody heavy chain.

[0257] The terms "vector" includes "cloning vectors" and "expression vectors" and refers to a vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Preferably, and unless specified otherwise, the term “vector” refers to expression vectors.

[0258] The term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle and encodes at least an exogenous nucleic acid. The vector and / or particle can be utilized for the purpose of transferring a nucleic acid of interest into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Useful viral vectors include vectors based on retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). Recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0259] The terms “of the disclosure” as used herein are intended to refer to all aspects and embodiments disclosed and / or claimed herein. Any aspects, items or embodiments referred to herein as being “disclosed herein” or “described herein” areto be understood as being aspects, items or embodiments “of the disclosure” or “according to the disclosure”.

[0260] The term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either”, “one of”, “only one of”, or “exactly one of”.

[0261] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0262] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within this disclosure. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0263] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0264] The invention will now be described in more details with reference to the following figures and examples. All literature and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the foregoing description, the examples are to be consideredillustrative or exemplary and not restrictive. All embodiments disclosed herein can be combined unless the context clearly dictates otherwise.

[0265] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al, 2012, Cold Spring Harbor Laboratory Press).

[0266] DETAILED DESCRIPTION OF THE INVENTION

[0267] Compared to autologous products, which need to be manufactured individually for each patient, allogenic cell therapies based on cells from healthy donors and expanded in larger batches as off-the-shelf product could offer faster and less expensive access for patients to adoptive cell therapy (ACT) treatment. However, the challenge of HLA-mismatch between donor and patient needs to be overcome to avoid the risk of graft vs. host disease as well as immune rejection of the product limiting persistence and efficacy.

[0268] Allogeneic, off-the-shelf ACT products can be designed from various immune cells subsets, such as a|3 or yb T cells.

[0269] a|3 T cells are well suited for allogeneic, off-the-shelf use because their biology, activation pathways, and effector functions are well characterized and allow for reproducible engineering, expansion, and manufacturing from a single donor source. Their compatibility with precise gene-editing approaches enables the stable introduction of therapeutic receptors and other functional modules in a standardized manner appropriate for large-scale production.

[0270] However, endogenous a|3 TCRs can cause graft-versus-host disease, and donor-derived HLA molecules induce host-versus-graft immune rejection. Additional measures are required to prevent TCR mispairing and ensure that all residual native TCR components are eliminated. Engineered persistence and resistance to inhibitory signals may also be needed to maintain function in vivo. When these hurdles are addressed, a|3 T cells can be converted into safe, durable, and highly standardized allogeneic off-the-shelf cellular therapeutics.

[0271] yb T cells lend themselves to an allogeneic, off-the-shelf platform because they recognize target cells through non-H LA-restricted mechanisms and therefore do not trigger classical graft-versus-host disease. Their innate-like recognition pathways and rapid effector activity support the generation of standardized donor-derived cell banks suitable for repeated clinical dosing.

[0272] To deploy yb T cells as allogeneic products, several hurdles must nevertheless be addressed. Their persistence in vivo can be limited by immunosuppressive cytokines. Endogenous inhibitory pathways and susceptibility to exhaustion mayreduce durability. Additionally, donor-derived yd cells may still be subject to host-versus-graft clearance due to donor-derived HLA molecules. Once these barriers are overcome, y5 T cells can be developed as safe, durable, and functionally robust allogeneic off-the-shelf cellular therapeutics.

[0273] The present inventors have developed a versatile engineering technology based on targeted genomic integration of large transgene inserts to generate a platform for development of off-the-shelf ACT products, which is applicable to several T cell subsets.

[0274] As discussed above, to achieve optimal efficacy, an allogenic cell product requires a higher level of genetic engineering as compared to autologous ACT products. The present inventors have built multi-edited product candidates comprising at least two gene knock-outs (including B2M, to reduce or avoid host-versus-graft disease) combined with targeted knock-ins of two long inserts encoding antigen binding receptor chains, a single-chain HLA-I fusion protein and optionally CD8 chains, into the respective loci. Integration and expression of five ORFs was achieved with very high efficiency. The modular nature of the gene knock-out(KO) / knock-in (KI) pairs allows for versatile adjustments of product design.

[0275] The engineered cells combine three approaches for optimal allogeneic use: 1. Targeting: Insertion of antigen binding receptor, e.g. TCR / CAR, to direct activity against tumor target. CD8 co-expression may be required for CD8-dependent TCRs and / or in y5 T cells, which are mostly CD8 negative. 2. Cloaking: Allogenic effector cells need to be shielded from the patient immune system by inhibition of HLA expression through knock-out of B2M. At the same time, NK cell mediated rejection (missing self) needs to be suppressed. 3. Armoring: Additional gene edits, particularly in y5 T cells, may be introduced to improve functionality in suppressive tumor microenvironment and / or expansion / persistence and homing to tumor site.

[0276] In a first aspect, the present disclosure relates to an immune cell comprising a) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

[0277] The terms “immune cell” is used herein to include any cell that is involved in the immune response, including but not limited to leukocytes such as lymphocytes (e.g., T cells, B cells, and natural killer cells), macrophages, dendritic cells, neutrophils, monocytes, and other antigen-presenting cells, and any precursor cells of the foregoing. Preferably, the immune cell is a primate cell, more preferably a human cell. These cells may be naturally occurring, genetically modified, or artificially engineered for therapeutic, diagnostic, or research purposes. Preferably, the term “immune cell” refers to a lymphocyte, such as a T cell or T cell progenitor. Preferably, the T cell is ahuman T cell. Preferably, the T cell is a T cell isolated from a human or a descendent thereof. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD3-positive, CD4-positive and / or CD8-positive, CD4-positive helper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like. Preferably, the T cell is a gamma delta (yb) or alpha beta (a|3) T cell. The term “yb T cell” includes Vb2 cells, such as y9b2 T cells, and Vb1 T cells. Typically, the immune cells are allogeneic to the patient which they are administered to. However, in some instances, the immune cells may be autologous to the patient.

[0278] The immune cell comprises a genetically engineered inactivation of at least one, preferably of all copies of the B2M (beta-2 microglobulin, [32m) gene. Preferably, as used throughout the present disclosure, the genetically engineered inactivation is a genetically engineered disruption. Preferably, the genetically engineered disruption leads to a B2M knockout, which may refer to minimal or no expression of the endogenous B2M protein. A “knock-out” (KO) refers to a genetic engineering technique in which a specific nucleic acid sequence, such as a protein-encoding gene, is essentially inactivated or rendered non-functional (e.g. in terms of protein expression) by deleting, disrupting, or mutating its sequence. This preferably prevents the expression of the gene's protein product and effectively eliminates its function in the cell.

[0279] In other words, the immune cell is preferably “B2M deficient”. The immune cell may also be referred to as “B2M - / -“. Since B2M is an essential component of all HLA class I molecules, its inactivation preferably prevents the proper assembly and surface expression of all natural HLA class I proteins. Without wishing to be bound by theory, it is envisaged that by eliminating HLA class I expression, the engineered immune cells become "invisible" to recipient CD8 T cells in the treated patient, making them more suitable for use as universal donor cells in allogeneic cell therapy.

[0280] In the context of the present disclosure, the genetically engineered inactivation of the B2M gene preferably:

[0281] - reduces or eliminates endogenous MHC-I expression, reducing recognition by host CD8+T cells,

[0282] - reduces alloreactivity, thereby improving suitability for allogeneic or “off-the-shelf” cell therapies,

[0283] - prevents unwanted peptide presentation, thereby allowing the fusion HLA- I / B2M construct to control all peptide presentation, and / or

[0284] - prevents MHC-I dimers, thereby ensuring clean display of engineered fusion protein only.The B2M NCBI Gene ID is 567. An exemplary B2M coding sequence (NCBI Ref Seq: NM_004048.4, SEQ ID NO: 35) and the B2M protein sequence (UniProt Acc. No. P61769, SEQ ID NO: 36) are shown in Table 2 below. There may be many single nucleotide polymorphisms (SNPs) or other variants of the B2M gene and protein; as will be understood by those of skill in the art, the present disclosure is applicable to any such SNPs and other variants.

[0285] The immune cells can be engineered to disrupt the B2M gene such that no functional endogenous B2M protein is produced, or that (essentially) no B2M protein is expressed at all. “Non-functional” B2M proteins may include truncations, deletions, point mutations and insertions which preferably prevents their biological functions, specifically pairing with HLA class I molecules.

[0286] Any suitable technique for disrupting one, two or all copies of the B2M gene can be used; exemplary techniques are disclosed herein and are within the level of skill in the art based on the teachings herein and the teachings known in the art.

[0287] Preferably, the genetically engineered inactivation of the at least one B2M gene in the immune cell may preferably be achieved through insertion of at least one heterologous nucleic acid sequence(s) into the B2M gene locus, thereby disrupting the endogenous B2M gene. That is, the heterologous nucleic acid encoding the fusion protein, and / or the heterologous nucleic acid encoding the antigen binding protein, and / or any other heterologous nucleic acid described herein, and optionally further heterologous nucleic acid sequences such as regulatory sequences, may be knocked into the B2M gene locus, such that the B2M gene is knocked out.

[0288] A “knock-in” (KI) refers to a specific genetic engineering technique in which a targeted DNA sequence is inserted into a precise location within the genome. Unlike knockouts, which disrupt or delete a gene, knock-ins involve adding new genetic material to enhance or alter the function of a cell. This can include adding transgenes, functional proteins, or regulatory sequences. The term includes “replacement knock-ins”, which replace an endogenous nucleic acid sequence with a new or modified nucleic acid sequence, and “addition knock-ins”, which introduce a new nucleic acid sequence (e.g., an ORF or gene encoding a transgenic TCR, CAR, or cytokine) at a specific genomic locus without replacing the original nucleic acid sequence. Knock-ins are typically achieved using targeted gene editing technologies like CRISPR-Cas9, Cas12a, or TALENs. These tools create a double-strand break (DSB) at a specific locus, and the new genetic material is inserted through homology-directed repair (HDR).

[0289] In the context of the present disclosure, the B2M gene is preferably disrupted through a knock-in of a heterologous protein-encoding nucleic acid sequence, which disrupts and knocks out the B2M gene such that essentially no, or no functional, B2M protein is expressed by the immune cell. Various constructs and genetic engineering strategies are disclosed herein. For instance, the heterologous nucleic acid sequenceknocked or inserted into the B2M gene locus can encode a heterologous CD8 protein, such as a CD8 a and / or [3 chain, a heterologous antigen-binding protein (such as a TCR) or antigen-binding protein domain, such as a TCR a chain and / or [3 chain, or a heterologous fusion protein such as a single-chain HLA-E fusion protein as described elsewhere herein.

[0290] A genetically engineered inactivation, specifically disruption, in the B2M or any other gene can be achieved through targeted knock-in or knock-out using adeno-associated viruses (AAVs) and CRISPR / Cas technology. In this method, AAVs deliver a repair template into cells. The Cas protein, preferably Cas12a or Cas12i, creates a double-strand break at the target gene, and the cell's repair mechanisms, such as homology-directed repair (HDR), then use the repair template (which may contain homologous flanking sequences) to insert a desired heterologous nucleic acid sequence(s) (for knock-in) or disrupt the gene (for knock-out).

[0291] Alternative genetically engineered inactivation techniques are also available and applicable to the invention as described herein. Such methods include, e.g., gene silencing or knock-downs using siRNA or shRNA or any other suitable techniques. Any method is applicable as long as it disrupts B2M gene expression (e.g. at the DNA or RNA level) and, thereby, preferably the presence of endogenous HLA class I on the cell surface.

[0292] HLA-I fusion protein

[0293] The immune cells of the present disclosure are engineered to comprise at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein.

[0294] In other words, the preferably B2M deficient immune cells are engineered to recombinantly express an HLA class I fusion protein. Thus, the immune cells as used herein express one or more HLA class I fusion proteins in a B2M deficient genetic background. The B2M deficient cells recombinantly expressing a HLA class I fusion protein are nevertheless deficient in normal B2M expression or function in that the cells do not express endogenous wild type B2M protein capable of forming a non-covalently associated heterodimer with any HLA class I a chain on the cell surface. Since B2M is considered an essential structural component of all classical HLA class I molecules, without it HLA class I molecules cannot fold, assemble, traffic to the cell surface, or remain stable. As a result, in B2M deficient cells, endogenous HLA-I is not, or is essentially not, present on the cell’s surface.

[0295] The term “fusion protein” or “HLA class I fusion protein” or “single chain HLA class I fusion protein” (abbreviated “scHLA”, for instance “scHLA-E" for “single-chain HLA-E") as used herein refers to a fusion protein comprising at least a portion of aB2M protein covalently linked, either directly or via a linker sequence, to at least a portion of an HLA class I protein.

[0296] HLA (Human Leukocyte Antigen) class I proteins are a critical part of the immune system, specifically involved in presenting intracellular peptides (typically derived from proteins within the cell) to cytotoxic T cells (CD8+ T cells). The term “HLA” can be used interchangeably with the term MHC (Major Histocompatibility Complex) herein. The MHC is a gene complex found in the genome of all vertebrates. It encodes proteins responsible for antigen presentation to the immune system, enabling immune cells to distinguish between "self1and "non-self." HLA is the name given to the MHC system in humans. In other words, HLA proteins are the human-specific MHC proteins. As stated above, the terms are nevertheless used interchangeably.

[0297] HLA class I proteins are thought to play a key role in distinguishing healthy cells from infected or cancerous cells. HLA class I molecules generally consist of two main components: the HLA class I a (alpha) chain (also referred to as “heavy chain”) and [32-microglobulin ([32m), encoded by the B2M gene. The a chain, a transmembrane glycoprotein, forms the backbone of the HLA class I molecule. This a chain has three distinct extracellular domains, each with a specific function. The a1 and a2 domains form a peptide-binding groove where endogenous peptides, typically 9-11 amino acids in length, are loaded. This groove is highly variable due to polymorphisms in HLA genes, allowing the immune system to recognize a diverse range of antigens. The a3 domain, on the other hand, interacts with CD8 co-receptors on cytotoxic T cells and helps stabilize the HLA class I molecule.

[0298] The second component, [32-microglobulin ([32m), is a non-polymorphic protein that stabilizes the HLA class I a chain but does not span the cell membrane. Together, these two components enable HLA class I molecules to perform their primary function: presenting intracellular peptides on the cell surface. If a cell is infected by a virus or has undergone malignant transformation, it will typically present abnormal or non-self peptides. These peptides are then recognized by CD8+ T cells, leading to an immune response aimed at destroying the infected or cancerous cells.

[0299] HLA class I proteins are expressed on nearly all nucleated cells, which enables broad immune surveillance.

[0300] As discussed previously, the heavy chain of HLA class I requires [32-microglobulin ([32m) for correct folding, assembly, and stable surface expression. Genetic disruption of B2M therefore typically abolishes endogenous surface HLA class I, which preferably effectively removes the principal antigenic determinants responsible for host T cell-mediated rejection.

[0301] However, complete loss of endogenous HLA class I expression exposes donor cells to innate immune recognition, as NK cells detect the absence of self-HLA through “missing-self” surveillance mechanisms. As a result, B2M knockout, while preventingT cell-mediated allo-responses, simultaneously renders the engineered cells highly susceptible to NK-cell-mediated cytotoxicity. This reaction must be mitigated through additional engineering measures, such as the introduction of engineered single-chain HLA molecules that restore inhibitory signalling without reinstating polymorphic T cell epitopes.

[0302] Accordingly, although the removal of HLA class I via B2M disruption prevents classical host-versus-graft disease, the resulting sensitivity to NK surveillance constitutes a second immunological barrier that requires deliberate correction in the design of allogeneic off-the-shelf cell products.

[0303] In the context of the present disclosure, the transgenic HLA-I fusion protein is preferably capable of binding at least one inhibitory receptor on NK cells, thereby preferably inducing NK cell inhibition. Said inhibitory receptor may be NKG2A.

[0304] While HLA-A, HLA-B, and HLA-C are classical HLA class I a chains, there are also non-classical HLA class I a chains, including HLA-E, HLA-F, and HLA-G. These non-classical molecules differ from their classical counterparts in terms of function, expression patterns, and polymorphism.

[0305] Classical HLA class I molecules are highly polymorphic and play a central role in presenting antigens to cytotoxic CD8+ T cells. They are expressed on almost all nucleated cells. In contrast, non-classical HLA molecules exhibit limited polymorphism and have more specialized immunoregulatory roles, along with restricted expression patterns.

[0306] In the context of the present disclosure, the HLA-I protein, specifically the “portion” of the HLA-I protein that is covalently linked to the portion of the B2M protein, preferably comprises or consists of an HLA class I a chain or a variant or derivative thereof.

[0307] The HLA-I fusion protein may thus comprise at least a portion of B2M and at least a portion of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G, preferably an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G a chain or a variant or derivative thereof. Preferably, the HLA a chain does not contain the leader sequence (or signal sequence) of the HLA class I a chain (leaderless HLA a chain).

