Cd277 targeting costimulatory receptor

Abstract

The present invention relates to an immune cell expressing a primary immune receptor comprising a target cell binding domain; and a costimulatory immune receptor comprising a CD277 binding domain. The costimulatory immune receptor may further comprise a hinge and / or transmembrane domain, and an intracellular costimulatory signalling domain. The immune cell may be a human immune cell, more preferably a human T cell, human Natural Killer (NK) cell, human Antigen Presenting Cell (APC), more preferably an αβ T-cell or a γδ T- cell. The present invention can be used in the prevention or treatment of cancer, infectious disease and / or autoimmune disease.

Description

[0001] CD277 TARGETING COSTIMULATORY RECEPTOR

[0002] Technical field

[0003] The invention is in the field of immunology and cell therapy. In particular, the invention relates to engineered immune cells with modified immune receptor(s), and to the use of said engineered immune cells in medical treatments, particularly in the treatment of cancer, infectious disease, and autoimmune disease.

[0004] Background of the invention

[0005] The use of engineered immune cells for the treatment of various diseases is a rapidly progressing field. Most advancements have been made in the field of cancer treatment using engineered T cells, like CAR T cells or TCR-engineered T cells, but also other immune subsets, like NK cells or macrophages, are being used. Currently the use of engineered immune cells in non-malignant diseases is explored as well.

[0006] To date the most impressive clinical response has been observed for CAR T cells in hematological malignancies, with durable remissions and long persistence of engineered T cells in the patients. However, for tumors of solid origin, engineered T cell therapies have been less successful, due to the more immune suppressive tumor microenvironment. Many different concepts are explored to increase the potency of the engineered T cells against solid tumors [1], One of these concepts is the use of an additional chimeric costimulatory receptor, that will provide the engineered T cells with a strong “signal 2” and thereby increasing their potency. The majority of these chimeric costimulatory receptors have a similar design, an extracellular protein domain that can bind a ligand, a transmembrane domain, and an intracellular signaling domain of a costimulatory molecule. The extracellular ligand binding domain is very important for its function and preferably this ligand is overexpressed on target cell of interest.

[0007] However, there remains a need to identify new and improved costimulatory receptors and / or to develop new approaches to improve the efficacy of engineered immune cells. It is an objective of the present disclosure to meet one or more of the above or other needs in the art.

[0008] Summary of the invention The present inventors explored the use of CD277 targeting chimeric costimulatory receptors to improve efficacy of engineered T cells. CD277 is overexpressed on tumor cells of diverse origin and on surrounding stroma cells [2],

[0009] CD277, also known as butyrophilin 3A (BTN3A), has three isoforms (BTN3A1-3) that have highly conserved extracellular domains but differ in their intracellular part. CD277 has been described as an inhibitory molecule for apT cells [3] and one of the isoforms, BTN3A1, is important, together with BTN2A1 , for the activation of y952T cells [4-6], This activation of y952T cells by the BTN3A1 / BTN2A1 complex is mediated by phosphoantigens, either endogenous or exogenous / microbe derived, that bind the intracellular B30.2 domain of BTN3A1. The levels of endogenous phophoantigens can be elevated by the use of aminobisphosphonate compounds, like pamidronate or zoledronate, which inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway, leading to the accumulation of IPP in the cytosol [7],

[0010] Several antibodies have been described to modulate the immune response to CD277, with most reports focusing on the modulation of y952T cells [2, 8] and one describing effects on a T cells as well [3], For modulating y952T cells the epitope recognized by the antibody clone plays a role for it function, these antibodies can either be agonistic or antagonistic, while for the described effect on apT cells all tested epitopes result in increased activation.

[0011] Based on two reported antibodies, the present inventors designed antiCD277-costimulatory receptors and tested them next to a tumor reactive y952TCR in engineered T cells. The inventors establish that in this context the epitope bound by the antiCD277-scFv is important to further enhance the T cell function. In two different tumor xenograft models, the present inventors show superior tumor control for engineered T cells co-expressing the tumor reactive y952TCR and the anti-CD277-costimulatory receptor, compared to engineered T cells expressing the tumor reactive y952TCR only.

[0012] Detailed description of the invention

[0013] The present disclosure relates to an immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain, preferably wherein said immune receptor is a costimulatory immune receptor. The (costimulatory) immune receptor may be expressed by an immune cell, e.g. together with a primary immune receptor.

[0014] Accordingly, the present disclosure provides for an immune cell expressing

[0015] - a first (or primary) immune receptor preferably comprising a target cell binding domain; and - a second (or costimulatory) immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain.

[0016] It was surprisingly found, in comparison to expression of only a primary immune receptor, that the (co-)presence of such immune receptor that binds CD277 and / or a BTN2A1 lowers the threshold of immune cell activation and can improve fitness as well as proliferation capacity of the immune cell.

[0017] CD277 (also known as Butyrophilin-like protein 3 (BTN3A) and BTN2A1 are members of the butyrophilin family of transmembrane I proteins, which is a group of immunoregulatory proteins involved in various immune system functions. 8 members (BTN1A1, BTN2A1 / 2A2, BTN3A1 / 3A2 / 3A3, MOG, and BTNL2) are located in the major histocompatibility complex (MHC) class I region of human chromosome 6. CD277 is a cell surface receptor protein encoded by the BTN3A gene, e.g. in humans. It has three isoforms: BTN3A1 (SEQ ID NO:1), BTN3A2 (SEQ ID NO:2), and BTN3A3 (SEQ ID NO:3).

[0018] It is found that BTN2A1 and / or CD277 (particularly isoform BTN3A1) binding is important for recognizing intracellular phosphoantigens that originate either from microbial pathogens or from a dysregulated mevalonate pathway in the case of stressed or malignant cells, e.g. as occurring in cancer, infectious disease or auto-immune disease. These phosphoantigens bind to the intracellular domain B30.2 of BTN3A1 resulting in the formation of a complex between the intracellular domains of BTN3A1 and BTN2A1.

[0019] The CD277 binding domain may bind CD277 as represented by an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The CD277 binding domain preferably binds CD277 at an epitope that does not overlap with the y952TCR binding site on CD277 which may refer to amino acid positions corresponding to 37, 39, 44, 94, 96, 98, 104, 105 and / or 107 in any one of SEQ ID NO:1-3. In other words, The CD277 binding domain preferably does not bind CD277 at amino acid positions corresponding to 37, 39, 44, 94, 96, 98, 104, 105 and / or 107 in any one of SEQ ID NO: 1-3. This may prevent interference with the y952TCR binding site on CD277. Accordingly, the binding epitope preferentially does not comprise residues Lys-37 (or Arg-37), Val-39, Arg-44, Lys-94, Leu-96, Tyr-98, Phe-104, Tyr-105, and / or Lys-107 of any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0020] The binding epitope preferentially comprises residues Leu-5, Ser-8, Asp-20, Pro-22, His-24, Phe-26, Pro-27, Thr-28, Met-29, Ser-30, Glu-32, Thr-33, Tyr-50, Ala-51, Asp-52, Gly-53, Lys- 54, Glu-55, Leu-71 , Arg-72, Asp-73, Thr-76, Ala-77, Lys-79, Ala-81, and / or Arg-83 of any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the CD277 binding domain preferably binds CD277 at amino acid positions corresponding to 5, 8, 20, 22, 24, 26, 27, 28, 29, 30, 32, 33, 50, 51 , 52, 53, 54, 55, 71 , 72, 73, 76, 77, 79, 81 , and / or 83 of any one of SEQ ID NO:1 , 2, and 3.

[0021] In an exemplary embodiment, the (exogenous) primary immune receptor comprising the target cell binding domain can be or comprise an op T-cell receptor or extracellular domain thereof, a Chimeric Antigen Receptor (CAR) or extracellular domain thereof, a y<5 T-cell receptor or extracellular domain thereof, or more preferably a Y952 T-cell receptor or extracellular domain thereof. In addition to the primary receptor, a costimulatory immune receptors, as well-known by the skilled person, can provide additional signals, i.e. in addition to signaling from the primary receptor, in order to activate the immune cell. The present disclosure shows superior efficacy for engineered immune cells co-expressing a target cell binding immune receptor and an anti-CD277-costimulatory receptor, compared to immune cells expressing only the target cell binding immune receptor.

[0022] The present second or costimulatory immune receptor may be a chimeric receptor and / or may comprise: i) an extracellular domain that binds CD277 and / or BTN2A1 , preferably an extracellular domain that binds CD277 or specifically isoform BTN3A1 thereof; ii) a hinge and / or transmembrane domain; iii) an intracellular (costimulatory) signalling domain; and / or iv) a signal peptide, e.g. to ensure cell surface expression, such as a CD33 signal peptide.

[0023] The extracellular domain that binds CD277 may be an extracellular domain that binds CD277 as represented by an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with any one of SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3. The binding epitope preferentially comprises residues Ser30-Thr33, Tyr50-Lys54, and Arg72- Asp73 of any one of SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, and / or preferably does not comprise residues Tyr98-Tyr105. In other words, the extracellular domain that binds CD277 may be an extracellular domain that binds CD277 at amino acid positions corresponding to 30, 31 , 32, 33, 50, 51 , 52, 53, 54, 72, and / or 73 of any one of SEQ ID NO:1, 2, and 3, and / or preferably not at amino acid positions corresponding to 98, 99, 100, 101, 102, 103, 104, and / or 105 of any one of SEQ ID NO:1 , 2, and 3 (such as Tyr-98, Gln-100, Asp-101, Asp-103, Phe-104, Tyr-105). The extracellular domain that binds CD277 preferably binds CD277 at an epitope that does not overlap with the y952TCR binding site on CD277 and / or does not overlap with amino acid positions corresponding to 37, 39, 44, 94, 96, 98, 104, 105 and / or 107 in any one of SEQ ID NO:1-3. In other words, The extracellular domain that binds CD277 preferably does not bind CD277 at amino acid positions corresponding to 37, 39, 85, 94, 96, 98, 104, 105 and / or 107 in any one of SEQ ID NO: 1-3. This may prevent interference with the y952TCR binding site on CD277. Accordingly, the binding epitope preferentially does not comprise residues Lys-37 (or Arg-37), Val-39, Arg-44, Lys-94, Leu-96, Tyr-98, Phe-104, Tyr-105, and / or Lys-107 of any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0024] The binding epitope preferentially comprises residues Leu-5, Ser-8, Asp-20, Pro-22, His-24, Phe-26, Pro-27, Thr-28, Met-29, Ser-30, Glu-32, Thr-33, Tyr-50, Ala-51, Asp-52, Gly-53, Lys- 54, Glu-55, Leu-71 , Arg-72, Asp-73, Thr-76, Ala-77, Lys-79, Ala-81, and / or Arg-83 of any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the extracellular domain that binds CD277 preferably binds CD277 at amino acid positions corresponding to 5, 8, 20, 22, 24, 26, 27, 28, 29, 30, 32, 33, 50, 51 , 52, 53, 54, 55, 71, 72, 73, 76, 77, 79, 81, and / or 83 of any one of SEQ ID NO:1 , 2, and 3.

[0025] The “hinge” domain may refer to a (hydrophilic) region which is between the target binding domain and the transmembrane domain. The (costimulatory) receptor of the disclosure may comprise a hinge domain but is it also possible to leave out such a hinge.

[0026] The transmembrane domain may be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membranebound or transmembrane protein. The transmembrane domain may be derived for example from CD8alpha or CD28.

[0027] In the present disclosure, the hinge and / or transmembrane (and / or intracellular) domain(s) preferably is or comprises one of CD8aHTM, CD8aH -CD28TM, lgG4H-CD28TM, and lgG4H-CD8aTM, preferably (a domain of) CD28. For example, the hinge and / or transmembrane domain may be or comprise a hinge domain comprising an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NQ:30 and / or having a length of 10-25 or 12, 13, 14, 15, or 16 amino acids; a transmembrane domain comprising an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:31 and / or having a length of IQ- 40 or 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids; an intracellular domain comprising an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:32 and / or having a length of 20-60 or 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , or 52 amino acids; an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:55 and / or having a length of 30-60 or 40-50 amino acids (“CD8aHinge”); an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:56 and / or having a length of 15-35 or 20-30 amino acids (“CD8aTransmembrane”); and / or an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:57 and / or having a length of 10-25 or 15-20 amino acids (“lgG4Hinge”).