[0308] Preferably, the HLA class I a chain may be selected from HLA-E, HLA-F and HLA-G.

[0309] Therefore, the HLA-I fusion protein may comprise at least a portion of B2M and at least a portion of HLA-E, HLA-F or HLA-G. In certain preferred embodiments, the HLA-I fusion protein comprises a leader sequence (or signal peptide) covalently linked to the at least a portion of B2M and at least a portion of an HLA a chain. The leader sequence can be the leader sequence of the B2M protein, the leader sequence of anHLA a chain protein or the leader sequence of other secretory proteins. The HLA-I fusion protein may comprise a B2M protein with its leader sequence removed, and / or an HLA a chain protein with its leader sequence removed.

[0310] As discussed above, the HLA class I a chain may preferably be selected from a non-classical HLA class I a chain, in particular HLA-E, HLA-F and HLA-G.

[0311] HLA-E (UniProt ID: P13747) primarily interacts with immune receptors on natural killer (NK) cells, particularly the CD94 / NKG2A inhibitory receptor. It presents peptides derived from the leader sequences of classical HLA molecules and plays a role in regulating NK cell activity by signaling whether a cell should be "spared" or attacked. HLA-E is expressed on many nucleated cells and can contribute to immune evasion by tumors or viruses.

[0312] In preferred embodiments, the fusion protein comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to the HLA-E amino acid sequence of SEQ ID NO: 37 as shown in Table 2 below, preferably to the non-underlined portion of those sequences in Table 2, i.e. excluding any signal or leader sequence:

[0313] HLA-F (Uni-Prot ID: P30511) may function as an open conformer capable of interacting with classical HLA molecules and immune receptors. It may help modulate NK and T cell activity and could play a role in immune surveillance and regulation. HLA-F has a dynamic expression pattern and can localize intracellularly or on the cell surface under certain conditions, such as during immune activation.

[0314] The fusion protein may comprise or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of the HLA-F amino acid sequences of any one of SEQ ID NO: 38, 39 or 40 as shown in Table 2 below, preferably to any one of the non-underlined portion of those sequences, i.e. excluding any signal or leader peptide.

[0315] HLA-G (UniProt ID: P17693 ) is crucial for immune tolerance, particularly at the maternal-fetal interface, where it inhibits NK cells, T cells, and antigen-presenting cells to prevent fetal rejection. HLA-G is also involved in tumor immune evasion and tolerance induction in transplantation. Its expression is restricted to specific tissues, including the placenta, thymus, and other immune-privileged sites.

[0316] The fusion protein may comprise or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of the HLA-G amino acid sequences of any one of SEQ ID NO: 41 or 42 as shown in Table 2 below, preferably to the non-underlined portion of those sequences in table 2, i.e. not taking into account the (underlined) signal sequence:Hence, the fusion protein may comprise at least a portion of a non-classical HLA-I a chain, preferably HLA-E, HLA-F or HLA-G, more preferably HLA-E, which is covalently linked, either via a linker sequence or directly, to at least a portion of a B2M protein.

[0317] Preferably, the “portion” of the B2M protein is functional, i.e. retains the biological functionalities of the full-length B2M protein in terms of folding and pairing with the HLA-I a chain on the cell surface. The fusion protein may in particular comprise a full-length HLA-I a chain covalently linked, either directly or via a linker sequence, to a full-length B2M protein. The resulting fusion protein may also be referred to as a “single-chain fusion protein” or “single-chain HLA-E / scHLA-E” (if HLA-E is used as the HLA-I a chain) herein.

[0318] Preferably, the immune cell further expresses a peptide ligand that is presented by the fusion protein on the surface of said cell. Said peptide ligand can be covalently linked to at least a portion of said HLA-I a chain, such that the fusion protein preferably does not present any other peptides on the cell surface. Alternatively, the peptide ligand may be expressed by the immune cell, but not be covalently linked to the fusion protein.

[0319] HLA-I fusion proteins may be designed with covalently attached peptide ligands to facilitate folding to form a stable, surface-expressed complex. Without a tightly bound peptide, the heavy chain can be unstable, prone to misfolding, and may be loaded with endogenous peptides, leading to variable expression and unpredictable receptor engagement. Covalent attachment preferably ensures that the engineered HLA-I fusion protein folds correctly, traffics efficiently, and remains structurally stable at the cell surface. It may also prevent peptide exchange, ensuring a uniform, non-polymorphic inhibitory signal, which may be beneficial for consistent NK-cell evasion in allogeneic therapeutic cell products.

[0320] The HLA-I fusion protein preferably binds, in its binding cleft, the peptide ligand that is covalently attached to the fusion protein, either directly or via a linker sequence. In such embodiments, the fusion protein may preferably not bind other peptides.

[0321] Alternatively, the covalently linked peptide ligand may be cleaved via a built-in protease cleavage site, and the cleaved peptide ligand can bind to the peptide binding cleft of fusion protein. Alternatively, the peptide ligand occupying the peptide binding cleft of the HLA-I fusion protein is produced by the intracellular antigen processing pathway, in which the peptide ligand is produced by proteasome, transported to and loaded onto the HLA-I fusion protein in the endoplasmic reticulum.

[0322] The peptide ligand may comprise or consist of a HLA leader peptide, such as a HLA class I molecule (i.e., HLA-A, -B, and -C) or even from a HLA-G leader peptide, specifically if the HLA-I fusion protein a chain is HLA-E, or any other suitable peptide that can be bound by the HLA-I protein portion of choice. In view of the above, immunecells of the present disclosure may include a fusion protein comprising or consisting of a HLA class I a chain covalently linked, either directly or via a linker sequence, to a) at least a portion of a B2M protein and b) a peptide ligand. The fusion protein may have the general structure:

[0323] PeptideLigand-linker-B2M-linker- HLAa

[0324] HLAa stands for a HLA class I a chain.

[0325] The fusion protein preferably comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NO 1 or 2 as shown in Table 2 below:

[0326] The heterologous nucleic acid encoding the fusion protein preferably comprises or consists of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NO: 43 or 44 as shown in Table 2 below:

[0327] The heterologous nucleic acid encoding the fusion protein typically additionally encodes a signal peptide, which is cleaved off during processing.

[0328] SignalPeptide-PeptideLigand-linker-B2M-linker-HLAa

[0329] HLA-I fusion proteins of the present disclosure may comprise B2M and / or HLA a chain variants or fragments, as long as the resulting fusion protein is functional, i.e. possesses normal HLA class I functions, e.g., forming proper secondary structure of the HLA-I / B2M heterodimer on the cell surface and engaging the inhibitory receptors on the surface of NK cells. Additionally, a “functional” fusion protein preferably binds and presents the covalently attached peptide in its peptide binding cleft.

[0330] “Functional” HLA-I variants may share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence identity with the naturally occurring HLA a chain amino acid sequence, e.g. of HLA-E, HLA-F or HLA-G as disclosed herein and shown in Table 2 below, preferably without the signal sequence.

[0331] “Functional” B2M variants may share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with the naturally occurring B2M amino acid sequence of SEQ ID NO: 36 shown in Table 2 below, preferably without the (undesigned) signal sequence. .

[0332] It will be understood that the HLA-I portion and the B2M portion of the fusion protein in its membrane-bound form may or may not each comprise a signal peptide. Signal peptides are typically cleaved off during protein processing before and during transport to the cell surface. Signal peptides in any of the sequences disclosed hereincan be easily identified using SignalP 6.0 (Nature Biotechnology (2022), doi: 10.1038 / s41587-021-01156-3) or similar prediction tools.

[0333] In some embodiments, neither the HLA-I a chain nor the B2M portion of the fusion protein in its membrane-bound form comprises a signal peptide. As noted above, the heterologous nucleic acid encoding the fusion protein typically additionally encodes a signal peptide, which is cleaved off during intracellular protein processing:

[0334] SignalPeptide-PeptideLigand-linker-B2M-linker-HLAa

[0335] In such preferred embodiments, after the signal peptide has been cleaved off, the membrane-bound form of the single chain HLA-I fusion protein comprises the following elements:

[0336] PeptideLigand-linker-B2M-linker-HLAa

[0337] In such preferred embodiments, the “HLAa” stands for a HLA class I a chain, preferably a HLA-E a chain.

[0338] In some embodiments, the HLA-I a chain of the fusion protein in its membranebound form does not comprise a signal peptide, but its B2M portion does comprise a signal peptide.

[0339] The HLA-I fusion protein can be expressed from any suitable gene locus in the immune cell. In some embodiments, the HLA-I fusion protein is inserted into the B2M gene locus (targeted knock-in), such that the immune cells do not or essentially do not express endogenous B2M, but express a HLA-I fusion protein comprising a HLA-I a chain and B2M. In such settings, the heterologous nucleic acid(s) encoding the HLA-I fusion protein is / are inserted into the genome of the immune cell, preferably into the B2M locus, for stable expression. In this way, the B2M loci are disrupted by inserting in the B2M loci the nucleic acid(s) encoding the HLA-I fusion protein to disrupts normal B2M expression and preclude the formation of wild type HLA class I proteins but permits expression of the HLA-I fusion protein of choice on the surface of the otherwise B2M deficient cells.

[0340] Put differently, the such immune cells express B2M only in the context of the HLA-I fusion protein. To this end, the at least one nucleic acid sequence(s) encoding said fusion protein may be inserted into the at least one B2M gene locus, preferably all B2M gene loci. In this way, the heterologous nucleic acid sequence(s) encoding the fusion protein is knocked in and the B2M gene is knocked out.

[0341] The B2M knockout strategy is designed to reduce immune rejection in allogeneic settings by disrupting or impairing the surface expression of endogenous HLA class I proteins, as B2M is typically necessary for stabilizing the HLA a chain. While the absence of endogenous HLA-I expression is believed to protect engineered cells from CD8+ T cell-mediated responses, it also makes them susceptible torejection by Natural Killer (NK) cells. NK cells, which are part of the innate immune system, identify and induce apoptosis in cells that lack HLA class I expression. However, the HLA-I a chain in the fusion protein can pair with the covalently attached B2M portion, enabling stable expression of the fusion protein on the cell surface. This expression is thought to reduce or prevent NK cell-mediated killing by binding to inhibitory receptors on NK cells. These receptors, such as CD94 / NKG2, recognize HLA class I a chains and inhibit NK cell-induced apoptosis in cells that do not express endogenous HLA class I proteins. Thus, the HLA-I fusion protein acts as a ligand for these inhibitory NK cell receptors.

[0342] The fusion protein thus preferably possesses normal HLA class I functions, e.g., forming proper secondary structure of the HLA-I / B2M heterodimer on the cell surface and engaging the inhibitory receptors on the surface of NK cells.

[0343] As an alternative to the B2M knock-in, the nucleic acid sequence(s) encoding the fusion protein can be inserted into and expressed from any other suitable gene loci in the immune cell, some of which are exemplified herein, as long as the immune cell is B2M deficient, i.e. comprises a genetically engineered inactivation of at least one, preferably all, endogenous B2M genes in its genome.

[0344] The nucleic acid sequence(s) encoding the HLA-I fusion protein can be operably linked to suitable elements regulating the expression of said proteins. Exemplary promoters are described herein. In embodiments of the present disclosure, the expression of the HLA-I fusion protein is regulated by the endogenous B2M regulatory sequence(s) located at the B2M locus. Alternatively, the expression of the HLA-I fusion protein can be regulated by heterologous regulatory elements that are inserted into the B2M locus.

[0345] CD8

[0346] The immune cell of the present disclosure may further comprise at least one heterologous nucleic acid sequence(s) encoding a CD8 a and / or CD8 [3 chain.

[0347] Specifically, the heterologous nucleic acid sequence(s) may encode only at least one CD8 a chain, or the heterologous nucleic acid sequence(s) may encode at least one CD8 a and at least one CD8 [3 chain.

[0348] CD8 functions as a co-receptor on the surface of T cells. It plays a role in T cell activation and target cell recognition, particularly in the context of MHC class I-mediated antigen presentation. Adding heterologous CD8 can stabilize the TCR-MHC-I interaction, improving the overall strength and efficiency of the immune response. CD8 is typically composed of two subunits, either a and [3 chains (in the case of CD8a[3) or just a single a chain (CD8aa). However, some TCRs that can be used according to the present disclosure are CD8-independent, and so an engineered immune cell expressing such a TCR do not require a heterologous CD8.The CD8 a and / or [3 chains may be selected from any CD8 a or [3 chain amino acid sequence known in the art. The CD8 a chain may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to any one of the sequences disclosed under UniProt ID: P01732. Specifically, the CD8 a chain may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NO: 45, 46 or 47 shown in Table 2 below, preferably to any one of the non-underlined portions of those sequences in Table 2, i.e. excluding any signal or leader sequences.

[0349] The CD8 [3 chain may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to any of the amino acid sequences disclosed under UniProt ID: P10966. Specifically, the CD8 [3 chain may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55 shown in Table 2 below, preferably to any one of the non-underlined portions of said sequences in Table 2, i.e. excluding any signal or leader sequence.

[0350] It will be understood that the CD8 in its membrane-bound form will typically not comprise a signal peptide. Signal peptides are typically cleaved off during protein processing before and during transport to the cell surface. Signal peptides in any of the sequences disclosed herein can be easily identified using SignalP 6.0 or similar prediction tools.

[0351] The CD8 a and / or [3 chain can be expressed from any suitable gene locus in the immune cell. In some embodiments, the CD8 a and / or [3 chain is inserted into the B2M gene locus for a targeted knock-in as described above in the context of the HLA-I fusion protein knock-in.

[0352] Thus, the at least one nucleic acid sequence(s) encoding said CD8 a and / or CD8 [3 chain may be inserted into the B2M gene locus. In this way, said heterologous nucleic acid sequence(s) is knocked in and the B2M gene is knocked out. The at least one nucleic acid sequence(s) encoding said CD8 aand / or CD8 [3chain can be inserted into the B2M gene locus in addition to the nucleic acid sequence(s) encoding the HLA-I fusion protein, or any other heterologous nucleic acid sequence(s) described herein.

[0353] As an alternative to the B2M replacement knock-in, the nucleic acid sequence(s) encoding the CD8 a and / or CD8 [3 chain can be inserted into and expressed from any other suitable gene locus in the immune cell, some of which are exemplified herein, as long as the immune cell is B2M deficient, i.e. comprises a genetically engineered inactivation of at least one, preferably all, endogenous B2M genes in its genome.

[0354] If the CD8 and TCR or CD8 and HLA-I fusion protein are expressed from the same gene locus, the coding ORFs can be arranged in any suitable form, e.g. CD8[3-CD8a-TCR[3-TCRa, or CD8a-CD8[3-TCR[3-TCRa or any other suitable arrangement.In preferred embodiments, a heterologous nucleic acid sequence encoding a HLA-E fusion protein according to SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 , and a CD8 a and CD8 [3 chain is knocked into the B2M gene locus.

[0355] In preferred embodiments, a heterologous nucleic acid sequence encoding a HLA-E fusion protein according to SEQ ID NO: 2, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2, and a CD8 a and CD8 [3 chain is knocked into the B2M gene locus.

[0356] In any embodiment described herein, the open reading frame encoding the HLA-E fusion protein and CD8 a and [3 chains is preferably knocked in-frame into the coding region of the B2M gene. The genes of the open reading frame are preferably arranged in the following order: 2A-FusionProtein-2A-CD8a-2A-CD8[3. 2A cleavage sites are short peptide motifs that mediate ribosomal skipping during translation, thereby allowing multiple polypeptides to be produced from a single contiguous open reading frame without requiring proteolytic processing. In particular, 2A peptides enable the co-translational separation of upstream and downstream protein sequences with high efficiency, ensuring that each of the HLA-E fusion protein, CD8a, and CD8[3 chains is generated as an individual polypeptide despite being encoded within a single polycistronic transcript. The use of 2A sequences may therefore facilitate coordinated expression of all three components in substantially equimolar amounts, while maintaining the compact overall structure of the knock-in cassette. The skilled person is aware of alternative options to achieve expression of the fusion protein, CD8 sequences and TCR sequences from the desired genomic loci.

[0357] Additional knock-outs

[0358] In addition to the B2M knock-out, the immune cell may further comprise a genetically engineered inactivation of a gene associated with any one of NK cell inhibition, T cell exhaustion, and / or graft-versus-host disease. Preferably, the immune cell comprises a genetically engineered inactivation of a TGFBR2 gene, a TRAC gene, a TRBC1 and / or TRBC2 gene, a CIITA gene, a CISH gene, a PD-1 gene, a CTLA-4 gene, a NKG2A gene, a Cbl-b gene, a Reg-1 gene, a PTPN2 gene, a LAG3 gene, a TIM3 gene and / or a FAS gene.

[0359] TGFBR2

[0360] The person skilled in the art is capable of selecting suitable additional knock-outs depending on the chosen platform, desired applications, and specific functional requirements of the engineered cell. Such additional knock-outs may be introduced, for example, to enhance cellular fitness, reduce alloreactivity, eliminate endogenous signaling pathways, improve persistence, or prevent undesired interactions with host tissues or therapeutic modalities. The selection of further gene disruptions will depend on the intended use and inherent limitations of the modifiedcell. In this context, the skilled person can readily determine which endogenous genes, pathways, or surface molecules are advantageous to disrupt in order to optimize safety, stability, efficacy, or manufacturability of the engineered cellular product.