[0028] The (cytoplasmic) signaling domain (or the intracellular signaling domain) generally is (co-) responsible for activation of at least one of the normal effector functions of the immune cell in which the immune receptor is expressed. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the immune receptor to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or enhances immune cell effector functions.

[0029] Generally, immune cell activation can be mediated by two distinct classes of signaling sequences, firstly those that initiate antigen-dependent primary activation through the primary receptor (primary signaling domain) and secondly those that act to provide a secondary or costimulatory signal (secondary or costimulatory signaling domain). Examples of intracellular co-stimulatory signaling domains that are suitable in the present disclosure are CD27, CD28, 4-1 BB (CD137), 0X40, CD30, CD40, , ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, DAP12, DAP10, , or functional part of any of those. The present disclosure preferably does not use a CD3 stimulatory signaling domain, CD3 gamma, delta, epsilon, or zeta signaling domain, or a CD3 zeta stimulatory signaling domain.

[0030] The intracellular signalling domain preferably is or comprises one of 4-1 BB, BAFF-R, CD40, 0X40 (CD134), TACI (CD267), and CD28, preferably 4-1 BB. For example, the intracellular signalling domain may be or comprise 4-1 BB - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:33 and / or having a length of 20-60 or 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 amino acids;

[0031] BAFF-R

[0032] - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:51 and / or having a length of 60-100 or 70-90 amino acids;

[0033] CD40

[0034] - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:51 and / or having a length of 60-100 or 70-90 amino acids;

[0035] 0X40 (CD 134)

[0036] - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:53 and / or having a length of 20-60 or 30-40 amino acids;

[0037] TACI (CD267)

[0038] - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:54 and / or having a length of 80-120 or 90-115 amino acids; and / or

[0039] CD28

[0040] - an amino acid sequence having at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:32 and / or having a length of 20-60 or 30-50 amino acids.

[0041] A "signal peptide" may be incorporated and refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0042] In a preferred embodiment of the present disclosure, the extracellular domain that binds CD277 and / or BTN3A1 comprises

[0043] - an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 4 and / or an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:5 (also referred herein as “CTX2026”);

[0044] - an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:6 and / or an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:7 (also referred herein as “103.2”);

[0045] - an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 58 and / or an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:59 (also referred herein as “A3A”); and / or

[0046] - an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 60 and / or an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:61 (also referred herein as “4G2”). The skilled person knows that polypeptides having a different amino acid sequence can have the same activity and that it is therefore possible to introduce modifications in the VL, VH and / or CDRs as depicted in SEQ ID Nos 4, 5, 6 and 7 according to the disclosure without loss of activity of the polypeptide (e.g. to substitute a certain amino acid by another one).

[0047] For example, the following amino acids can be exchanged for one another (conservative substitutions):

[0048] Ala, Ser, Thr, Gly (small aliphatic, nonpolar or slightly polar residues)

[0049] Asp, Asn, Glu, Gin (polar, negatively charged residues and their amides)

[0050] His, Arg, Lys (polar, positively charged residues)

[0051] Met, Leu, lie, Vai (Cys) (large aliphatic, nonpolar residues)

[0052] Phe, Ty, Trp (large aromatic residues)

[0053] (refer for example to Schulz, G. E. et al, Principles of Protein Structure, Springer- Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W.H. Freeman & Co., San Francisco, 1984)

[0054] In a preferred embodiment, the extracellular domain that binds CD277 and / or BTN3A1 (or BTN2A1) is a single chain variable fragment (scFv), V-Nar (i.e. (humanized) shark variable domain), or VhH, preferably an scFv. Said scFv may comprise

[0055] - a light chain variable domain comprising an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 4 and / or a heavy chain variable domain comprising an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:5 (also referred herein as “CTX2026”); and / or

[0056] - a light chain variable domain comprising an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:6 and / or a heavy chain variable domain comprising an amino acid sequence having at least 70, 80, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:7 (also referred herein as “103.2”).

[0057] The extracellular domain may bind CD277 and / or BTN3A1 (or BTN2A1) with an affinity as determined by a dissociation constant KD lower than 1x1 O'6M, preferably lower than 1x10-7M, more preferably lower than 1x10-8M, more preferably lower than 5x10-9M, more preferably lower than 1x10-9M, even more preferably lower than 8x1 O'10M, even more preferably lower than 6x1 O'10M, yet even more preferably lower than 4x1 O'10M, yet even more preferably lower than 2x1 O'10M, yet even more preferably lower than 1x1 O'10M, yet even more preferably lower than 5x1 O'11M or lower than 1x1 O'11M, most preferably lower than 5x1 O'12M or lower than 1x1 O’12M.

[0058] The present disclosure can be used in the context of inter alia treatment of cancer, infectious disease and / or autoimmune disease. Accordingly, the target cell binding domain of the first or primary immune receptor preferably is chosen from:

[0059] - a cancer cell binding domain;

[0060] - an infected cell binding domain; and / or

[0061] - auto-antibody producing B cell binding domain, autoreactive T cell binding domain, or autoantigen presenting cell binding domain.

[0062] A “target cell binding domain” refers to the region of the (primary) immune receptor that specifically binds to an antigen on a target cell, e.g. to a tumor associated antigen (TAA) or tumor specific antigen (TSA).

[0063] In a preferred embodiment, the cancer cell binding domain is a domain that binds a tumor- associated antigen (e.g. specifically expressed or overexpressed by tumor cells), preferably a domain that binds BTN2A1, BTN3A1, BTN2A1 / BTN3A complex, BTN2A1 / BTN3A1 complex, Annexin-A2, EPCR, CD1, CD19, CD21 , CD22, CD24, CD27, CD30, CD33, CD36, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, CD123, mesothelin, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PD-L1 (CD274), Her2 / neu (CD340), Her3, EGFR, PDGFR, SLAMF7, VEGFR1, VEGFR2, DR5, TF, GD2, GD3, Glypican 3, PTHR2 or neoantigen peptide-HLA complex and / or wherein Treg antigen is CTLA4 or CD25. Particularly preferred are BTN2A1, CD38 or Her2 / neu (CD340).