[0361] In certain embodiments, a knock-out of the TGF-[3 receptor type II (TGFBR2, UniProt ID: P37173) is particularly envisaged for yb T cells. yb T cells can be highly responsive to cytokine cues within the tumor microenvironment, and the immunosuppressive cytokine TGF-[3 is known to potentially inhibit their activation, proliferation, cytotoxicity, and tissue infiltration. Engagement of TGF-[3 with its receptor complex, including TGFBR2, induces canonical SMAD-dependent signaling pathways that might downregulate effector functions of yb T cells, including expression of key cytotoxic mediators and pro-inflammatory cytokines. As many solid tumors overexpress and secrete TGF-[3 to create an immunosuppressive niche, yb T cells entering such environments may be particularly susceptible to functional silencing.

[0362] Accordingly, disruption of TGFBR2 in yb T cells preferably reduces or prevents initiation of TGF-[3-mediated signaling and thereby renders the engineered cells resistant to TGF-[3-induced suppression. Such resistance can support enhanced persistence, cytotoxicity, and metabolic fitness of yb T cells in TGF-(3-rich tissues. For these reasons, TGFBR2 knock-out is specifically contemplated as an additional or preferred genetic modification in engineered yb T-cell platforms. The TGFBR2 NCBI Gene ID is 7048. The TGFBR2 coding sequence (NCBI Ref Seq: NM_003242.6, SEQ ID NO: 56) and the TGFBR2 protein sequence (UniProt ID: P37173, SEQ ID NO: 57) are shown in Table 2 below. There may be many single nucleotide polymorphisms (SNPs) or other variants of the TGFBR2 gene and protein; as will be understood by those of skill in the art, the present disclosure is applicable to any such SNPs and other variants.

[0363] Advantageously, the knock-out of TGBFR2- specifically in yb T cells - can be combined with the knock-in of a heterologous nucleic acid encoding the antigen binding protein, in particular a TCR, into the TGFBR2 gene.

[0364] TRAC, TRBC1, TRBC2

[0365] In certain embodiments, knock-out of the endogenous T-cell receptor a-chain constant region (TRAC) together with the T-cell receptor [3-chain constant region genes (TRBC1 and TRBC2, collectively also referred to as TRBC1 / 2) is particularly relevant for allogeneic a|3 T-cell platforms. The endogenous a|3 T-cell receptor (TCR) is thought to be a major driver of alloreactivity, as recognition of mismatched HLA molecules on recipient cells can trigger graft-versus-host responses. Disruption of TRAC prevents expression of the endogenous TCRa chain, thereby eliminating surface expression of a functional a|3 TCR heterodimer. However, the present inventors determined that because the [3-chain can be encoded by either TRBC1 or TRBC2, additional knock-out of both TRBC loci is required to fully abrogateendogenous TCR[3 expression and avoid the formation of residual or mispaired TCR complexes.

[0366] Simultaneous elimination of TRAC, TRBC1 and TRBC2 according to the present disclosure therefore ensures complete removal of the endogenous a|3 TCR from the engineered cell population. This prevents alloreactive signaling through the native TCR, thereby reducing or eliminating the risk of graft-versus-host disease when the modified cells are administered allogeneically. Moreover, removal of endogenous TCR chains may reduce competition for CD3 complexes and other shared signaling components, which may facilitate stable surface expression and functional signaling of any introduced antigen-binding protein, such as a CAR or engineered TCR. For these reasons, the present inventors determined that a combined knock-out of TRAC, TRBC1 and TRBC2 is particularly advantageous and is specifically contemplated for allogeneic a|3 T-cell platforms to enhance safety, uniformity, and functional performance of the engineered cells.

[0367] The combined knock-out of B2M, TRAC, TRBC1 and TRBC2 is also referred to as a “triple” knock-out herein (since both TRBC1 and TRBC2 encode the constant region segments of the TCR [3 chain).

[0368] Advantageously, the knock-out of TRAC - specifically in a[3T cells - can be combined with the targeted knock-in of a heterologous nucleic acid encoding the antigen binding protein (or any other protein of interest), in particular a TCR, into the TRAC gene. The TRBC1 and TRBC2 genes can then additionally be knocked through disruption without knocking in any further heterologous nucleic acid sequence.

[0369] Further knockouts

[0370] The CIITA gene encodes the class II transactivator (UniProt ID: P33076), a transcription factor that regulates the expression of MHC class II molecules on antigen-presenting cells. A CIITA knockout can reduce MHC class II expression, potentially enhancing immune evasion of engineered cells from CD4+ T cell responses.

[0371] The CISH gene encodes the cytokine inducible SH2-containing protein, a negative regulator of cytokine signaling that modulates immune cell responses (UniProt ID: Q9NSE2). A CISH knockout can enhance immune cell activation and persistence by removing this negative feedback, improving their ability to respond to cytokines and strengthening their anti-tumor efficacy.

[0372] The PD-1 gene encodes the programmed cell death protein 1, an immune checkpoint receptor that negatively regulates T cell activation and immune responses, particularly in tumors (UniProt ID: Q15116). A PD-1 knockout in immune cells can enhance their anti-tumor activity by preventing the inhibitory signals from PD-1 andallowing the immune cells to maintain stronger and longer-lasting responses against cancer cells.

[0373] The CTLA-4 gene encodes cytotoxic T-lymphocyte antigen 4, an immune checkpoint receptor that inhibits T cell activation by competing with CD28 for binding to B7 molecules on antigen-presenting cells (UniProt ID: P16410). A CTLA-4 knockout in T cells can enhance T cell activation and proliferation, improving their anti-tumor efficacy by removing inhibitory signals and allowing sustained T cell responses against tumors.

[0374] The NKG2A gene encodes the natural killer cell granule protein 2A, an inhibitory receptor expressed on natural killer (NK) cells and some T cells, which binds to HLA-E molecules to inhibit immune responses (UniProt ID: P26715). An NKG2A knockout can enhance immune cell activity by preventing inhibition through the HLA-E / NKG2A axis, improving their ability to target and kill tumor cells, particularly in HLA-E-expressing tumors.

[0375] The CBLB gene encodes the Cbl-b E3 ubiquitin ligase, a negative regulator of T cell activation that controls T cell signaling by promoting the degradation of signaling molecules and attenuating immune responses (UniProt ID: Q13191). Cbl-b knockout in T cells can enhance T cell activation and proliferation by removing this inhibitory signal, leading to stronger and prolonged anti-tumor responses, as Cbl-b normally dampens TCR signaling.

[0376] The ZC3H12A gene encodes the endonuclease Regnase-1, which recognizes and cleaves a stem loop structure in the 3'UTR of mRNAs involved in cytokine signaling and inflammation. Removing this inhibitory mechanism by ZC3H12A knockout may enhance and prolong activity of allogenic anti-tumor T cells.

[0377] The PTPN2 gene encodes protein tyrosine phosphatase non-receptor type 2, an enzyme that negatively regulates immune cell signaling by dephosphorylating key signaling molecules, thus controlling T cell activation and inflammation (UniProt ID: P17706). PTPN2 knockout in allogeneic cell therapy may enhance immune cell activation and function, potentially improving the therapeutic efficacy of engineered cells by reducing negative feedback and boosting immune responses against tumors or pathogens while also potentially improving the persistence of the engineered cells in the host.

[0378] The LAG3 gene encodes lymphocyte-activation gene 3, an immune checkpoint receptor that inhibits T cell activation and function, particularly in response to antigen stimulation (UniProt ID: P18627). LAG3 knockout in allogeneic cell therapy can enhance T cell activation and persistence by removing this inhibitory signal, leading to more robust immune responses and improved therapeutic efficacy, especially in cancer immunotherapy, by preventing immune suppression within the tumor microenvironment.The TIM3 gene encodes T-cell immunoglobulin and mucin-domain containing-3, an immune checkpoint receptor that negatively regulates T cell activation and plays a role in promoting immune tolerance, particularly in chronic infections and tumors. TIM3 knockout in allogeneic cell therapy can enhance T cell responses by removing this inhibitory checkpoint, improving the efficacy of engineered T cells, particularly in overcoming immune evasion mechanisms in the tumor microenvironment and enhancing their persistence and functionality.

[0379] The FAS gene encodes the Fas cell surface death receptor, which is involved in the regulation of programmed cell death (apoptosis) through activation of the extrinsic apoptotic pathway (UniProt ID: P25445). FAS knockout in allogeneic cell therapy can enhance the survival and persistence of engineered cells by preventing apoptosis induced by Fas signaling, thereby improving their efficacy and reducing the risk of unwanted cell death in the therapeutic setting.

[0380] Preferably, the immune cell comprises genetically engineered inactivation of all copies of the TGFBR2, TRAC, TRBC1, TRBC2, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3 and / or FAS genes, respectively, depending on the engineering approach. The immune cell may comprise only one of the described knock-outs, or may combine several knock-outs. Preferably, said genetically engineered inactivation, preferably disruption, causes a knockout of the affected genes, i.e. TGFBR2, TRAC, TRBC1, TRBC2, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3, respectively. A preferred target for the additional genetically engineered inactivation, preferably disruption, is the TGFBR2 gene. Another preferred target for the additional genetically engineered inactivation, preferably disruption, are the TRAC and optionally TRBC1 and TRBC2 genes.

[0381] Again, the genetically engineered inactivation, preferably disruption, of the selected gene can be achieved through targeted knock-in or knock-out using adeno-associated viruses (AAVs) and CRISPR / Cas technology. In this method, AAVs deliver a repair template into cells. The Cas protein, preferably Cas12a or Cas12i, creates a double-strand break at the target gene, and the cell's repair mechanisms, such as homology-directed repair (HDR), then use the repair template (which may contain homologous sequences) to insert a desired sequence (for knock-in) or disrupt the gene (for knock-out).

[0382] Alternative genetically engineered inactivation techniques are also available and applicable to the invention as described herein. Such methods include, e.g., gene silencing or knock-downs using siRNA or shRNA or any other suitable techniques. Any method is applicable as long as it disrupts TGFBR2, TRAC, TRBC1 / 2 (or any other of the aforementioned) gene expression (e.g. at the DNA or RNA level) and preferably the presence of endogenous HLA class I on the cell surface.

[0383] Cytokine supportIn some embodiments, the immune cell described herein may further require, utilize, or benefit from cytokine support to maintain, enhance, or stabilize one or more functional properties of the engineered cell. As used herein, “cytokine support” refers to the provision, expression, or activity of one or more cytokines, cytokine receptor components, cytokine signaling modulators, or cytokine-related fusion proteins that preferably contribute to the survival, expansion, persistence, differentiation state, cytotoxicity, or effector function of the engineered immune cell.

[0384] Extrinsic cytokine support

[0385] In some embodiments, the immune cell may be cultured, expanded, activated, or otherwise maintained in the presence of exogenously supplied cytokines. Suitable cytokines may include, without limitation, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or combinations thereof. Extrinsic cytokine supplementation may support:

[0386] - enhanced proliferation or expansion during manufacturing;

[0387] - maintenance of a less-differentiated phenotype;

[0388] - improved persistence or survival during or after administration; or

[0389] - augmentation of cytotoxic or effector activity.

[0390] In certain embodiments, cytokine support may be provided intermittently, continuously, or in a staged manner depending on the manufacturing or therapeutic context.

[0391] In certain embodiments, extrinsic cytokine support may be provided to the patient during therapy by administering suitable cytokines, such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or combinations thereof.

[0392] Intrinsic cytokine support

[0393] In some embodiments, the immune cell may comprise one or more heterologous nucleic acid sequences encoding a cytokine, cytokine subunit, cytokine receptor component, or cytokine-related fusion protein. Such intrinsic support may be designed to provide autocrine or paracrine stimulation, thereby reducing or eliminating the need for systemic cytokine administration. Examples of intrinsic cytokine support may include, but are not limited to:

[0394] - engineered expression of IL-2, IL-12, IL-15, IL-18, or IL-21;

[0395] - expression of a modified cytokine that is membrane-tethered or has restricted spatial activity;- expression of a cytokine-receptor fusion or signaling enhancer (e.g., IL-15 / IL-15Ra fusion complexes);

[0396] - expression of signaling adaptors that amplify cytokine receptor pathways.

[0397] In some embodiments, intrinsic cytokine support may be under the control of an endogenous or heterologous promoter, a constitutive promoter, an inducible element, or a promoter responsive to cellular activation, antigen recognition, environmental cues, or synthetic regulatory networks.

[0398] In view of the above, the immune cell may therefore further comprise at least one heterologous nucleic acid sequence(s) encoding a cytokine of cytokine-receptor fusion protein, wherein said cytokine is selected from of interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21. Said cytokines may be expressed in soluble or membrane-bound form.

[0399] Interleukin-2 (IL-2, UniProt ID: P60568) is a cytokine that plays a role in regulating immune responses by promoting the growth, differentiation, and activation of T cells. It is essential for T cell proliferation, survival, and effector function.

[0400] In allogeneic cell therapy, IL-2 is commonly used to boost the expansion and persistence of engineered T cells, such as TCR-T cells or CAR-T cells, after they are infused into patients. By providing IL-2, the activity of these therapeutic cells is enhanced, leading to stronger anti-tumor responses and immune activation. Additionally, IL-2 can help to overcome immune suppression in the tumor microenvironment, improving the efficacy of the therapy.

[0401] Interleukin-15 (IL-15, UniProt ID: P40933) is a cytokine that plays a role in the activation, proliferation, and survival of T cells, NK cells, and other immune cells, particularly enhancing the persistence and memory formation of CD8+ T cells and NK cells.

[0402] In allogeneic cell therapy, IL-15 is often used to enhance the long-term survival and functionality of engineered T cells, such as TCR-T and CAR-T cells. IL-15 can support immune cell expansion and help to maintain the persistence of these cells in the patient's body, enabling them to sustain anti-tumor responses over time. By reducing immune exhaustion and promoting memory-like properties, IL-15 contributes to improving the effectiveness and durability of therapeutic cell therapies.

[0403] Interleukin-18 (IL-18, UniProt ID: Q14116) is a pro-inflammatory cytokine that enhances the production of IFN-y and stimulates the activation and proliferation of T cells, NK cells, and macrophages. It plays a role in innate immunity and adaptive immune responses, particularly in cytokine release and immune cell activation.In allogeneic cell therapy, IL-18 can be utilized to boost the activity and effector functions of engineered immune cells such as TCR-T and CAR-T cells. By enhancing IFN-y production, IL-18 improves the immune response against tumors, helps to overcome the immune suppression in the tumor microenvironment, and can promote better cytotoxic activity of therapeutic cells. This cytokine is particularly valuable in enhancing anti-tumor immunity and improving therapeutic efficacy in adoptive cell transfer therapies.

[0404] Interleukin-21 (IL-21) is a cytokine that regulates the immune system by promoting the activation, differentiation, and survival of T cells, B cells, and NK cells. It plays a critical role in enhancing T cell responses and antibody production while also supporting the development of memory T cells (UniProt ID: Q9HBE4).

[0405] In allogeneic cell therapy, IL-21 is used to enhance the persistence, proliferation, and effector function of engineered T cells, such as TCR-T and CAR-T cells. It improves the cytotoxic activity of therapeutic T cells, helps to overcome immune suppression in the tumor microenvironment, and may contribute to the development of long-term memory T cells that persist in the host. IL-21 is particularly useful in boosting the anti-tumor immunity of adoptively transferred T cells, increasing the overall efficacy of cell therapies.

[0406] The immune cells of the present disclosure may be engineered to express one or more of the cytokines in soluble or membrane-bound form. Preferred constructs are described in PCT / US2023 / 066367 and PCT / US2023 / 066314, which are incorporated by reference herein. Cytokine (interleukin) armouring may be useful for some yb T-cell platforms because cytokine support can ensure optimal activation, persistence, and cytotoxic function of yb T cells. The tumor microenvironment is often cytokine-poor and immunosuppressive, which can limit the expansion and activity of unmodified yb T cells after adoptive transfer. Engineered expression of supportive cytokines (e.g., IL-2, IL-7, IL-15 or IL-21) may provide cell-intrinsic stimulation that enhances survival and effector function, improves resistance to suppressive signals, and reduce the need for external cytokine administration. Accordingly, cytokine armouring might be particularly advantageous for strengthening the performance of certain yb T-cell therapies.