[0064] In addition or alternatively, the infected cell binding domain is a domain that binds BTN2A1, BTN3A1 , BTN2A1 / BTN3A complex, BTN2A1 / BTN3A1 complex, MIC (MICA and MICB) and ULBP (ULBP1-ULBP6), MR1, CD1, or viral peptide-H LA-complex.

[0065] In addition or alternatively, the auto-antibody producing B cell binding domain, autoreactive T cell binding domain, or autoantigen presenting cell binding domain is a domain that binds CD19, CD83, TRBC1, TRBC2, or autoantigen peptide-HLA complex such as

[0066] - proinsulin C peptide (-HLA complex), e.g. for type I diabetes mellitus;

[0067] - citrullinated vimentin peptide (-HLA complex), e.g. for rheumatoid arthritis; or

[0068] - SmD peptide (-HLA complex), e.g. for Systemic Lupus Erythematosus.

[0069] The immune cell according to the present disclosure, i.e. expressing a first (or primary) immune receptor comprising a target cell binding domain and / or a second (or costimulatory) immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain, can for example be a human immune cell, more preferably a human T cell, human Natural Killer (NK) cell, human Antigen Presenting Cell (APC), human monocyte, human macrophage, or more preferably a (human) op T-cell or a yb T-cell. Also envisaged are op T-cells with yb TCRs (TEGs) which combine the strong proliferation capacity of op T cells (which are active even in late stage cancer patients, with the broad tumor- reactivity of yb TCRs.

[0070] In a particularly preferred embodiment, the present disclosure relates to a combination of an immune cell subset expressing CD4 and an immune cell subset expressing CD8, both subsets expressing the first (or primary) immune receptor comprising a target cell binding domain (as described herein); and the second (or costimulatory) immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain (as described herein). The ratio between the number of cells in the subset expressing CD4 and the number of cells in the subset expressing CD8 may be between 0.1 : 1 and 1 : 0.1 , preferably between 0.3 : 1 and 1 : 0.3.

[0071] Preferably the combination relates to an op or yb T-cell subset expressing CD4 and an op or yb T-cell subset expressing CD8, most preferably a combination of a op T-cell subset expressing CD4 and a op T-cell subset expressing CD8.

[0072] The immune cell (combination) according to the present disclosure may be autologous, or allogenic to the patient receiving the treatment. For example, the immune cell(s) are donor cells. The patient preferably is a mammalian or human patient.

[0073] The immune cell(s) according to the present disclosure can be used in therapy, particularly in the prevention or treatment of cancer, infectious disease and / or autoimmune disease. The prevention or treatment may comprise administering the immune cell(s) to a patient in need thereof.

[0074] The cancer may for example be leukaemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, non-Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumour, stomach cancer, or esophageal cancer.

[0075] The infectious disease may be or may be caused by e.g. bacterial infection, fungal infection, viral infection (e.g. COVID-19, or Hanta virus) (which can cause) sepsis, pneumonia, meningitis, acute respiratory distress syndrome, necrotizing fasciitis. The auto-immune disease may be chosen e.g. from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, psoriasis, Hashimoto’s thyroiditis, Sjogren’s syndrome, autoimmune hepatitis, pemphigus vulgaris and graft versus host disease after allogeneic stem cell transplantation or rejection of a transplant.

[0076] The auto-immune disease may be chosen for example from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, psoriasis, Hashimoto’s thyroiditis, Sjogren’s syndrome, autoimmune hepatitis, pemphigus vulgaris and graft versus host disease after allogeneic stem cell transplantation or rejection of a transplant.

[0077] Co-administration of aminobisphosphonates, such as pamidronate or zoledronate, may increase the intracellular levels of phosphoantigens, e.g. in tumor cells or infected cells, which can further contribute to recognition of target cells by the immune cells (within the context of a y<5 TCR as first signal) according to the present disclosure and / or further contribute to activation of said immune cells.

[0078] The immune cell(s) of the present disclosure may be comprised in a pharmaceutical composition. A pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier. A carrier, as used herein, means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration.

[0079] A pharmaceutical composition comprising therapeutically effective population(s) of cells according to the present disclosure may include pharmaceutically acceptable excipient(s) (carrier or diluents). Excipients included in the formulations will have different purposes depending, for example, on the nature of the construct, the (sub)population of immune cells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-injection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0080] A pharmaceutical composition comprising therapeutically effective population(s) of cells according to the present disclosure may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, without limitation, intratumoral, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra- articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0081] In general, a composition may be administered that comprises between about 1 x 104and about 1 x 1O10immune cells. In addition or alternatively, the composition may comprise between 0.1-10, or 1 -100, 10-1000 mg aminobisphosphonate. In most cases, the composition may comprise between about 1 x 105and about 1 x 109immune cells, from about 5 x 105to about 5 x 108immune cells, or from about 1 x 106to about 1 x 107immune cells. A physician may ultimately determine appropriate dosages to be used.

[0082] The present disclosure also provides for a method for producing the immune cell(s) according to the present disclosure, the method comprising

[0083] - introducing, in an immune cell, a nucleic acid or nucleic acid combination encoding a first (or primary) immune receptor comprising a target cell binding domain and a second (or costimulatory) immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain as described herein; or - introducing, in an immune cell having a first (or primary) immune receptor comprising a target cell binding domain, a nucleic acid or nucleic acid combination encoding a second (or costimulatory) immune receptor comprising a CD277 binding domain and / or a BTN2A1 binding domain as described herein. Accordingly, the immune cell(s) can be genetically modified in vitro to express the first (or primary) immune receptor and second (or costimulatory) receptor as disclosed herein. These engineered cells may be expanded in vitro to a therapeutically effective population of expressing cells. In cellular therapy these engineered cells may be infused to a recipient in need thereof as a pharmaceutical composition (as disclosed herein). The infused cells in the recipient may be able to kill (or at least stop growth of) cancerous / infected cells expressing the antigen which is recognized by the first (or primary) immune receptor as disclosed herein. The recipient may be the same subject from which the cells were obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).

[0084] The immune cell(s) may be a human T cell or human NK cell, more preferably an op T-cell or a yb T-cell. The nucleic acid or nucleic acid combination that encode the first and / or second immune receptor may be mRNA that can be translated directly when introduced in the cytoplasm of e.g. a T cell, for example via transfection. The nucleic acid or nucleic acid combination may be comprised in a DNA vector or in a viral vector. Introduction of the nucleic acid or nucleic acid combination may be via transfection or transduction methods depending on what type of nucleic acid or nucleic acids are used. It is understood that depending on what type of genetic construct or constructs are used, the genetic construct may consist of DNA or RNA.

[0085] General definitions

[0086] In the present disclosure, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0087] T cells, or T lymphocytes, belong to a group of white blood cells named lymphocytes, which play a role in cell-mediated immunity. T cells originate from hematopoietic stem cells in the bone marrow, mature in the thymus (that is where the T is derived from), and gain their full function in peripheral lymphoid tissues. During T-cell development, CD4"CD8" T-cells (negative for both the CD4 and CD8 co-receptor) are committed either to an op (alpha beta) or y<5 (gamma delta) fate as a result of an initial p or 5 TCR gene rearrangement. Cells that undergo early p chain rearrangement express a pre-TCR structure composed of a complete p chain and a pre-TCRa chain on the cell surface. Such cells switch to a CD4+CD8+state, rearrange the TCRa chain locus, and express an apTCR on the surface. CD4"CD8" T cells that successfully complete the y gene rearrangement before the p gene rearrangement express a y6TCR and remain CD4"CD8". (Claudio Tripodo et al. Gamm delta T cell lymphomas Nature Reviews Clinical Oncology 6, 707-717 (December 2009). The T cell receptor associates with the CD3 protein to form a T cell receptor complex. T cells, i.e. expressing an apTCR or a yQTCR, express the T cell receptor complex on the cell surface. The yCT-cells constitute about 1-5% of the total population of T cells. The extracellular region of a T cell receptor chain comprises a variable region. The variable region of a T cell receptor chain three complementarity determining regions (CDR1 , CDR2, CDR3) are located. These regions are in general the most variable and contribute to diversity among TCRs. CDR regions are composed during the development of a T-cell where so-called Variable-(V), Diverse-(D), and Joining-(J)-gene segments are randomly combined to generate diverse TCRs. The constant region of a T cell receptor chain, i.e. being either an alpha, beta, gamma or delta chain, does not substantially vary. Similarly, the framework regions of a T cell receptor chain, i.e. being either an alpha, beta, gamma or delta chain, do not substantially vary either.

[0088] “apT cells” or “alpha beta T cells” may be defined with respect of function as T lymphocytes that express an apTCR, which recognises peptides bound to MHC molecules (major histocompatibility complex), which are expressed on the surface of various cells. MHCs present peptides derived from the proteins of a cell. When for example a cell is infected with a virus, the MHC will present viral peptides, and the interaction between the apTCR and the MHC-complex activates specific types of T-cells which initiate and immune responses to eliminate the infected cell. Hence, a T cells may be functionally defined as being cells capable of recognizing peptides bound to MHC molecules. apT-cells may be identified using an antibody specific for the op T-cell receptor such as described below (e.g. the BW242 antibody that is specific for a human op TCR). apT cells may be selected from peripheral blood for example via the CD3 antigen, as the large majority of T cells have the apTCR. Such a selection will also include ybT-cells. From such selected cells, the nucleic acid (or amino acid) sequence corresponding to the aT-cell receptor chain and the pT-cell receptor chain may be determined. Hence, apT-cells may also be defined as being cells comprising a nucleic acid (or amino acid) sequence corresponding to the aT-cell receptor chain and / or the PT-cell receptor chain.

[0089] “ybT cells” or “gamma delta T cells” represent a small subset of T cells for which the antigenic molecules that trigger their activation is largely unknown. Gamma delta T cells may be considered a component of adaptive immunity in that they rearrange TCR genes to produce junctional diversity and will develop a memory phenotype. However, various subsets may also be considered part of the innate immunity where a restricted TCR is used as a pattern recognition receptor. For example, Vy9 / V<52 T cells are specifically and rapidly activated by a set of non-peptidic phosphorylated isoprenoid precursors, collectively named phosphoantigens. ybT-cells may be identified using an antibody specific for the y<5 T-cell receptor. Antibodies suitable for FACS are widely available. Conditions are selected, such as provided by the antibody manufacturer that allows the selection of negative and / or positive cells. Examples of antibodies that may be suitable are available from BD Pharmingen (BD, 1 Becton Drive, Franklin Lakes, NJ USA), ybTCR-APC (clone B1 , #555718) or as available from Beckman Coulter, pan-ybTCR-PE (clone IMMU510, # IM1418U). Also, from such selected cells, the nucleic acid (or amino acid sequence) sequence corresponding to the yT cell receptor chain and / or the ST cell receptor chain may be determined. Hence, y<5T cells may also be defined as being cells comprising a nucleic acid (or amino acid) sequence corresponding to a yT-cell receptor chain and / or a 52T-cell receptor chain. Natural Killer cells (NK cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation. NK cells do not express T-cell antigen receptors (TOR) or Pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcyRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL / 6 mice. Up to 80% of human NK cells also express CD8.

[0090] “Engineered cells" refers herein to cells having been engineered, e.g. by the introduction of an exogenous nucleic acid sequence or specific alteration of an endogenous gene sequence. An exogenous nucleic acid sequence that is introduced may comprise a wild type sequence of any species that may be modified. An engineered cell may comprise genetic modifications such as one or more mutations, insertions and / or deletions in an endogenous gene and / or insertion of an exogenous nucleic acid (e.g. a genetic construct) in the genome. An engineered cell may refer to a cell in isolation or in culture. Engineered cells may be "transduced cells" wherein the cells have been infected with e.g. an engineered virus. For example, a retroviral vector may be used, such as described in the examples, but other suitable viral vectors may also be contemplated such as lentiviruses. Non-viral methods may also be used, such as transfections or electroporation of DNA vectors. DNA vectors that may be used are transposon vectors. Engineered cells may thus also be “stably transfected cells” or “transiently transfected cells”. Transfection refers to non-viral methods to transfer DNA (or RNA) to cells such that a gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposomal or lipoplex transfection of nucleic acids. Such a transfection may be transient, but may also be a stable transfection wherein cells can be selected that have the gene construct integrated in their genome.