[0407] However, cytokine armouring is also envisaged for certain a|3 T cells. Importantly, cytokine armouring may not be required in embodiments where the engineered yb or a|3 T cells already receive sufficient cytokine support from the manufacturing process, the therapeutic regimen, or the tissue environment. For example, when exogenous cytokines such as IL-2, IL-7, IL-15, or IL-21 are routinely administered as part of the treatment protocol, or when the target tissue naturally provides adequate cytokine levels to sustain T-cell persistence and effector function, additional cell-intrinsic cytokine expression may be unnecessary. Furthermore, T-cell platforms that are engineered for enhanced intrinsic fitness, reduced exhaustion, orprolonged survival may maintain adequate activity without cytokine armouring. In some settings, cytokine armouring may also be avoided to limit the risk of excessive T-cell expansion, uncontrolled proliferation, or local tissue inflammation. Accordingly, cytokine armouring is optional and may be omitted when external cytokine support or intrinsic cellular properties are sufficient to achieve the desired therapeutic function.

[0408] In some embodiments, cytokine armouring can be achieved with heterologous membrane-bound cytokines, or membrane-bound cytokine-receptor fusion proteins. In embodiments of the present disclosure, the immune cell comprises a heterologous nucleic acid encoding a membrane-bound interleukin-15 fusion protein.

[0409] The heterologous membrane-bound IL-15 fusion protein preferably comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 58 as shown in Table 2 below.

[0410] The heterologous nucleic acid encoding the membrane-bound IL-15 fusion protein preferably comprises or consists of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NO: 59 or 60 as shown in Table 2 below:

[0411] The at least one heterologous nucleic acid sequence(s) encoding said cytokine (fusion protein), e.g. the membrane-bound IL-15 fusion protein described herein, may be inserted into the B2M gene locus. In this way, said heterologous nucleic acid sequence(s) is knocked in and the B2M gene is knocked out. The at least one nucleic acid sequence(s) encoding said cytokine (fusion protein) can be inserted into the B2M gene locus in addition to the nucleic acid sequence(s) encoding the HLA-I fusion protein, the CD8 a / or [3 chain, or any other heterologous nucleic acid sequence(s) described herein.

[0412] As an alternative to being knocked into the B2M gene locus, the heterologous nucleic acid sequence(s) encoding cytokine (fusion protein), e.g. the membranebound IL-15 fusion protein described herein, can be inserted into and expressed from any other suitable gene locus in the immune cell, some of which are exemplified herein, e.g. TRAC or TGBR2, as long as the immune cell is B2M deficient, i.e. comprises a genetically engineered inactivation of at least one, preferably all, endogenous B2M genes in its genome.

[0413] Antigen binding protein

[0414] The antigen binding protein preferably comprises three complementarity determining regions (CDR) CDRal, CDRa2 and CDRa3, for instance on a first polypeptide. The antigen binding protein preferably comprises three CDRs CDRbl, CDRb2, and CDRb3, for instance on a second polypeptide. The antigen binding protein therefore preferably comprises six complementarity determining regions (CDR) CDRal, CDRa2, CDRa3, CDRbl, CDRb2, and CDRb3. Preferably, saidantigen binding construct is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), or a derivative or fragment of a TCR or CAR.

[0415] A “derivative” or “fragment” of a TCR or CAR is preferably functional and retains the binding specificity of the TCR or CAR that it is derived from.

[0416] The first polypeptide preferably comprises or consists of a TCR a-chain variable domain (Va) or a derivative or fragment thereof, and / or the second polypeptide preferably comprises or consists of a TCR [3-chain variable domain (V|3) or a derivative or fragment thereof. The first and / or said second polypeptide may further comprise a TCR constant domain.

[0417] Preferred CDR, variable and constant domain sequences are listed in Table 2 below.

[0418] It is envisaged that the antigen binding protein is preferably capable of specifically binding to a target peptide presented by an MHC-I molecule. Said target peptide may be a CT45, PRAME, COL6A3, NY-ESO-1, MAGE-A3, KLK3, KLK4, KK-LC-1, MART-1, CEA, HA-1, KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide.

[0419] In preferred embodiments, the antigen binding protein is a TCR that specifically binds to a PRAME peptide presented on MHC-I and has the CDRs as disclosed in PCT / EP2018 / 057482.

[0420] In preferred embodiments, the TCR specifically binds to a target peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 67).

[0421] In preferred embodiments, the TCR comprises the CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 3, 4, 5, 6, 7 and 8 as shown in Table 2 below.

[0422] The TCR may comprise a TCR a variable domain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9 as shown in Table 2 below, and / or a TCR [3 variable domain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10 as shown in Table 2 below.

[0423] In further preferred embodiments, the antigen binding protein is a TCR that specifically binds to a COL6A3 peptide presented on MHC-I and has the CDRs as disclosed in PCT / EP2018 / 080176.

[0424] Preferably, the TCR may comprise the CDRs comprising or consisting of the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15 and 16 as shown in Table 2 below.Said TCR may comprise a TCR a variable domain as comprised in the amino acid sequence of SEQ ID NO: 17 as shown in Table 2 below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the non-underlined sequence portion shown in Table 2 below, and / or may comprise a TCR [3 variable domain as comprised in the amino acid sequence shown below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the non-underlined sequence portion shown in Table 2 below.

[0425] In other preferred embodiments, the TCR may comprise the CDRs comprising or consisting of the amino acid sequence of SEQ ID NO: 19, 20, 21 , 22, 23 or 24 as shown in Table 2 below.

[0426] Said TCR may comprise a TCR a variable domain as comprised in the amino acid sequence of SEQ ID NO: 25 as shown in Table 2 below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the non-underlined sequence portion shown in Table 2 below, and / or may comprise a TCR [3 variable domain as comprised in the amino acid sequence of SEQ ID NO: 26 as shown in Table 2 below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the nonunderlined sequence portion shown in Table 2 below.

[0427] In further preferred embodiments, the antigen binding protein is a TCR that specifically binds to a CT45 peptide presented on MHC-I and has the CDRs as disclosed in PCT / EP2022 / 071104.

[0428] In preferred embodiments, the TCR comprises the CDRs comprising or consisting of SEQ ID NO: 27, 28, 29, 30, 31 and 32 as shown in Table 2 below.

[0429] Said TCR may comprise a TCR a variable domain as comprised in the amino acid sequence of SEQ ID NO: 33 as shown in Table 2 below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the non-underlined sequence portion shown in Table 2 below, and / or may comprise a TCR [3 variable domain as comprised in the amino acid sequence of SEQ ID NO: 34 as shown in Table 2 below, or an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity thereto, preferably to the nonunderlined sequence portion shown in Table 2 below.

[0430] The at least one nucleic acid sequence(s) encoding said antigen binding protein may be inserted into any suitable gene locus, for instance the B2M locus or the locus of a gene that is involved in NK cell inhibition, T cell exhaustion, and / or graft-versus-host disease, as exemplified above. A preferred example is the TGFBR2 gene locus, specifically in the context of y5 T cells. Another preferred example is the TRAC genelocus, specifically in the context of a|3 T cells. In this way, said heterologous nucleic acid sequence(s) is knocked in and the targeted gene is knocked out.

[0431] In preferred embodiments, the immune cell may be a yd T cell expressing a heterologous a|3 T cell receptor, e.g. from the TRAC or TGFBR2 locus. In further preferred embodiments, the immune cell may be an a|3 T cell expressing a heterologous a|3 T cell receptor, e.g. from the TRAC locus.

[0432] Point mutations in the transgenic TCR

[0433] In a further aspect of the disclosure, the heterologous TCR comprises one or more amino-acid substitutions within the EWTQD (SEQ ID NO: 68) motif located in the constant (C|3) region of the human TCR [3-chain. This motif forms part of an epitope recognized by the anti-human TCR a / |3 monoclonal antibody clone BW242 / 412 (commercially available from Miltenyi Biotec), and point mutations within this region have been found to reduce or abolish binding of this antibody. Modification of this epitope provides an additional means of controlling differential antibody-mediated recognition or depletion of engineered T cells expressing the transgenic and / or endogenous TCR.

[0434] The EWTQD motif consists of five amino acids — glutamate (E), tryptophan (W), threonine (T), glutamine (Q), and aspartate (D) — located at contiguous positions in the TRBC constant domain. In preferred embodiments, the heterologous TCR [3-chain comprises a mutation at one or more residues within this motif. Such mutations may include conservative or non-conservative substitutions, provided that antibody binding to the native epitope is reduced.

[0435] In one embodiment, the mutation comprises substitution of the glutamate residue (E) at the first position of the EWTQD motif with a non-acidic residue, for example: E^A, E^G, E^Q, or E^K. Such substitutions alter the local charge distribution and may reduce recognition by BW242 / 412. In another embodiment, the mutation comprises modification of the threonine residue (T) of the motif, including: T—A, T— K, T—V, orT^M. These substitutions may alter hydrogen-bonding potential within the targeted epitope or interfere with binding through steric hindrance. In an additional embodiment, the heterologous TCR [3-chain comprises a mutation of the glutamine residue (Q), such as: Q^A, Q^E, Q^K, Q^N, or Q^R. Such substitutions may interfere with antibody recognition without impairing TCR stability. In yet another embodiment, the mutation affects the aspartate residue (D) at the final position of the motif. Suitable substitutions include: D^A, D^G, D^K, or D^R. By modifying the acidic residue into an uncharged or basic residue, these substitutions can reduce the binding affinity of the BW242 / 412 antibody.

[0436] In certain preferred embodiments, the heterologous TCR [3-chain comprises two or more simultaneous point mutations within the EWTQD motif. In a preferredembodiment, the EWTQD epitope is mutated to EWKAK (SEQ ID NO: 69), wherein E^E, W— >W, T— K, Q^A, and D^K. In other embodiments, the EWTQD epitope is mutated to AWMQR (SEQ ID NO: 70), wherein E^A, W— >W, T^M, Q^A, and D^R.

[0437] Such combinations can strongly diminish or fully eliminate binding by BW242 / 412, while maintaining proper TCR folding and expression. Additionally, the predicted low MHC-II presentation of the mutated epitope indicates a favourable low immunogenicity, leading to reduced host-versus-graft disease and clearance.

[0438] Other multi-mutation variants are also contemplated, such as: EWTQA (SEQ ID NO: 71), EWSKD (SEQ ID NO: 72), AWTQD (SEQ ID NO: 73), EATKD (SEQ ID NO: 74), or EWAQN (SEQ ID NO: 75), and the skilled person will appreciate that additional combinations may be employed provided they reduce antibody binding relative to the wild-type EWTQD motif.

[0439] Modification of the EWTQD epitope reduces or abolishes binding of BW242 / 412, thereby allowing selective detection and depletion of endogenous versus transgenic TCR expressing T cells. These mutations therefore constitute an additional layer of control over receptor recognition, mispairing behaviour, and selective depletion profiles and favourably act together with the TRAC / TRBC1 / TRBC2 knockouts to improve the quality of the final T cell product with respect to residual expression of endogenous TCR and potential graft vs. host reactivity. The point mutations described herein are thus particularly relevant for the allogeneic a|3 T cells.

[0440] Regulatory elements

[0441] Any of the heterologous protein-encoding nucleic acid sequences described herein may be operably linked to at least one suitable regulatory element, such as a heterologous or endogenous promoter. As such, the at least one nucleic acid sequence(s) encoding the fusion protein, the at least one nucleic acid sequence(s) encoding the antigen binding protein and / or optionally the at least one nucleic acid sequence(s) encoding the CD8 a and / or CD8 [3 chain and / or the at least one nucleic acid sequence(s) encoding the cytokine may be operably linked to a heterologous or endogenous promoter, preferably a B2M, EFl alpha, EF1alpha-HTLV, MSCV, MND or MND-HTLV promoter.

[0442] B2M promoter

[0443] The B2M promoter drives the expression of the B2M (Beta-2-Microglobulin) gene.

[0444] The B2M promoter may comprise or consist of a nucleic acid sequence according any one of SEQ ID NO: 61 or 62 as shown in Table 2 below, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 61 or 62.EF1a promoter (constructs)

[0445] The EF1a (Elongation Factor 1 Alpha) promoter is a strong, constitutive promoter derived from the EF1a gene, which encodes an essential protein involved in the elongation phase of protein synthesis.

[0446] The EF1a -HTLV promoter is a hybrid promoter which combines elements of the EF1a promoter and the Human T-cell leukemia virus (HTLV) promoter. The EF1a-HTLV promoter may comprise or consist of a nucleic acid sequence according to SEQ ID NO: 63 as shown in Table 2 below, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 63.

[0447] MSCV promoter

[0448] The MSCV promoter is a strong, constitutive promoter derived from the Murine Stem Cell Virus. The MSCV promoter may comprise or consist of a nucleic acid sequence according to SEQ ID NO: 64 as shown in Table 2 below, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 64.

[0449] MND promoter (constructs)

[0450] The MND promoter is a strong, synthetic constitutive promoter, which combines elements from the Moloney Murine Leukemia virus (MMLV) and the myeloproliferative sarcoma virus (MPSV). The MND promoter may comprise or consist of a nucleic acid sequence according to SEQ ID NO: 65 as shown in Table 2 below, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 65.

[0451] The MND-HTLV promoter is a hybrid promoter which combines elements from the MND promoter and the Human T-cell leukemia virus (HTLV) promoter. The MND-HTLV promoter may comprise or consist of a nucleic acid sequence according to SEQ ID NO: 66 as shown in Table 2 below, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity SEQ ID NO: 66:

[0452] Any heterologous protein-encoding nucleic acid sequence described herein that is inserted into the B2M gene locus, for instance the nucleic acid(s) encoding the HLA-I fusion protein and optionally the nucleic acid(s) encoding CD8 a and / or [3 , and / or the nucleic acid(s) encoding the cytokine (fusion protein), may be operably linked to the endogenous B2M promoter.

[0453] In embodiments of the present disclosure, the at least one nucleic acid sequence(s) inserted into the B2M gene locus may thus be operably linked to the endogenous B2M promoter. In further embodiments of the present disclosure, the at least one nucleic acid sequence(s) inserted into the TRAC gene locus may be operably linked to a heterologous MND or B2M promoter. In further embodiments theat least one nucleic acid sequence(s) inserted into the TGFBR2 locus is / are operably linked to a heterologous MND-HTLV promoter.

[0454] Specific embodiments

[0455] In view of the above, the present disclosure provides immune cells comprising 1) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein, wherein said heterologous nucleic acid sequence(s) is / are inserted into the B2M gene locus, leading to a genetically engineered inactivation of the at least one B2M gene; said heterologous nucleic acid(s) further optionally encoding at least one CD8 a and / or P chain, and 2) at least one heterologous nucleic acid encoding an antigen binding protein, wherein said heterologous nucleic acid(s) is / are inserted into the TGFBR2 gene locus, or the TRAC gene locus, leading to a genetically engineered inactivation of said TGFBR2 or TRAC gene, respectively.

[0456] In some embodiments, the immune cell is a yb T cell, and the at least one heterologous nucleic acid encoding the antigen binding protein is inserted into the TGFBR2 gene locus. In some embodiments, the immune cell is a yb T cell, and the at least one heterologous nucleic acid encoding the antigen binding protein is inserted into the TRAC gene locus.

[0457] In some embodiments, the immune cell is a a|3 T cell, and the at least one heterologous nucleic acid encoding the antigen binding protein is inserted into the TRAC gene locus. In such embodiments, the a|3 T cell preferably comprises a genetically engineered inactivation of at least one, preferably all, TRBC1 and / or TRBC2 gene(s).

[0458] In preferred embodiments, the immune cell described herein comprises and preferably expresses at least the following heterologous nucleic acids:

[0459] a TCR a variable domain, or a derivative or fragment thereof;

[0460] a TCR p variable domain, or a derivative or fragment thereof;

[0461] a HLA-I a chain fusion protein;

[0462] optionally a CD8 a chain, or a derivative or fragment thereof;

[0463] optionally a CD8 p chain or a derivative or fragment thereof;

[0464] optionally an interleukin or interleukin fusion protein, wherein said interleukin is optionally selected from interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21.Preferably, the immune cell may comprise and preferably express at least the following heterologous nucleic acids:

[0465] a TCR a variable domain, or a derivative or fragment thereof;

[0466] a TCR p variable domain, or a derivative or fragment thereof;

[0467] a HLA-I a chain fusion protein, preferably a HLA-E a chain fusion protein, more preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1 or 2;

[0468] a CD8 a chain, or a derivative or fragment thereof; and

[0469] a CD8 p chain or a derivative or fragment thereof. The immune cell further comprises a genetically engineered inactivation of at least one, preferably all, B2M gene(s), and preferably of at least one, preferably all, TRAC gene(s) and / or TGFBR2 gene(s). Allogeneic ap T cell products

[0470] In preferred embodiments of the present disclosure, the immune cell is an ap T cell, more preferably an allogeneic ap T cell.

[0471] As described throughout the present disclosure, the allogeneic ap T cell preferably comprises a) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

[0472] The fusion protein is preferably a HLA-I fusion protein, more preferably a HLA-E fusion protein as described herein. The antigen binding protein is preferably a TCR as described herein, preferably an ap TCR comprising or consisting of a TCR a chain and a TCR p chain. The allogeneic ap T cell preferably further comprises at least one heterologous nucleic acid encoding CD8 as described herein. Preferably, the CD8 comprises or consist of a CD8 a chain and a CD8 p chain.