[0091] The terms “binds”, “specifically binds” or “specific for” with respect to an target-binding domain, e.g. of immune receptor, refer to a target binding domain which recognizes and binds to a specific target, e.g. antigen, but does not substantially recognize or bind other molecules in a sample or given context. As will be clear to a skilled person, there are several methods to determine the KD of a binding domain. For example, the KD for a given target / ligand can be determined experimentally using LigandTracer, e.g. by using live cells (expressing the ligand, or target), and adding (microliters of) domain directly and performing dissociation analysis through refreshing the medium, or through a process called binding affinity analysis, wherein the general process comprises the steps of: Preparing a range of concentrations of the target in a buffer solution;

[0092] Preparing a fixed concentration of the domain in the same buffer solution; Mixing of each concentration of the ligand with the fixed concentration of the domain; Incubating the mixture for a specific period of time to allow binding to occur;

[0093] Separating the unbound ligand from the bound domain using a technique such as filtration or centrifugation;

[0094] Measuring the amount of bound ligand in each sample, typically by using a labeled ligand or by detecting the amount of unbound ligand;

[0095] Plotting the data as the fraction of bound domain versus the concentration of the ligand;

[0096] Using a mathematical model, such as the Langmuir isotherm or the Scatchard plot, to determine the KD value from the binding data.

[0097] As used herein, the term “identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" perse has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. For example NCBI Nucletide Blast with standard settings (blastn, https: / / blast.ncbi.nlm.nih.gov / ). Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0098] As an illustration, by a nucleotide sequence or amino acid sequence having at least, for example, 95% "identity" to a reference sequence, it is intended that the nucleotide sequence or amino acid sequence is identical to the reference sequence except that there may be up to five point mutations per each 100 nucleotides or amino acids of the reference sequence. In other words, to obtain a nucleotide sequence or amino acid sequence being at least 95% identical to a reference sequence, up to 5% of the nucleotides or amino acids in the reference sequence may be deleted and / or substituted with another nucleotide or amino acid, and / or a number of nucleotides or amino acids up to 5% of the total nucleotides or amino acids in the reference sequence may be inserted into the reference sequence. Preferably, the sequence identity refers to the sequence identity over the entire length of the sequence. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids or nucleotides) are referred to.

[0099] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0100] Methods of carrying out the conventional techniques used in methods of the present invention will be evident to the skilled worker, and are disclosed for example in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

[0101] Brief description of the Figures

[0102] Figure 1 Design and expression of anti-CD277-costim molecules. A) Schematic overview of the chimeric costimulatory receptor, containing an anti-CD277 scFv, CD28 hinge and TM peptide, and intracellular signaling domain of 4-1BB. B) The two selected anti-CD277 scFv, 103.2 (PDB: 4f9p) and CTX2026 (PDB: 6xlq), bind to the extracellular domain of CD277 in distinct orientation. C) Surface expression of the tumor reactive y952TCR CI5 and the anti- CD277-costim molecules after retroviral transduction and selection of a T cells was analyzed by FACS using anti-pan-ybTCR-PECy7 and CD277ec-streptavidin-PE tetramers on a BD LSRFortessa.

[0103] Figure 2 antiCD277-costim 103.2-41 BB enhances the killing potential of tumor reactive T cells. Luciferase transduced tumor cell lines, RPMI-8226 and SCC9, were co-cultured with TCR or TCR-costim transduced T cells in indicated effector-to-target ratio in absence or presence of 10 pM pamidronate (PAM). After 20 hours luciferin was added to the samples and luminescence was measured. The luminescence signal of untreated tumor cells was used as reference for 100% living cells. Data represents mean ± SD, n=2.

[0104] Figure 3 antiCD277-costim 103.2-41 BB enhances proliferation in low antigen conditions.

[0105] Transduced T cells (either CD4+ or CD8+) were labeled with cell trace violet and incubated with RPMI-8226 or SCC9 cells for six days in the absence or presence of 100 pM pamidronate. The cells were analyzed by flow cytometry.

[0106] Figure 4 antiCD277-costim 103.2-41 BB, but not 103.2-CD28 enhances T cell function.

[0107] A) Transduced CD4+ T cells were cocultured with SCC9 or Fadu cells for 20 hours in the absence or presence of 10 pM or 100 pM pamidronate in a 1:1 effectortarget ratio. IFNy level in co-culture supernatants was determined by ELISA. Significance was calculated using Two Way ANOVA. *P <0.05, **P < 0.01 , ***P < 0.001, ****P < 0.0001. B) RPMI-8226-lucGFP cells were co-cultured with TCR or TCR-costim transduced T cells in indicated effector-to-target ratio in presence of 10 pM pamidronate. After 20 hours luciferin was added to the samples and luminescence was measured. The luminescence signal of untreated tumor cells was used as reference for 100% living cells. Data represents mean ± SD, n=2.

[0108] Figure 5 antiCD277-costim 103.2-41 BB enhances the killing potential of both CD4+ and CD8+ tumor reactive T cells, without harming healthy cells. A 3D bone marrow niche was constructed by culturing RPMI-8226 tumor cells and mesenchymal stroma cells in Matrigel. After four days, CD4+ and CD8+ engineered T cells were added on top of a trans-well membrane and pamidronate (10 pM) was added to the media. Six days after adding the T cell, tumor cells and stroma cells were quantified by FACS. (A) Percentage killed tumors cells compared to Mock treatment (LM1 T cells). (B) absolute number of MSCs in the well, lower dotted line (7 10A4 cells) represents number of seeded MSCs and the upper dotted line represents mean number of MSCs in the mock treatment. Bars represent mean ± SD, symbols represent individual wells (n>4). Significance was calculated using One Way ANOVA. *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0109] Figure 6 CI5-103.2-41 BB T cells prevent tumor outgrowth in multiple myeloma xenograft model. NSG mice were irradiated on day -1 and injected intravenously (i.v.) with 5 x 106RPMI 8226-luciferase cells at day 0. On day 1 and 7 the mice were injected i.v. with 1 x 107engineered T cells (n=10 per group). On day 1 and every three weeks after mice were administered IL-2. A) Tumor burden was determined weekly using bioluminescence imaging (BLI) B) individual BLI traces of mice treated with CI5-103.2-41 BB T cells. C) at least twice a week mouse survival was assessed, by monitoring weight loss and symptoms of disease. Significance was calculated by log-rank (Mantel-Cox) test; *P <0.05, **P < 0.01 , ***P < 0.001 , ****p < 0.0001.