[0473] The TCR a and p chains may be encoded by the same or different heterologous nucleic acid molecules, although it is preferred that they are encoded by the same heterologous nucleic acid molecule. The CD8 a and p chains may be encoded by the same or different heterologous nucleic acid molecules, although it is preferred that they are encoded by the same heterologous nucleic acid molecule. In preferred embodiments, the HLA-E fusion protein and CD8 a and p chains are encoded by the same heterologous nucleic acid, where the genes are preferably separated by 2Aelements as described above. The TCR a and [3 chains are preferably encoded by a different heterologous nucleic acid, and are equally separated by 2A elements.

[0474] The heterologous nucleic acids encoding the HLA-E fusion protein and CD8 a and p chains, and the heterologous nucleic acid encoding the TCR a and p chains are preferably provided in the form of suitable constructs, which are designed as homology templates targeting the desired gene insertion sites. Such constructs are preferably provided in the form of suitable vectors, e.g. AAVs.

[0475] Specifically, the allogeneic ap T cell may thus preferably comprises a) a genetically engineered disruption of at least one, preferably all, B2M genes, preferably leading to a B2M knockout, b) a heterologous nucleic acid encoding a HLA-E fusion protein as described herein, and c) a heterologous nucleic acid encoding a TCR a chain and a TCR p chain. Additionally, the allogeneic ap T cell may preferably further comprise d) a heterologous nucleic acid encoding a CD8 a chain and a CD8 p chain. Further, the allogeneic ap T cell preferably further comprises a genetically engineered disruption in the TRAC and optionally the TRBC1 and TRBC2 gene(s), preferably leading to a TRAC and optionally TRBC1 and TRBC2 knockout. Finally, the heterologous TCR p-chain preferably further comprises at least one or more amino-acid substitutions within the EWTQD motif located in its constant (CP) region.

[0476] The genetically engineered inactivation of the at least one B2M gene and at least one TRAC, TRBC1 and TRBC2 genes is preferably achieved using CRISPR / Cas technology. The gene sequences at the B2M, TRAC, and TRBC1 / 2 loci are cut and disrupted by the Cas enzyme. Preferably, the HLA-E fusion protein transgene, and optionally the CD8 a and p chain transgenes, are inserted into the B2M locus. Preferably, the HLA-E fusion protein and CD8 a and p chain transgenes are operably linked to and controlled by the endogenous B2M promoter. Preferably, the TCR a and P chain transgenes are inserted into the TRAC locus. Preferably, the TCR a and p chain transgenes are operably linked to and controlled by a B2M promoter (fragment) according to SEQ ID NO: 61 or 62, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 61 or 62. Preferably, the heterologous TCR comprises point mutations in its constant domain, changing the EWTQD motif (SEQ ID NO: 68) to EWKAK (SEQ ID NO: 69) or AWMQR (SEQ ID NO: 70).

[0477] The HLA-E fusion protein preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 43 or 44, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 43 or 44.

[0478] The CD8 a chain preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 45.The CD8 [3 chain preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 48, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 48.

[0479] The TCR preferably specifically binds to an MHC-I presented target peptide selected from a CT45, PRAME, COL6A3, NY-ESO-1, MAGE-A3, KLK3, KLK4, KK-LC-1, MART-1, CEA, HA-1, KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide. For instance, the TCR may specifically bind to an MHC-I presented PRAME peptide comprising or consisting of the amino acid sequence according to SEQ ID NO: 67. Such TCRs may comprise the CDRs comprising or consisting of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7 and / or 8. Preferably, such TCRs may comprise a TCR a variable domain according to SEQ ID NO: 9 or 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 9 or 10. However, any other TCR, preferably any TCR exemplified herein, is also contemplated.

[0480] Taken together, the allogeneic a|3 T-cell product disclosed herein provides multiple structural and functional advantages arising from a series of coordinated genetic and molecular design improvements. Optimized nucleic acid constructs ensure controlled and high-level expression of the transgenic TCR and, in certain embodiments, co-expression of CD8, thereby enhancing receptor function and product consistency. An improved immune-evasion strategy combines B2M knockout with knock-in of a single-chain HLA-E fusion protein engineered for enhanced surface expression, enabling effective protection from host-versus-graft immune responses and supporting prolonged in vivo persistence. Additionally, targeted disruption of the TRAC, TRBC1 , and TRBC2 loci eliminates endogenous TCR expression and prevents mispairing with the introduced TCR chains, thereby increasing safety and functional uniformity. The transgenic TCR further comprises a defined mutation permitting selective removal of residual endogenous TCR-expressing cells through antibody-based enrichment, resulting in a highly standardized, fully defined cell product. Collectively, these modifications yield an allogeneic a|3 T-cell platform with enhanced safety, functional performance, product homogeneity, and manufacturing robustness.

[0481] Allogeneic y5 T cell products

[0482] In further preferred embodiments of the present disclosure, the immune cell is a y5 T cell, more preferably an allogeneic y5 T cell.

[0483] As described throughout the present disclosure, the allogeneic y5 T cell preferably comprises a) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-Iprotein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

[0484] The fusion protein is preferably a HLA-I fusion protein, more preferably a HLA-E fusion protein as described herein. The antigen binding protein is preferably a TCR as described herein, preferably an a|3 TCR comprising or consisting of a TCR a chain and a TCR [3 chain. The allogeneic a|3 T cell preferably further comprises at least one heterologous nucleic acid encoding CD8 as described herein. Preferably, the CD8 comprises or consist of a CD8 a chain and a CD8 [3 chain.

[0485] The TCR a and [3 chains may be encoded by the same or different heterologous nucleic acid molecules, although it is preferred that they are encoded by the same heterologous nucleic acid molecule. The CD8 a and [3 chains may be encoded by the same or different heterologous nucleic acid molecules, although it is preferred that they are encoded by the same heterologous nucleic acid molecule. In preferred embodiments, the HLA-E fusion protein and CD8 a and [3 chains are encoded by the same heterologous nucleic acid, where the genes are preferably separated by 2A elements as described above. The TCR a and [3 chains are preferably encoded by a different heterologous nucleic acid, and are equally separated by 2A elements.

[0486] Specifically, the allogeneic yb T cell may thus preferably comprises a) a genetically engineered disruption of at least one, preferably all, B2M genes, preferably leading to a B2M knockout, b) a heterologous nucleic acid encoding a HLA-E fusion protein as described herein, and c) a heterologous nucleic acid encoding a TCR a chain and a TCR [3 chain. Additionally, the allogeneic yb T cell may preferably further comprise d) a heterologous nucleic acid encoding a CD8 a chain and a CD8 [3 chain. Further, the allogeneic yb T cell preferably further comprises a genetically engineered disruption in the TGBFR2 gene, preferably leading to a TGBFR2 knockout.

[0487] The genetically engineered inactivation of the at least one B2M gene and at least one TGBFR2 gene is preferably achieved using CRISPR / Cas technology. The gene sequences at the B2M, and TGBFR2 loci are cut and disrupted by the Cas enzyme. Preferably, the HLA-E fusion protein transgene, and optionally the CD8 a and [3 chain transgenes, are inserted into the B2M locus. Preferably, the HLA-E fusion protein and CD8 a and [3 chain transgenes are encoded by the same heterologous nucleic acid and are operably linked to and controlled by the endogenous B2M promoter. Preferably, the TCR a and [3 chain transgenes are inserted into the TGBFR2 locus. Preferably, the TCR a and [3 chain transgenes are operably linked to and controlled by an MND promoter fragment according to SEQ ID NO: 65, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 65. Alternatively, in equally preferred embodiments, the TCR a and [3 chain transgenes are targeted to and inserted into the TRAC locus. Preferably, the TCR a and [3 chain transgenes are operably linked to and controlled by a B2M promoter (fragment) according to SEQ ID NO: 61 or 62, or a nucleic acid sequencehaving at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 61 or 62.

[0488] The heterologous nucleic acids encoding the HLA-E fusion protein and CD8 a and [3 chains, and the heterologous nucleic acid encoding the TCR a and p chains are preferably provided in the form of suitable constructs, which are designed as homology templates targeting the desired gene insertion sites. Such constructs are preferably provided in the form of suitable vectors, e.g. AAVs.

[0489] The HLA-E fusion protein preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 43 or 44, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 43 or 44.

[0490] The CD8 a chain preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 45, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 45.

[0491] The CD8 p chain preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 48, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 48.

[0492] The TCR preferably specifically binds to an MHC-I presented target peptide selected from a CT45, PRAME, COL6A3, NY-ESO-1, MAGE-A3, KLK3, KLK4, KK-LC-1, MART-1, CEA, HA-1, KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide. For instance, the TCR may specifically bind to an MHC-I presented PRAME peptide comprising or consisting of the amino acid sequence according to SEQ ID NO: 67. Such TCRs may comprise the CDRs comprising or consisting of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7 and / or 8. Preferably, such TCRs may comprise a TCR a variable domain according to SEQ ID NO: 9 or 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 9 or 10. However, any other TCR, preferably any TCR exemplified herein, is also contemplated.

[0493] Further exemplary engineering strategies for allogeneic y5 T cell products are shown in the Table below:

[0494] TRAC B2M* TGFbR Name wt scHLA-E-CD8 (MND-P)TCR V1 wt (MND-P)-CD8 (MND-P)TCR V2 wt CD8-HLA-E (MND-P)TCR V3 (B2M-P)-TCR (MND-P)-CD8 wt V4

[0495] (B2M-P)-TCR CD8 wt V5

[0496] (B2M-P)-TCR scHLA-E-CD8 wt V6 (EFIa-P)-TCR (MND-P)-CD8 wt V7

[0497]

[0498] (MND-P)-TCR (MND-P)-CD8 wt V8(EFIa-P)-TCR (MND-P)-CD8 KO V9 (MND-P)-TCR (MND-P)-CD8 KO V10

[0499] wt scHLA-E-CD8 (MND-HTLV- V11 P)TCR-mblL15

[0500] wt scHLA-E-CD8a- (MND-P)TCR V12 mblL15

[0501] wt scHLA-E-TCR (MND-HTLV- V13

[0502]

[0503] P)CD8-mblL15

[0504] *if not indicated otherwise, constructs inserted into the B2M locus used the endogenous (“endo”) B2M promoter.

[0505] (Pharmaceutical) composition

[0506] In a further aspect, the present disclosure provides a (pharmaceutical) composition comprising the immune cell.

[0507] The term “pharmaceutical composition” shall have the meaning as defined elsewhere herein. The pharmaceutical composition may be provided in soluble or cryopreserved form and may be thawed before use. The pharmaceutical composition may comprise suitable pharmaceutically acceptable carrier(s), excipient(s) and / or stabilizer(s). Exemplary suitable pharmaceutically acceptable carriers include any isotonic carrier such as, for example, saline (about 0.90% w / v of NaCI in water, about 300 mOsm / L NaCI in water, or about 9.0 g NaCI per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. The pharmaceutical composition may further include excipients such as dimethyl sulfoxide (DMSO) and dextran 40. In some instances, the pharmaceutically acceptable composition may be supplemented with human serum albumin.Kit

[0508] In a further aspect, the present disclosure provides a kit comprising the (pharmaceutical) composition or the immune cell. Optionally, the kit may further comprise at least one further therapeutic or co-therapeutic agent. For instance, the kit may additionally comprise IL-2.

[0509] The kit may be a kit of parts and may comprise separate containers holding the (pharmaceutical) composition or immune cell, and optionally further therapeutic or co-therapeutic agents.

[0510] Methods and Uses

[0511] In a further aspect, the present disclosure provides the immune cell, (pharmaceutical) composition or kit for use in medicine. Specifically, the present disclosure provides the immune cell(s) or (pharmaceutical) composition or kit for use in treating cancer, an autoimmune disease, or an infection.

[0512] In a further aspect, the present disclosure accordingly also provides a method of treating cancer, an autoimmune disease, or an infection, said method comprising administering the immune cell(s) or (pharmaceutical) composition or kit to a patient in need thereof.

[0513] The uses and methods of the present disclosure may further comprise administering said immune cell(s), (pharmaceutical) composition or kit and, additionally, at least one further therapeutic or co-therapeutic agent or treatment.

[0514] The immune cell(s) is / are preferably administered in a therapeutically effective amount and according to a suitable treatment regimen to the patient.

[0515] The methods and uses of the present disclosure may comprise administering said immune cell(s) or pharmaceutical composition to the patient in a single dose.

[0516] The methods and uses of the present disclosure may comprise administering said immune cell(s) or pharmaceutical composition intravenously to the patient.

[0517] The methods and uses of the present disclosure may comprise administering said immune cell(s) or pharmaceutical composition and additionally administering interleukin-2, interleukin-15, or both to the patients.

[0518] In some embodiments of the methods and uses of the present disclosure, the treatment does not require administration of interleukin-15.ASPECTS

[0519] The invention of the present disclosure is defined, inter alia, by the following aspects: 1. An immune cell comprising

[0520] a) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

[0521] 2. The immune cell of aspect 1, wherein said immune cell further expresses a peptide that is presented by the fusion protein on the cell surface.

[0522] 3. The immune cell of aspect 2, wherein said peptide is covalently linked to at least a portion of said HLA-I protein.

[0523] 4. The immune cell of aspect 3, wherein said fusion protein does not present any other peptides on the cell surface.

[0524] 5. The immune cell of any one of the preceding aspects, wherein said portion of the HLA-I protein comprises or consists of an HLA class I a chain or a variant or derivative thereof.

[0525] 6. The immune cell of aspect 5, wherein said HLA class I a chain is selected from HLA-E, HLA-F and HLA-G.

[0526] 7. The immune cell of any one of the preceding aspects, wherein said immune cell comprises a genetically engineered inactivation of all copies of the B2M gene, preferably causing a B2M knockout.

[0527] 8. The immune cell of any one of the preceding aspects, wherein said immune cell is a human cell.

[0528] 9. The immune cell of aspect 8, wherein said immune cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably an alpha beta or gamma delta T cell, such as a V deltal T Cells or a V delta2 T cell.

[0529] 10. The immune cell of any one of the preceding aspects, wherein said genetically engineered inactivation of the at least one B2M gene is achieved through insertion of the at least one heterologous nucleic acid sequence(s) into the B2M gene locus.11. The immune cell of any one of the preceding aspects, further comprising d) at least one heterologous nucleic acid sequence(s) encoding a CD8 alpha and / or CD8 beta chain.

[0530] 12. The immune cell of any one of the preceding aspects, wherein said at least one nucleic acid sequence(s) encoding said fusion protein according to b) and / or said at least one nucleic acid sequence(s) encoding an antigen binding protein according to c) and / or said at least one nucleic acid sequence(s) encoding a CD8 alpha and / or CD8 beta chain according to d) is / are inserted into the B2M gene locus.

[0531] 13. The immune cell of any one of the preceding aspects, further comprising e) a genetically engineered inactivation of a gene associated with any one of NK cell inhibition, T cell exhaustion, and / or graft-versus-host disease, preferably selected from a TGFBR2 gene, a TRAC gene, a TRBC gene, a CIITA gene, a CISH gene, a PD-1 gene, a CTLA-4 gene, a NKG2A gene, a CBLB gene, a REG1 gene, a PTPN2 gene, a LAG3 gene, a TIM3 gene and / or a FAS gene.

[0532] 14. The immune cell of aspect 13, wherein said immune cell comprises a genetically engineered inactivation of all copies of theTGFBR2, TRAC, TRBC1 , CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1 PTPN2, LAG3, TIM3 and / or FAS genes, respectively.

[0533] 15. The immune cell of aspect 13 or 14, wherein the genetically engineered inactivation of the TGFBR2, TRAC, TRBC, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1 , PTPN2, LAG3, TIM3 and / or FAS genes causes a knockout of TGFBR2, TRAC, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3 and / or FAS, respectively.

[0534] 16. The immune cell of any one of the preceding aspects, further comprising at least one heterologous nucleic acid sequence(s) encoding or enhancing the expression of interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21.

[0535] 17. The immune cell of any one of the preceding aspects, wherein

[0536] said at least one nucleic acid sequence(s) encoding said fusion protein according to b) and optionally said at least one nucleic acid(s) encoding said CD8 alpha and / or beta chain according to d) is / are inserted into the B2M gene locus; and said at least one nucleic acid sequence(s) encoding said antigen binding protein according to c) is / are inserted into the TGFBR2 locus.

[0537] 18. The immune cell of any one of the preceding aspects, whereinsaid at least one nucleic acid sequence(s) encoding said fusion protein according to b) and optionally said at least one nucleic acid(s) encoding said CD8 alpha and / or beta chain according to d) is / are inserted into the B2M gene locus; and said at least one nucleic acid sequence(s) encoding said antigen binding protein according to c) is / are inserted into the TRAC locus and / or TRBC locus.

[0538] 19. The immune cell of any one of the preceding aspects, wherein said at least one nucleic acid sequence(s) encoding said fusion protein according to b) said at least one nucleic acid sequence(s) encoding an antigen binding protein according to c) and / or optionally said at least one nucleic acid sequence(s) encoding a CD8 alpha and / or CD8 beta chain according to d) is / are operably linked to a heterologous or endogenous promoter, preferably a B2M, EF1 alpha, EF1alpha-HTLV, MSCV, MND or MND-HTLV promoter.

[0539] 20. The immune cell of any one of the preceding aspects, wherein said at least one nucleic acid sequence(s) inserted into the B2M gene locus is / are operably linked to the endogenous B2M promoter.

[0540] 21. The immune cell of any one of the preceding aspects, wherein said at least one nucleic acid sequence(s) inserted into the TRAC gene locus is / are operably linked to a heterologous MND or B2M promoter.

[0541] 22. The immune cell of any one of the preceding aspects, wherein said fusion protein comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

[0542] 23. The immune cell of any one of the preceding aspects, wherein said nucleic acid sequence(s) encoding said fusion protein comprise(s) or consist(s) of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence according to SEQ ID NO: 11 or SEQ ID NO: 12.

[0543] 24. The immune cell of any one of the preceding aspects, wherein said antigen binding protein comprises six complementarity determining regions (CDR) CDRal, CDRa2, CDRa3, CDRbl, CDRb2, and CDRb3, wherein said CDRal, CDRa2 and CDRa3 are preferably comprised by a first polypeptide, and said CDRbl , CDRb2, and CDRb3 are preferably comprised by a second polypeptide..

[0544] 25. The immune cell of any one of the preceding aspects, wherein said antigen binding construct is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 26. The immune cell of any one of the preceding aspects, wherein said first polypeptide comprises or consists of a TCR alpha-chain variable domain (Valpha) or a derivative or fragment thereof.27. The immune cell of any one of the preceding aspects, wherein said second polypeptide comprises or consists of a TCR beta-chain variable domain (Vbeta) or a derivative or fragment thereof.

[0545] 28. The immune cell of any one of the preceding aspects, wherein said first and / or said second polypeptide further comprises a TCR constant domain.

[0546] 29. The immune cell of any one of aspects 26 to 28, wherein said immune cell is a yb T cell expressing a heterologous a|3 T cell receptor.

[0547] 30. The immune cell of any one of the preceding aspects, wherein said immune cell comprises and preferably expresses at least the following heterologous nucleic acids: a TCR alpha chain variable domain, or a derivative or fragment thereof; a TCR beta chain variable domain, or a derivative or fragment thereof; a HLA-I a chain fusion protein; and optionally at least one of the following additional heterologous ORFs: a CD8 alpha chain, or a derivative or fragment thereof;

[0548] a CD8 beta chain or a derivative or fragment thereof;

[0549] an interleukin, optionally selected from interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21.

[0550] 31. The immune cell of aspect 30, wherein said immune cell comprises and preferably expresses at least the following heterologous nucleic acids: a TCR alpha chain variable domain, or a derivative or fragment thereof; a TCR beta chain variable domain, or a derivative or fragment thereof; a HLA-I a chain fusion protein; a CD8 alpha chain, or a derivative or fragment thereof; and a CD8 beta chain or a derivative or fragment thereof;

[0551] 32. The immune cell of any one of the preceding aspects, wherein said antigen binding protein is capable of specifically binding to a target peptide presented by an MHC-I molecule.

[0552] 33. The immune cell of aspect 32, wherein said target peptide is a CT45, PRAME, COL6A3, NY-ESO-1, MAGE-A3, KLK3, KLK4, KK-LC-1, MART-1, CEA, HA-1, KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide.

[0553] 34. The immune cell of any one of aspects 24 to 33, wherein

[0554] said CDRal comprises or consists of an amino acid sequence according to SEQ ID NO: 3;

[0555] said CDRa2 comprises or consists of an amino acid sequence according to SEQ ID NO: 4;

[0556] said CDRa3 comprises or consists of an amino acid sequence according to SEQ ID NO: 5;said CDRbl comprises or consists of an amino acid sequence according to SEQ ID NO: 6;

[0557] said CDRb2 comprises or consists of an amino acid sequence according to SEQ ID NO: 7; and

[0558] said CDRb3 comprises or consists of an amino acid sequence according to SEQ ID NO: 8.

[0559] 35. The immune cell of any one of aspects 26 to 34, wherein said TCR alpha-chain variable domain (Valpha) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 9.

[0560] 36. The immune cell of any one of aspects 27 to 35, wherein said TCR beta-chain variable domain (Vbeta) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0561] 37. The immune cell of any one of the preceding aspects, wherein heterologous nucleic acid sequence(s) is / are introduced into said immune cell through adeno-associated vectors (AAVs) and a CRISPR / Cas system.

[0562] 38. The immune cell of aspect 37, wherein said CRISPR / Cas system comprises a Cas protein selected from Cas9 or a variant or derivative thereof, or Cas12 or a variant or derivative thereof.

[0563] 39. The immune cell of aspect 38, wherein said Cas9 variant is selected from SpCas9, SaCas9, St1Cas9, NmCas9, or CjCas9.

[0564] 40. The immune cell of aspect 39, wherein said Cas12 variant is selected from Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12e (C2c5), Cas12f (Cas14), or Cas12i; preferably Cas12a (Cpf1) or Cas12i.

[0565] 41. A (pharmaceutical) composition comprising the immune cell of any one of the preceding aspects, an optionally a pharmaceutically acceptable carrier or excipient.

[0566] 42. A kit comprising the immune cell or (pharmaceutical) composition of any one of the previous aspects, and optionally at least one further therapeutic or co-therapeutic agent.

[0567] 43. The immune cell(s) or (pharmaceutical) composition or kit of any one of the preceding aspects for use in medicine.

[0568] 44. The immune cell(s) or (pharmaceutical) composition or kit of any one of the preceding aspects for use in treating cancer, an autoimmune disease, or an infection.45. A method of treating cancer, an autoimmune disease, or an infection, said method comprising administering the immune cell(s) or (pharmaceutical) composition or kit of any one the preceding aspects to a patient in need thereof.

[0569] 46. The use or method of any one of aspect 43 to 45, comprising administering said immune cell(s), (pharmaceutical) composition or kit and, additionally, at least one further therapeutic or co-therapeutic agent or treatment.EXAMPLES

[0570] Example 1: Allogeneic y6 T cells

[0571] yb T cells were obtained, activated and expanded as described in PCT / US2018 / 062442 or using standard protocols known in the art. Cas12a-containing RNPs were transfected into the cells by electroporation, followed by infection with recombinant Adeno-associated virus (AAV) vectors providing homology templates for sequence Knockin to construct B2M deficient yb T cells with transgenic TCRs, HLA-E fusion proteins and optionally CD8 and / or membrane-bound IL-15. Two different AAVs were used to deliver donor templates targeting the human B2M and TGFBR2 or TRAC gene and carrying the sequences encoding transgenic TCR, CD8, fusion protein or membrane-bound IL15, respectively (see description of the Figures).

[0572] The yb T cell products evaluated in the Examples comprised the following sequences:

[0573] Transgene Encoded sequence

[0574] SEQ ID NO:

[0575] HLA-E Fusion Protein 1 or 2

[0576] CD8a 45

[0577] CD8[3 48

[0578] TCRa 76

[0579] TCR[3 77

[0580] IL-15 fusion protein 58

[0581]

[0582] Several construct designs were tested. Typically, several genes were delivered per each construct, each separated by 2A peptides, with two constructs being used for each yb T cell product (see Figures Legends for details on construct design). In the in vitro tumor cell killing experiments (FIG. 9) and in vivo study (FIG. 10), constructs V1 (2A-scHLA-E-A2-CD8a-2A-CD8[3 into the B2M locus and MND-TCR[3-2A-TCRa in TGFBR2 locus) and V6 (B2M-TCR[3-2A-TCRa into the TRAC locus and scHLA-E-2A-CD8a-2A-CD8[3 into the B2M locus) were used.

[0583] Example 1.1: Expression of TCR, CD8, IL-15 and HLA-E fusion proteins in B2M deficient cellsEngineered y5 T cells were stained and subjected to flow cytometry to evaluate the expression of B2M, TCR, CD8, IL-15 and HLA-E fusion proteins.

[0584] Example 1.2: Mixed lymphocyte reaction

[0585] B2M KO yd T cells were generated according to the methods described previously and subsequently co-cultured with both HLA-matched and HLA-mismatched peripheral blood mononuclear cells (PBMCs). Activation of T cells from these PBMC was assessed by measuring proliferation and CD25 expression. The B2M knock-out cells elicited a significantly reduced CD8+ T cell activation when cocultured with H LA-mismatched PBMCs compared to wildtype cells (FIG. 4A), indicating reduced allorejection. Notably, the B2M knock-out had no detectable effect on CD4+ T cell activation (FIG. 4B), demonstrating selective modulation of immune responses.

[0586] Example 1.3: NK killing assay

[0587] Engineered y5 T cells were co-cultured with expanded, activated NK cells. Target cell killing was assessed using flow cytometry after 24 of coculture. B2M KO T cells were more susceptible to killing by the NK cells than unengineered controls. Expression of an HLA-E fusion protein (“HLA-E”) significantly reduced NK cell killing compared to B2M knock-out (KO) only T cells, indicating an effective inhibition of missing-self recognition. HLA negative K562 cells served as a control to confirm NK cell-mediated killing.

[0588] Example 1.4: In vitro T cell killing assay

[0589] Engineered y5 T cells were added to cells presenting the cognate HLA:peptide antigen for the transgenic ab TCR (“targets”) at an effector: target ratio of 1 :1 or 1 :2 in the absence or presence of 10 ng / ml TGF-a. Target cell killing was monitored using a time lapse imaging system measuring fluorescent from the RFP expressing target cells. After the initially provided target cells had been killed, the effector T cells were transferred to a new plate of preseeded target cells to assess their ability to repeatedly attack the tumor cells. After each round of coculture, the media was analyzed for interferon gamma secreted by the effector T cells. At the end of the experiment, the effector cells were enumerated by flow cytometry to measure persistence and proliferation.

[0590] Example 1.5: In vivo study

[0591] y5 T cells were evaluated in an in vivo tumor model using NOG mice. The objective was to assess the efficacy and persistence cells produced with different engineering strategies. The tumor model utilized the Hs695T cell line, with 1 million cells mixed with Matrigel.Three treatment groups were established: Group 1 received unedited y5 T cells, administered intravenously to four mice. The treatment included IL-2 at 15,000 III and IL-15 at 28,000 IU, or IL-2 only, administered subcutaneously three times per week, and a single dose of 2e7 y5 T cells on day 7 after tumor injection. Group 2 received TCR / CD8-scHLA-E-TGFbR-KO y5 T cells (construct setup V1), with the same administration route, group size, cytokine treatment, and dose as Group 1. Group 3 received TCR / CD8-scHLA-E y5 T cells (construct setup V6), following the same treatment protocol.

[0592] Hs695T cells were injected subcutaneously on day 0. y5 T cell treatment treatment started on day 7. Cytokines were administered 3 times a week for 5 weeks in total starting on day 7.

[0593] Readouts for the study included tumor growth measurement to assess efficacy and blood persistence analysis via flow cytometry to evaluate the persistence of y5 T cells in the bloodstream.

[0594] Example 2: Allogeneic a T cells

[0595] Example 2.1: Preparation: Peripheral blood mononuclear cells (PBMC) from healthy donors were activated in cytokine supplemented T- cell expansion media commonly used in the field (eg. TEXMACS, Optimizer, etc.) with immobilized anti-CD3 and soluble anti-CD28 antibodies for two days. The activated cells were engineered by electroporation with Cas12-based RNPs targeting TRAC, TRBC and B2M genetic loci followed by infection with recombinant AAVs delivering repair templates with partial homology to the genomic sequence flanking the cleaved sites within the TRAC and B2M loci. The repair templates contained the sequences of the transgenic TCR chains with point mutations to prevent binding by abTCR antibody to facilitate residual endogenous TCR expressing cells, HLA-E fusion protein and CD8 chains. After the engineering step, the cells were expanded in cytokine supplemented media for five days. For depletion of residual cells expressing the endogenous TCR, the cells were transferred into MACS separation buffer, incubated with abTCR-Biotin antibody (Miltenyi, Cat# 130-113-529) and anti-streptavidin beads (Miltenyi, Cat# 130-048-102) and passed through an LD column (Miltenyi Biotec). After depletion, the cells were cultivated for another three days in medium including cytokines. Success of expansion and engineering was evaluated by cell counting and flow cytometric analysis of TCR (endogenous and transgenic), CD8, B2M and HLA-E. Transgenic TCR was detected through the use of specific Vb antibody.

[0596] The a|3 T cell products evaluated in the Examples comprised the following sequences:Transgene Encoded sequence

[0597] SEQ ID NO:

[0598] HLA-E Fusion Protein 1 or 2

[0599] CD8a 45

[0600] CD8P 48

[0601] TCRa 76

[0602] TCRP 77

[0603]

[0604] Several genes were delivered per each construct, each separated by 2A peptides, with two constructs being used for each a|3 T cell product. The constructs were designed as follows: 2A-scHLA-E-A2-CD8a-2A-CD8[3 into the B2M locus, and B2M(promoter)-TCR[3-A2-TCRa into the TRAC locus.

[0605] Example 2.2: Expression of TCR, CD8, IL-15 and HLA-E fusion proteins in B2M deficient cells

[0606] Engineered a|3 T cells were stained and subjected to flow cytometry to evaluate the expression of B2M, TCR, CD8, and HLA-E fusion proteins. Results showed that a|3 T cells can be extensively multiplex-engineered by inserting five ORFs into two knockout loci with high editing efficiencies. Heterologous TCRs, CD8 chains, and an HLA-E fusion protein were successfully integrated at the intended sites, and the resulting edited cells expanded vigorousl -over 100-fold- demonstrating that complex, multi-gene engineering is compatible with strong T-cell proliferation (FIG: 12).

[0607] Example 2.3: Mutations in TCR constant domain for enhanced selective depletion of endogenous T cell receptors.

[0608] Point mutations were introduced into the EWTQD motif in the transgenic TCR [3-chain by standard genetic engineering methods. Cells were subjected to magnetic-activated cell sorting / MACS (Miltenyi Biotec) according to the supplier’s protocol with the anti-a[3 TCR antibody clone BW242 / 412 (Mitenyi), and analyzed using flow cytometry.

[0609] Results indicate that introducing defined mutations into the transgenic TCR [3-chain constant region prevent recognition by the BW242 / 412 antibody used to detect and deplete residual endogenous TCR-expressing cells. Among the designs tested, the EWKAK mutant displayed the strongest expression of the transgenic TCR and the lowest level of mispaired TCRs, indicating superior characteristics (FIG. 13, 14).Example 2.4: Disruption of the TBRC1 and TBRC2 genes

[0610] Guide RNAs for Cas12 were designed to target identical sites in the TBRC1 and TBRC2 loci using Chop Chop and CRISPIC. Simultaneous knockout of TRAC and TRBC1 / 2 markedly diminished endogenous TCR expression and strongly reduced TCR chain mispairing. With both loci disrupted, endogenous TCR-positive cells were largely eliminated, and introduction of a mutant transgenic TCR at the TRAC locus led to only rare mispairing events (FIG. 15, 16). Recovered cell products retained viability or proliferative capacity (FIG. 17). Any residual mixed TCRs were further minimized by depletion of the few remaining endogenous TCR-expressing cells using MACS as described above, demonstrating an effective strategy to prevent mispairing and improve the fidelity of transgenic TCR expression (FIG. 18).

[0611] Example 2.5: NK killing assay

[0612] Engineered ab T cells were co-cultured with expanded, activated NK cells. Target cell killing was assessed using flow cytometry after 24 of coculture. B2M KO T cells were more susceptible to killing by the NK cells than unengineered controls. Expression of an HLA-E fusion protein (“HLA-E”) significantly reduced NK cell killing compared to B2M knock-out (KO) only T cells, indicating an effective inhibition of missing-self recognition. HLA negative K562 cells served as a control to confirm NK cell-mediated killing.

[0613] The results show that knocking out both B2M and TRAC heightened susceptibility of engineered T cells to NK-cell-mediated killing, confirming the expected loss of HLA-l-mediated protection. Introducing an HLA-E fusion protein effectively reversed this sensitivity: T cells carrying the HLA-E knock-in exhibited substantially increased resistance to NK cytotoxicity compared with B2M / TRA C-deficient cells, demonstrating that HLA-E expression can restore NK-cell evasion in fully engineered T-cell products (FIG. 19).

[0614] Example 2.6: In vitro T cell killing assay

[0615] Engineered ab T cells were added to cells presenting the cognate HLA:peptide antigen for the transgenic ab TCR (“targets”) at an effector: target ratio of 1:1. Target cell killing was monitored using a time lapse imaging system measuring fluorescent from the RFP expressing target cells. After the initially provided target cells had been killed, the effector T cells were transferred to a new plate of preseeded target cells to assess their ability to repeatedly attack the tumor cells. After each round of coculture, the media was analyzed for interferon gamma secreted by the effector T cells.