[0110] Figure 7 CI5-103.2-41 BB T cells protect mice in a rechallenge multiple myeloma xenograft model. At day 55 7 mice from the CI5-103.2-41 BB T cell group were divided over 2 groups based on BLI signal. On day 59 4 mice were rechallenged by an i.v. injection of 5 x 6

[0111] 10 RPMI 8226-luciferase cells. Injections of IL-2 were maintained every 3 weeks. A) individual BLI traces of mice treated with CI5-103.2-41 BB T cells in the non-rechallenged (n=3) and rechallenged (n=4) groups. B) Comparison between the number of CI5-103.2-41 BB T cells in the bone marrow (per 10A6 cells) and blood (per ml) of the non-rechallenged (n=2) and rechallenged (n=4) . Bars represent mean T cell numbers, symbols depict value of an individual mouse.

[0112] Figure 8 CI5-103.2-41 BB T cells reduced tumor outgrowth rate in a head and neck tumor xenograft model. NSG mice were irradiated on day -1 and injected s.c. with 5 x 10A5 SCC9-lucGFP cells (in A) or 10A6 FaDu cells (in B and C) on day 0. On day 1 and 7, mice were injected with 10A7 engineered T cells; LM1 , CI5, and CI5-103.2-41 BB, 10 mice per group. IL-2 and pamidronate were administered every 3 weeks. A) To assess SCC9-lucGFP tumor outgrowth tumor burden was measured once a week using bioluminescent imaging. Statistical significance was calculated using mixed-effects model with repeated measures in GraphPad Prism 9.3.0 ****P <0.0001 . B) To assess Fadu tumor outgrowth tumor volume was measured twice a week. Statistical significance was calculated using mixed-effects model with repeated measures in GraphPad Prism 9.3.0 *P <0.05. Data represent mean ± SEM. At day 37 all surviving mice were sacrificed. Four mice treated with CI5 T cells and CISIOS.2-41 BB T cells were selected to analyze C) the number of tumor infiltrating engineered T cells and D) the number of engineered T cells in the bone marrow. Bars represent the mean number of T cells and symbols depict individual values. Significance was calculated using a Mann-Whitney U test in GraphPad Prism 9.3.0 *P <0.05.

[0113] Figure 9 Characterization of anti-CD277 hybridoma subclone supernatants revealed antibodies with distinct profiles. A) Hybridoma clone supernatants were used to stain UMev / tand UMeCD277cells and were detected using a secondary APC conjugated anti-mouse Ig antibody and analyzed by FACS. The fold change in MFI between the UMe^ and UMeCD277cells was plotted. B) RPMI-8226 cells were incubated with hybridoma clone supernatant, after 15 minutes scFv-103.2-HA was added. Cells were washed once, and incubated with anti-HA antibody to detect scFv103.2 binding. The cells were analyzed by FACS. The data is normalized to scFv103.2-HA binding alone. C) CL5-T cells were cocultured with FaDu cells and hybridoma clone supernatant in the presence of 30 pM pamidronate for 20 hours, as a control a condition without hybridoma clone supernatant was used (dotted line). IFNy production by CI5-T cells was determined using an IFNy ELISA.

[0114] Figure 10. Unlike CI5-NKG2D-41BB T cells, CI5-103.2-41 BB T cells control tumor outgrowth in mice A) NSG mice were irradiated on day -1 and injected intravenously (i.v.) with 5x 10A6 RPMI 8226-luciferase cells at day 0. On day 1 and 7 the mice were injected i.v. with 1 x 10A7 engineered T cells (n=10 per group). On day 1 and every three weeks after mice were administered IL-2. Tumor burden was determined weekly using bioluminescence imaging (BLI). Left panel contains data of an experiment with TEG-LM1, TEG001, TEG001- NKG2D-41BB T cells. Right panel contains data of a separate experiment with TEG-LM1, TEG001, TEG001-103.2-41 BB T cells. B) NSG mice were irradiated on day -1 and injected s.c. with 5 x 10A5 SCC9-lucGFP cells on day 0. On day 1 and 7, mice were injected with 10A7 engineered T cells; TEG-LM1, TEG001 , TEG001-NKG2D-41 BB T cells and TEG001- 103.2-41 BB, 10 mice per group. IL-2 and pamidronate were administered every 3 weeks. To assess SCC9-lucGFP tumor outgrowth tumor burden was measured once a week using bioluminescent imaging. Note: data of TEG-LM1, TEG001 , and TEG001-103.2-41 BB, was already shown in Figure 8A.

[0115] Figure 11 Changing the intracellular signaling domain of 103.2-costim does not affect efficacy. A) Schematic overview of the 103.2-costim designs where the intracellular signaling domain was derived from 41 BB, CD28, BAFF-R, CD40, 0X40, or TACI. B) T cells engineered with a Y952 TOR or Y952TCR co-expressing indicated 103.2-costim molecules were cocultured with SCC9 in a 1 :1 E:T ratio for 24 hours. The percentage of CD107 positive cells was determined by FACS. Displayed data mean and standard deviation of n=2 independent experiments with duplicated. Statistical analysis: one way ANOVA, followed by multiple comparisons by Holm-Sidak. *P <0.05, **P < 0.01 , ***P < 0.001. C) SCC9lucGFP cells were co-cultured with Y952TCR, Y952TCR-103.2-41 BB, or Y952TCR-103.2-CD28 transduced T cells in indicated effector-to-target ratio in absence pamidronate (pam). After 20 hours luciferin was added to the samples and luminescence was measured. The luminescence signal of untreated tumor cells was used as reference for 100% living cells. Data represents mean ± SD, n=2.