[0616] The results demonstrate that engineered allogeneic a|3 T cells expressing a transgenic TCR together with an HLA-E fusion protein and CD8a[3 maintained potent, antigen-specific cytotoxicity in vitro. When repeatedly challenged with tumor cell lines expressing the target antigen at high (Hs695T) or low (SNU475) density, thesemultiplex-edited T cells consistently eliminated tumor cells across multiple rounds, while control cultures showed continued tumor growth. Products from two independent donors showed similarly sustained, TCR-dependent killing, indicating robust and reproducible antitumor activity of the engineered allogeneic a|3 T cells (FIG. 20). Example 2.7: Target sensitivity and polyfunctionality

[0617] T2 cells were loaded with different concentrations of target peptide and cocultured with TCR-transduced allogenic effector T Cells at a ratio of 1 : 1. After 24 h of coculture, some wells were harvested and subjected to flow cytometry analysis to enumerate residual target cells and detect activation of effector cells. A clear dose dependence of cytotoxicity and cytokine production was observed with 1 nm peptide loading concentration being sufficient for robust responses.

[0618] Six hours later, additional wells containing T2 cells loaded with 100nm peptide were harvested and the effector T cells were stained for intracellular cytokine content. Production of IL2, IFN-gamma and TNF-alpha were analyzed simultaneously to determine the polyfunctionality of the T cell population.

[0619] Results indicate that engineered allogeneic a|3 T cells secreted strong, TCR-dependent IFN-y in response to tumor cells expressing the target antigen, even after repeated rounds of stimulation. Across multiple rechallenge cycles with Hs695T and SNU475 cells, the T-cell products maintained robust cytokine release while controls showed minimal activity. In dose-response assays, engineered T cells from multiple donors produced high levels of IFN-y and displayed potent antigen-dependent killing across increasing peptide concentrations (FIG. 21). They also mounted a polyfunctional response, with a large proportion of cells producing multiple cytokines, including IL-2, TNFa, and IFN-y, indicating a strong, multi-cytokine effector profile characteristic of highly functional allogeneic a|3 T-cell products (FIG. 22).

[0620] Example 2.8: In vivo study

[0621] ab T cells were evaluated in an in vivo tumor model using NOG mice. The objective was to assess the efficacy of the anti tumor response at different cell doses. The tumor model utilized the Hs695T cell line, with 1 million cells mixed with Matrigel.

[0622] Once the tumors had reached a volume around 100mm3, the mice were randomized and four treatment groups were established: Group 1 received 6 x106unedited ab T cells, administered intravenously. Group 2 - 4 received 6 x106, 3 x106and 1.5 x106TCR / CD8-scHLA-E T cells, respectively, using the same administration route.

[0623] Readouts for the study included tumor growth measurement to assess efficacy and blood persistence analysis via flow cytometry to evaluate the persistence of ab T cells in the bloodstream.All administered dose levels 1.5X106, 3x106, and 6x106cells per mouse effectively controlled tumor growth, whereas non-engineered control cells did not. These results show that the engineered T-cell products mediate strong, dose-independent tumor suppression in vivo (FIG. 23).SEQUENCES

[0624] Table 2

[0625] SEQ ID Name Sequence

[0626] NO:

[0627] 1 HLA-E fusion protein VMAPRTLVLG GGGSGGGGSG GGGSIQRRTP KIQVYSRHPA v1 (SEQ ID NO: 1) ENGKSNFLNC YVSGFHPSDI EVDLLKNGER IEKVEHSDLS FSKDWSFYLL YYTEFTPTEK DEYACRVNHV TLSQPKIVKW DRDMGGGGSG GGGSGGGGSG GGGSGSHSLK YFHTSVSRPG RGEPRFISVG YVDDTQFVRF DNDAASPRMV PRAPWMEQEG SEYWDRETRS ARDTAQIFRV NLRTLRGYYN QSEAGSHTLQ WMHGCELGPD GRFLRGYEQF AYDGKDYLTL NEDLRSWTAV DTAAQISEQK SNDASEAEHQ RAYLEDTCVE WLHKYLEKGK ETLLHLEPPK THVTHHPISD HEATLRCWAL GFYPAEITLT WQQDGEGHTQ DTELVETRPA GDGTFQKWAA VVVPSGEEQR YTCHVQHEGL PEPVTLRWKP ASQPTIPIVG IIAGLVLLGS VVSGAVVAAV IWRKKSSGGK GGSYSKAEWS DSAQGSESHS L

[0628] 2 HLA-E fusion protein VMAPRTLILG GGGSGGGGSG GGGSIQRRTP KIQVYSRHPA v2 (SEQ ID NO: 2) ENGKSNFLNC YVSGFHPSDI EVDLLKNGER IEKVEHSDLS FSKDWSFYLL YYTEFTPTEK DEYACRVNHV TLSQPKIVKW DRDMGGGGSG GGGSGGGGSG GGGSGSHSLK YFHTSVSRPG RGEPRFISVG YVDDTQFVRF DNDAASPRMV PRAPWMEQEG SEYWDRETRS ARDTAQIFRV NLRTLRGYYN QSEAGSHTLQ WMHGCELGPD GRFLRGYEQF AYDGKDYLTL NEDLRSWTAV DTAAQISEQK SNDASEAEHQ RAYLEDTCVE WLHKYLEKGK ETLLHLEPPK THVTHHPISD HEATLRCWAL GFYPAEITLT WQQDGEGHTQ DTELVETRPA GDGTFQKWAA VVVPSGEEQR YTCHVQHEGL PEPVTLRWKP ASQPTIPIVG IIAGLVLLGS VVSGAVVAAV IWRKKSSGGK GGSYSKAEWS DSAQGSESHS L

[0629] 3 CDRal SSNFYA

[0630] 4 CDRa2 MTLNGDE

[0631] 5 CDRa3 CALYNNNDMRF

[0632] 6 CDRbl SGHNS

[0633] 7 CDRb2 FNNNVP

[0634] 8 CDRb3 CASSPGSTDTQYF

[0635] 9 TOR alpha variable ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAK domain SPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDS ATYLCALYNNNDMRFGAGTRLTVKP

[0636] 10 TOR beta variable GVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGLE domain LLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDS AVYFCASSPGSTDTQYFGPGTRLTVL

[0637] 11 CDRal DRRSQS

[0638] 12 CDRa2 IYSNGD

[0639] 13 CDRa3 CAAYSGAGSYQLT

[0640] 14 CDRbl AMDHPY

[0641] 15 CDRb2 YYNGEE

[0642] 16 CDRb3 CASSVESSYGYT

[0643] 17 TCR alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL NCTYSDRRSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKG TKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ

[0644]

[0645] SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS TCR beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRC SPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFS AQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGT RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPD HVELSWVWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV SAEAWGRADCG FTSVSYQQG VLSATI LYE 1 LLG KATLYAVLVS ALVLMAMVKRKDF CDRal DRGSQS

[0646] CDRa2 IYSNGD

[0647] CDRa3 CAAYSGAGSYQLT

[0648] CDRbl AMDHPY

[0649] CDRb2 YYNGEE

[0650] CDRb3 CASSVESSYGYT

[0651] TCR alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKG TKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS TCR beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRC SPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFS AQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGT RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPD HVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV SAEAWGRADCG FTSVSYQQG VLSATI LYE 1 LLG KATLYAVLVS ALVLMAMVKRKDF CDRal DSSSTY

[0652] CDRa2 IFSNMDM

[0653] CDRa3 AEKETAGNKLT

[0654] CDRbl SNHLY

[0655] CDRb2 FYNNEI

[0656] CDRb3 ASTVQSPRTNEQF

[0657] TCR alpha variable EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGL domain QLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIY FCAEKETAGNKLTFGGGTRVLVKP TCR beta variable EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKV domain EFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDS AMYFCASTVQSPRTNEQFFGPGTRLTVL

[0658] B2M nucleic acid atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct sequence atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgtaa

[0659] B2M amino acid MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFL sequence NCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLY

[0660] YTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0661]

[0662] HLA-E, UniProt ID: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPR P13747 FISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDR ETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCEL GPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQ KSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKT HVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTE LVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPV TLRWKPASQPTIPIVGIIAGLVLLGSVVSGAWAAVIWRKKSSG GKGGSYSKAEWSDSAQGSESHSL HLA-F isoform 1 MAPRSLLLLLSGALALTDTWAGSHSLRYFSTAVSRPGRGEPR (SEQ ID NO: 38): YIAVEYVDDTQFLRFDSDAAIPRMEPREPWVEQEGPQYWEW TTGYAKANAQTDRVALRNLLRRYNQSEAGSHTLQGMNGCDM GPDGRLLRGYHQHAYDGKDYISLNEDLRSWTAADTVAQITQR FYEAEEYAEEFRTYLEGECLELLRRYLENGKETLQRADPPKAH VAHHPISDHEATLRCWALGFYPAEITLTWQRDGEEQTQDTEL VETRPAGDGTFQKWAAVWPPGEEQRYTCHVQHEGLPQPLIL RWEQSPQPTIPIVGIVAGLVVLGAVVTGAVVAAVMWRKKSSD RNRGSYSQAAV HLA-F isoform 2 MAPRSLLLLLSGALALTDTWAGSHSLRYFSTAVSRPGRGEPR YIAVEYVDDTQFLRFDSDAAIPRMEPREPWVEQEGPQYWEW TTGYAKANAQTDRVALRNLLRRYNQSEAGSHTLQGMNGCDM GPDGRLLRGYHQHAYDGKDYISLNEDLRSWTAADTVAQITQR FYEAEEYAEEFRTYLEGECLELLRRYLENGKETLQRAEQSPQ PTIPIVGIVAGLVVLGAVVTGAWAAVMWRKKSSDRNRGSYSQ AAV HLA-F isoform 3 MAPRSLLLLLSGALALTDTWAGSHSLRYFSTAVSRPGRGEPR YIAVEYVDDTQFLRFDSDAAIPRMEPREPWVEQEGPQYWEW TTGYAKANAQTDRVALRNLLRRYNQSEAGSHTLQGMNGCDM GPDGRLLRGYHQHAYDGKDYISLNEDLRSWTAADTVAQITQR FYEAEEYAEEFRTYLEGECLELLRRYLENGKETLQRADPPKAH VAHHPISDHEATLRCWALGFYPAEITLTWQRDGEEQTQDTEL VETRPAGDGTFQKWAAVWPPGEEQRYTCHVQHEGLPQPLIL RWEQSPQPTIPIVGIVAGLVVLGAVVTGAVVAAVMWRKKSSD RNRGSYSQAAAYSWSGNLMITWWSSLFLLGVLFQGYLGCLR SHSVLGRRKVGDMWILFFLWLWTSFNTAFLALQSLRFGFGFR RGRSFLLRSWHHLMKRVQIKIFD HLA-G isoform 1 MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRG (SEQ ID NO: 41): EPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEY WEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIG CDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQI SKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRAD PPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQT QDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGL PEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWR KKSSD

[0663]

[0664] HLA-G isoform 5 MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRG (SEQ ID NO: 42): EPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEY WEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIG CDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQI SKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRAD PPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQT QDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGL PEPLMLRWSKEGDGGIMSVRESRSLSEDL

[0665] HLA-E fusion protein atgagcagaa gcgtggccct ggccgtgctg gccttactgt ccctgagcgg v1 (A02) SEQ ID NO: cctggaggcc gtgatggcac ctcggacact ggtgctgggc ggcggaggaa 11 gcggcggcgg cgggagcggc ggcggcggct ccatccagag acgtactccc aagatccagg tgtactcccg ccaccctgcc gagaatggca agtccaactt cctcaactgc tacgtgtccg gcttccaccc ctctgacatc gaagtggacc tgctgaagaa cggtgagcgc atcgagaagg tggagcacag cgatctttcg ttttcaaaag attggtcctt ctacctgttg tattacacgg agttcacccc aaccgagaag gacgaatacg cttgtcgggt caaccatgtt accctgagcc agccgaagat tgtcaaatgg gaccgcgata tggggggggg cggaagcggc ggaggaggct ccggcggtgg cggaagcggg ggggggggca gcgggtctca cagcctgaag tatttccaca catccgtgtc tcgccccggg aggggggagc ctcggtttat ctccgtgggc tacgtcgacg atacccagtt cgtgaggttc gataacgacg ccgcttcccc acgaatggtg ccaagagccc cttggatgga acaggaaggc tctgagtact gggacaggga aacacggagt gccagggaca cagctcagat cttcagagtg aatctgagaa cactgagagg ctactacaac cagtccgaag ccggctccca caccctgcag tggatgcacg gctgcgaact ggggcccgac ggaaggtttc tgaggggcta cgagcaattc gcctacgatg gcaaggatta cctgaccctg aacgaggatc tgcggtcttg gacggccgtg gataccgccg ctcagatcag cgagcagaag agcaatgatg ccagcgaggc gagcaccag agagcctatc tggaggatac ttgtgtggaa tggctgcaca agtacctgga aaagggcaag gaaacactgc tgcacctgga accacccaag acccatgtga cacaccaccc catctctgac catgaggcaa cactgaggtg ctgggccctg ggattctacc ccgccgagat cacactgacc tggcaacagg acggcgaagg ccacacccag gatacagaac tcgtggagac tagacccgcc ggtgacggca ccttccagaa gtgggcagct gtggtggtgc caagcgggga agagcagagg tacacctgtc acgtgcagca tgagggcctg cctgagccag tgacactgag gtggaaacct gcctcccagc ctacaatccc aatcgtggga ataattgccg gcctggtgct gctgggaagt gtggtgtccg gcgccgtggt ggccgccgtc atctggagaa aaaagagctc tggcggcaag ggcgggtcct atagcaaagc agagtggagc gatagcgccc agggctcaga gagccactct ctg

[0666] HLA-E fusion protein atgagcagaa gcgtggccct ggccgtgctg gccttactgt ccctgagcgg v2 (cw3) SEQ ID NO: cctggaggcc gtgatggcac ctcggacact gatactgggc ggcggaggaa 12 gcggcggcgg cgggagcggc ggcggcggct ccatccagag acgtactccc aagatccagg tgtactcccg ccaccctgcc gagaatggca agtccaactt cctcaactgc tacgtgtccg gcttccaccc ctctgacatc gaagtggacc tgctgaagaa cggtgagcgc atcgagaagg tggagcacag cgatctttcg ttttcaaaag attggtcctt ctacctgttg tattacacgg agttcacccc aaccgagaag gacgaatacg cttgtcgggt caaccatgtt accctgagcc agccgaagat tgtcaaatgg gaccgcgata tggggggggg cggaagcggc ggaggaggct

[0667]

[0668] ccggcggtgg cggaagcggg ggggggggca gcgggtctca cagcctgaagtatttccaca catccgtgtc tcgccccggg aggggggagc ctcggtttat ctccgtgggc tacgtcgacg atacccagtt cgtgaggttc gataacgacg ccgcttcccc acgaatggtg ccaagagccc cttggatgga acaggaaggc tctgagtact gggacaggga aacacggagt gccagggaca cagctcagat cttcagagtg aatctgagaa cactgagagg ctactacaac cagtccgaag ccggctccca caccctgcag tggatgcacg gctgcgaact ggggcccgac ggaaggtttc tgaggggcta cgagcaattc gcctacgatg gcaaggatta cctgaccctg aacgaggatc tgcggtcttg gacggccgtg gataccgccg ctcagatcag cgagcagaag agcaatgatg ccagcgaggc cgagcaccag agagcctatc tggaggatac ttgtgtggaa tggctgcaca agtacctgga aaagggcaag gaaacactgc tgcacctgga accacccaag acccatgtga cacaccaccc catctctgac catgaggcaa cactgaggtg ctgggccctg ggattctacc ccgccgagat cacactgacc tggcaacagg acggcgaagg ccacacccag gatacagaac tcgtggagac tagacccgcc ggtgacggca ccttccagaa gtgggcagct gtggtggtgc caagcgggga agagcagagg tacacctgtc acgtgcagca tgagggcctg cctgagccag tgacactgag gtggaaacct gcctcccagc ctacaatccc aatcgtggga ataattgccg gcctggtgct gctgggaagt gtggtgtccg gcgccgtggt ggccgccgtc atctggagaa aaaagagctc tggcggcaag ggcgggtcct atagcaaagc agagtggagc gatagcgccc agggctcaga gagccactct ctg

[0669] CD8a isoform 1 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKC QVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLD TQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVC KCPRPVVKSGDKPSLSARYV CD8aisoform 2 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKC QVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLD TQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA GNRRRVCKCPRPVVKSGDKPSLSARYV CD8a isoform 3 MRNQAPGRPKGATFPPRRPTGSRAPPLAPELRAKQRPGERV MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKC QVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLD TQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVC KCPRPVVKSGDKPSLSARYV CD8p MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE isoform 1 (SEQ ID AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV NO: 48): EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITL GLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK

[0670]

[0671] CD8p isoform 2 MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITL GLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQLRLHPLEKC SRMDY

[0672] CD8p MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE isoform 3 (SEQ ID AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV NO: 50): EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGRRRRAR LRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT CD8p isoform 4 MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITL GLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQKFNIVCLKIS GFTTCCCFQILQISREYGFGVLLQKDIGQ CD8p isoform 5 MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITL GLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQPQGEGISGT FVPQCLHGYYSNTTTSQKLLNPWILKT CD8p isoform 6 MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGRRRRAR LRFMKQFYK CD8p isoform 7 MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKDFTNKQRI GFWCPATKRHRSVMSTMWKNERRDTFNPGEFNGC CD8p MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCE isoform 8 (SEQ ID AKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEV NO: 55): EQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKG TQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGLKGKVY QEPLSPNACMDTTAILQPHRSCLTHGS TGFBR2 nucleic acid atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg sequence gacgcgtatc gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt

[0673]

[0674] tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgcttctccaaag tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcaacc tgggaaaccg gcaagacgcg gaagctcatg gagttcagcg agcactgtgc catcatcctg gaagatgacc gctctgacat cagctccacg tgtgccaaca acatcaacca caacacagag ctgctgccca ttgagctgga caccctggtg gggaaaggtc gctttgctga ggtctataag gccaagctga agcagaacac ttcagagcag tttgagacag tggcagtcaa gatctttccc tatgaggagt atgcctcttg gaagacagag aaggacatct tctcagacat caatctgaag catgagaaca tactccagtt cctgacggct gaggagcgga agacggagtt ggggaaacaa tactggctga tcaccgcctt ccacgccaag ggcaacctac aggagtacct gacgcggcat gtcatcagct gggaggacct gcgcaagctg ggcagctccc tcgcccgggg gattgctcac ctccacagtg atcacactcc atgtgggagg cccaagatgc ccatcgtgca cagggacctc aagagctcca atatcctcgt gaagaacgac ctaacctgct gcctgtgtga ctttgggctt tccctgcgtc tggaccctac tctgtctgtg gatgacctgg ctaacagtgg gcaggtggga actgcaagat acatggctcc agaagtccta gaatccagga tgaatttgga gaatgttgag tccttcaagc agaccgatgt ctactccatg gctctggtgc tctgggaaat gacatctcgc tgtaatgcag tgggagaagt aaaagattat gagcctccat ttggttccaa ggtgcgggag cacccctgtg tcgaaagcat gaaggacaac gtgttgagag atcgagggcg accagaaatt cccagcttct ggctcaacca ccagggcatc cagatggtgt gtgagacgtt gactgagtgc tgggaccacg acccagaggc ccgtctcaca gcccagtgtg tggcagaacg cttcagtgag ctggagcatc tggacaggct ctcggggagg agctgctcgg aggagaagat tcctgaagac ggctccctaa acactaccaa atag

[0675] TGFBR2 amino acid MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNG sequence AVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV AVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLL PPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCA IILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAK LKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQ FLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRK LGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLT CCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRM NLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFG SKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETL TECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPE DGSLNTTK

[0676] Membrane-bound IL- NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM 15 KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTSSGGGSG GGGSGGGGSG GGGSGGGGSG GGTLQITCPP PMSVEHADIW VKSYSLYSRE RYICNSGFKR KAGTSSLTEC VLNKATNVAH WTTPSLKCIR DPALVHQRPA PPSTVTTAGV TPQPESLSPS GKEPAASSPS SNNTAATTAA IVPGSQLMPS

[0677]

[0678] KSPSTGTTEI SSHESSHGTP SQTTAKNWEL TASASHQPPGVYPQGHSDTT FWVLVVVGGV LACYSLLVTV AFIIFWVKSR QTPPLASVEM EAMEALPVTW GTSSRDEDLE NCSHHL

[0679] Membrane-bound IL- atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 15 v1 aactgggtga acgtgatctc cgacctgaag aagattgaag atctgatcca gtccatgcac attgacgcca ccctttacac cgagtcagat gtgcatccga gctgcaaggt caccgcgatg aagtgtttcc tgctggaact ccaagtcatc agcctcgaat ccggcgacgc ttcaattcac gacactgtgg agaacttgat cattctggcc aacaactcgc tgtcgtccaa tggaaacgtg accgagtccg ggtgcaaaga gtgcgaagaa ctcgaggaaa agaacatcaa ggagttcctg cagtccttcg tgcacatcgt gcagatgttt atcaacacta gctctggtgg tggttctggt gggggtggct ctggcggcgg gggatcaggc ggaggagggt ccggaggcgg aggctctggt gggggtactc tacagatcac gtgccctccc cccatgtccg tggaacacgc agacatctgg gtcaagagct acagcttgta ctccagggag cggtacattt gtaactctgg tttcaagcgt aaagccggca cgtccagcct gacggagtgc gtgttgaaca aggccacgaa tgtcgcccac tggacaaccc ccagtctcaa atgcattaga gaccctgccc tggttcacca aaggccagcg ccaccctcca cagtaacgac ggcaggggtg accccacagc cagagagcct ctccccttct ggaaaagagc ccgcagcttc atctcccagc tcaaacaaca cagcggccac aacagcagct attgtcccgg gctcccagct gatgccttca aaatcacctt ccacaggaac cacagagata agcagtcatg agtcctccca cggcaccccc tctcagacaa cagccaagaa ctgggaactc acagcatccg cctcccacca gccgccaggt gtgtatccac agggccacag cgacaccact ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaagtcaagg caaactcccc cgctggccag cgttgaaatg gaagccatgg aggctctgcc ggtgacttgg gggaccagca gcagagatga agacttggaa aactgctctc accaccta

[0680] Membrane-bound IL- atggactgga cctggatcct cttcttggtg gcagcagcca cgcgagtcca 15 v2 ctccaactgg gtgaatgtaa taagtgattt gaaaaaaatt gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac cccagttgca...

Claims

CLAIMS1. An immune cell comprisinga) a genetically engineered inactivation of at least one B2M gene; and b) at least one heterologous nucleic acid sequence(s) encoding a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of a human leukocyte antigen HLA-I protein; and c) at least one heterologous nucleic acid sequence(s) encoding an antigen binding protein.

2. The immune cell of claim 1, wherein said immune cell further comprises a peptide ligand that is presented by the fusion protein on the cell surface.

3. The immune cell of claim 2, wherein said peptide ligand is covalently linked to at least a portion of said HLA-I fusion protein.

4. The immune cell of claim 3, wherein said fusion protein does not present any other peptides on the cell surface.

5. The immune cell of any one of the preceding claims, wherein said portion of the HLA-I fusion protein comprises or consists of an HLA class I a chain or a variant or derivative thereof.

6. The immune cell of claim 5, wherein said HLA class I a chain is selected from HLA-E, HLA-F and HLA-G.

7. The immune cell of any one of the preceding claims, wherein said immune cell comprises a genetically engineered inactivation of all copies of the B2M gene, preferably causing a B2M knockout.

8. The immune cell of any one of the preceding claims, wherein said HLA-I fusion protein is capable of binding at least one inhibitory receptor on NK cells, thereby preferably inducing NK cell inhibition.

9. The immune cell of claim 8, wherein said immune cell is a human cell, preferably a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably an a[3or ybT cell, such as a V51T Cells or a V52 T cell.

10. The immune cell of any one of the preceding claims, wherein said genetically engineered inactivation of the at least one B2M gene is achieved through insertion of the at least one heterologous nucleic acid sequence(s) into the B2M gene locus.

11. The immune cell of any one of the preceding claims, further comprising d) at least one heterologous nucleic acid sequence(s) encoding a CD8 aand / or CD8 [3chain.

12. The immune cell of any one of the preceding claims, wherein said at least one nucleic acid sequence(s) encoding said fusion protein according to b) and / or said at least one nucleic acid sequence(s) encoding an antigen binding protein according to c) and / or said at least one nucleic acid sequence(s) encoding a CD8 aand / or CD8 [3chain according to d) is / are inserted into the B2M gene locus.

13. The immune cell of any one of the preceding claims, further comprising e) a genetically engineered inactivation of a gene selected from a TGFBR2 gene, a TRAC gene, a TRBC1 gene, a TRBC2 gene, a CIITA gene, a CISH gene, a PD-1 gene, a CTLA-4 gene, a NKG2A gene, a CBLB gene, a REG1 gene, a PTPN2 gene, a LAG3 gene, a TIM3 gene and / or a FAS gene.

14. The immune cell of claim 13, wherein said immune cell comprises a genetically engineered inactivation of all copies of the TGFBR2, TRAC, TRBC1, TRBC2, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1 PTPN2, LAG3, TIM3 and / or FAS genes, respectively.

15. The immune cell of claim 13 or 14, wherein the genetically engineered inactivation of the TGFBR2, TRAC, TRBC1, TRBC2, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3 and / or FAS genes causes a knockout of TGFBR2, TRAC, TRBC1, TRBC2, CIITA, CISH, PD-1, CTLA-4, NKG2A, CBLB, REG1, PTPN2, LAG3, TIM3 and / or FAS, respectively.

16. The immune cell of any one of the preceding claims, further comprising at least one heterologous nucleic acid sequence(s) encoding a cytokine or cytokine-receptor fusion protein, wherein said cytokine is selected from interleukin-2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21.

17. The immune cell of any one of the preceding claims, whereinsaid at least one nucleic acid sequence(s) encoding said fusion protein according to b) and optionally said at least one nucleic acid(s) encoding said CD8 alpha and / or beta chain according to d) is / are inserted into the B2M gene locus; and said at least one nucleic acid sequence(s) encoding said antigen binding protein according to c) is / are inserted into the TGFBR2 locus.

18. The immune cell of claim 17, wherein said immune cell is a y5 T cell.

19. The immune cell of any one of claims 1 to 16, whereinsaid at least one nucleic acid sequence(s) encoding said fusion protein according to b) and optionally said at least one nucleic acid(s) encoding said CD8 alpha and / or beta chain according to d) is / are inserted into the B2M gene locus; and said at least one nucleic acid sequence(s) encoding said antigen binding protein according to c) is / are inserted into the TRAC locus .

20. The immune cell of claim 18, wherein said immune cell is a a|3 T cell.

21. The immune cell of claim 19 or 20, wherein said immune cell further comprises a genetically engineered inactivation of at least one, preferably all, TRBC1 and TRBC2 genes.

22. The immune cell of any one of claims 19 to 21, wherein said immune cell is genetically engineered to comprise a heterologous T-cell receptor (TCR) comprising a TCR [3-chain that includes one or more amino-acid substitutions within the EWTQD (SEQ ID NO: 68) motif of the constant (C|3) region, the one or more substitutions preferably being selected such that binding of the anti-human TCR a / |3 monoclonal antibody clone BW242 / 412 is reduced or abolished.

23. The immune cell of claim 22, wherein the one or more amino-acid substitutions comprise substitution of the threonine (T) or glutamine (Q) residue of the EWTQD motif, optionally selected from T— >V, T— >M, Q— >A, D— >K, and / or D— >R.

24. The immune cell of claim 23, wherein the EWTQD motif is replaced with EWKAK (SEQ ID NO: 69) or AWMQR (SEQ ID NO: 70).

25. The immune cell of any one of the preceding claims, wherein said at least one nucleic acid sequence(s) encoding said fusion protein according to b) said at least one nucleic acid sequence(s) encoding an antigen binding protein according to c) and / or optionally said at least one nucleic acid sequence(s) encoding a CD8 aand / or CD8 [3chain according to d) is / are operably linked to a heterologous or endogenous promoter, preferably a B2M, EF1 alpha, EF1alpha-HTLV, MSCV, MND or MND-HTLV promoter or promoter fragment.

26. The immune cell of any one of the preceding claims, wherein said at least one nucleic acid sequence(s) inserted into the B2M gene locus is / are operably linked to the endogenous B2M promoter.

27. The immune cell of any one of the preceding claims, wherein said at least one nucleic acid sequence(s) inserted into the TRAC gene locus is / are operably linked to a heterologous MND or B2M promoter or promoter fragment.

28. The immune cell of any one of the preceding claims, wherein said fusion protein comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

29. The immune cell of any one of the preceding claims, wherein said nucleic acid sequence(s) encoding said fusion protein comprise(s) or consist(s) of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence according to SEQ ID NO: 43 or SEQ ID NO: 44.

30. The immune cell of any one of the preceding claims, wherein said antigen binding protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises three complementarity determining regions (CDR) CDRal , CDRa2 and CDRa3, and the second polypeptide comprises three CDRs CDRbl, CDRb2, and CDRb3.

31. The immune cell of any one of the preceding claims, wherein said antigen binding construct is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

32. The immune cell of any one of the preceding claims, wherein said first polypeptide comprises or consists of a TCR achain variable domain (Va) or a derivative or fragment thereof.

33. The immune cell of any one of the preceding claims, wherein said second polypeptide comprises or consists of a TCR beta-chain variable domain (V|3) or a derivative or fragment thereof.

34. The immune cell of any one of the preceding claims, wherein said first and / or said second polypeptide further comprises a TCR constant domain.

35. The immune cell of any one of claims 32 to 34, wherein said immune cell is a y5 T cell expressing a heterologous a|3 T cell receptor.

36. The immune cell of any one of the preceding claims, wherein said immune cell comprises and preferably expresses at least the following heterologous nucleic acids: a TCR achain variable domain, or a derivative or fragment thereof; a TCR pchain variable domain, or a derivative or fragment thereof; a HLA-I a chain fusion protein; and optionally at least one of the following::a CD8 achain, or a derivative or fragment thereof;a CD8 pchain or a derivative or fragment thereof;an interleukin or interleukin fusion protein, optionally selected from interleukin- 2, interleukin-12, interleukin-15, interleukin-18, and / or interleukin-21.

37. The immune cell of claim 36, wherein said immune cell comprises and preferably expresses at least the following heterologous nucleic acids: a TCR achain variable domain, or a derivative or fragment thereof; a TCR pchain variable domain, or a derivative or fragment thereof; a HLA-I a chain fusion protein; a CD8 achain, or a derivative or fragment thereof; and a CD8 pchain or a derivative or fragment thereof; 38. The immune cell of any one of the preceding claims, wherein said antigen binding protein is capable of specifically binding to a target peptide presented by an MHC-I molecule.

39. The immune cell of claim 38, wherein said target peptide is a CT45, PRAME, COL6A3, NY-ESO-1 , MAGE-A3, KLK3, KLK4, KK-LC-1 , MART-1 , CEA, HA-1 , KRAS G12D, TP53 R175H, WT1, HPV E6 / E7, or EBV LMP2 target peptide.

40. The immune cell of any one of claims 30 to 39, whereinsaid CDRal comprises or consists of an amino acid sequence according to SEQ ID NO: 3;said CDRa2 comprises or consists of an amino acid sequence according to SEQ ID NO: 4;said CDRa3 comprises or consists of an amino acid sequence according to SEQ ID NO: 5;said CDRbl comprises or consists of an amino acid sequence according to SEQ ID NO: 6;said CDRb2 comprises or consists of an amino acid sequence according to SEQ ID NO: 7; andsaid CDRb3 comprises or consists of an amino acid sequence according to SEQ ID NO: 8.

41. The immune cell of any one of claims 32 to 40, wherein said TCR achain variable domain (Va) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 9.

42. The immune cell of any one of claims 33 to 41, wherein said TCR [3chain variable domain (V|3) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 10.

43. The immune cell of any one of the preceding claims, wherein heterologous nucleic acid sequence(s) is / are introduced into said immune cell through adeno-associated vectors (AAVs) and a CRISPR / Cas system.

44. The immune cell of claim 43, wherein said CRISPR / Cas system comprises a Cas protein selected from Cas9 or a variant or derivative thereof, or Cas12 or a variant or derivative thereof.

45. The immune cell of claim 44, wherein said Cas9 variant is selected from SpCas9, SaCas9, St1Cas9, NmCas9, or CjCas9.

46. The immune cell of claim 45, wherein said Cas12 variant is selected from Cas12a (Cpf1), Cas12b (C2c1), Cas12d (CasY), Cas12e (C2c5), Cas12f (Cas14), or Cas12i; preferably Cas12a (Cpf1) or Cas12i.

47. A (pharmaceutical) composition comprising the immune cell of any one of the preceding claims, and optionally a pharmaceutically acceptable carrier or excipient.

48. A kit comprising the immune cell or (pharmaceutical) composition of any one of the previous claims, and optionally at least one further therapeutic or co-therapeutic agent.

49. The immune cell(s) or (pharmaceutical) composition or kit of any one of the preceding claims for use in medicine.

50. The immune cell(s) or (pharmaceutical) composition or kit of any one of the preceding claims for use in treating cancer, an autoimmune disease, or an infection.

51. A method of treating cancer, an autoimmune disease, or an infection, said method comprising administering the immune cell(s) or (pharmaceutical) composition or kit of any one the preceding claims to a patient in need thereof.

52. The use or method of any one of claim 49 to 51 , comprising administering said immune cell(s), (pharmaceutical) composition or kit and, additionally, at least one further therapeutic or co-therapeutic agent or treatment.

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