[0116] Figure 12 Changing the intracellular signaling domain of 103.2-costim does not affect efficacy. A) Schematic overview of the 103.2-41 BB designs where the hinge (H) and transmembrane (TM) sequence was altered, consisting either of CD28HTM, CD8aHTM, CD8aH-CD28TM, lgG4H-CD28TM, or lgG4H-CD8aTM. B) T cells engineered with a Y952 TCR or Y962TCR co-expressing indicated 103.2-HTM-41 BB molecules were cocultured with SCC9 in a 1 :1 E:T ratio for 24 hours. The percentage of CD107 positive cells was determined by FACS. Displayed data mean and standard deviation of n=2 independent experiments with duplicated. Statistical analysis: one way ANOVA, followed by multiple comparisons by Holm- Sidak. *P <0.05, **P < 0.01 , ***P < 0.001 , ***P < 0.0001.

[0117] Figure 13 Selection of the anti-CD277 scFV impacts funcionality of the chimera

[0118] A) Surface expression of the tumor reactive y952TCR and anti-CD277-costim molecules after retroviral transduction and selection of apT cells was analyzed by FACS using anti-pan- ybTCR-PECy7 and CD277ec-streptavidin-PE tetramers on a BD LSRFortessa. B) Transduced T cells (either CD4+ or CD8+) were labeled with cell trace violet and incubated with RPMI-8226 or SCC9 cells for six days in the absence or presence of 10 pM pamidronate. The cells were analyzed by flow cytometry. C) Transduced T cells were cocultured with SCC9 cells for 20 hours in the absence or presence of 10 pM pamidronate in a 1 :1 effectortarget ratio (5x 10A4 cells / well). IFNy level in co-culture supernatants was determined by ELISA.

[0119] Figure 14 Epitope of the anti-CD277 scFV is key for functionality of antiCD277-costim A) Binding of mAb 3A3 and mAb 4G2 to HEK293T-CD277KO reconstituted with BTN3A1- alanine mutants was determined using FACS. B) Residues important for binding of antiCD277 mAbs or the tumor reactivity of y952TCR were colored on the surface of BTN3A1 (pdb: 4l9p), in black residues with high confidence, in gray residues with lower confidence.

[0120] Figure 15 103-41 BB and CTX-41 BB improve activity of apTCR based therapies

[0121] A) T cells engineered with a TCR-WT1 or a TCR-WT1 co-expressing 103-41 BB or CTX2026-41 BB were cocultured with SCC9 loaded with 0.05 pg / ml WT1126-134 peptide in a 1 :1 E:T ratio for 24 hours. The percentage of CD107 positive cells was determined by FACS.

[0122] One way ANOVA (paired) with multiple comparison Tukey n=3 independent experiments with biological duplicates *P <0.05. B) Transduced T cells expressing apTCR-WT 1 or apTCR- WT 1 co-expressing 103-41 BB or CTX2026-41 BB were cocultured with SCC9 cells for 20 hours in the absence or presence of WT1126-134 peptide in a 1 :1 effectortarget ratio (5x 10A4 cells / well). IFNy level in co-culture supernatants was determined by ELISA.

[0123] Sequences referred to (part 1):

[0124] SEQ ID NO:1 :

[0125] >sp 10004811 BT3A1_HUIV1AN Butyrophilin subfamily 3 member Al Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD

[0126] SGKYLCYFQD GDFYEKALVE LKVAALGSDL HVDVKGYKDG GIHLECRSTG WYPQPQIQWS

[0127] NNKGENIPTV EAPVVADGVG LYAVAASVIM RGSSGEGVSC TIRSSLLGLE KTASISIADP

[0128] FFRSAQRWIA ALAGTLPVLL LLLGGAGYFL WQQQEEKKTQ. FRKKKREQEL REMAWSTMKQ

[0129] EQSTRVKLLE ELRWRSIQYA SRGERHSAYN EWKKALFKPA DVILDPKTAN PILLVSEDQR

[0130] SVQRAKEPQD LPDNPERFNW HYCVLGCESF ISGRHYWEVE VGDRKEWHIG VCSKNVQRKG

[0131] WVKMTPENGF WTMGLTDGNK YRTLTEPRTN LKLPKPPKKV GVFLDYETGD ISFYNAVDGS HIHTFLDVSF SEALYPVFRI LTLEPTALTI CPA

[0132] SEQ ID N0:2:

[0133] >sp | P78410 | BT3A2_HUI\ / lAN Butyrophilin subfamily 3 member A2

[0134] Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA

[0135] ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD

[0136] SGKYLCYFQD GDFYEKALVE LKVAALGSNL HVEVKGYEDG GIHLECRSTG WYPQPQIQWS

[0137] NAKGENIPAV EAPVVADGVG LYEVAASVIM RGGSGEGVSC IIRNSLLGLE KTASISIADP

[0138] FFRSAQPWIA ALAGTLPILL LLLAGASYFL WRQQKEITAL SSEIESEQEM KEMGYAATER

[0139] EISLRESLQE ELKRKKIQYL TRGEESSSDT NKSA

[0140] SEQ ID NO:3:

[0141] >sp 10004781 BT3A3_HUMAN Butyrophilin subfamily 3 member A3

[0142] Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA

[0143] ETMELRWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD

[0144] SGKYLCYFQD GDFYEKALVE LKVAALGSDL HIEVKGYEDG GIHLECRSTG WYPQPQIKWS

[0145] DTKGENIPAV EAPVVADGVG LYAVAASVIM RGSSGGGVSC IIRNSLLGLE KTASISIADP

[0146] FFRSAQPWIA ALAGTLPISL LLLAGASYFL WRQQKEKIAL SRETEREREM KEMGYAATEQ

[0147] EISLREKLQE ELKWRKIQYM ARGEKSLAYH EWKMALFKPA DVILDPDTAN AILLVSEDQR

[0148] SVQRAEEPRD LPDNPERFEW RYCVLGCENF TSGRHYWEVE VGDRKEWHIG VCSKNVERKK

[0149] GWVKMTPENG YWTMGLTDGN KYRALTEPRT NLKLPEPPRK VGIFLDYETG EISFYNATDG

[0150] SHIYTFPHAS FSEPLYPVFR ILTLEPTALT ICPIPKEVES SPDPDLVPDH SLETPLTPGL

[0151] ANESGEPQAE VTSLLLPAHP GAEVSPSATT NQNHKLQART EALY

[0152] SEQ ID NO:4:

[0153] >CTX2026_VL

[0154] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL

[0155] QPEDFATYYCQQATDFPPTFGGGTKVEIK

[0156] SEQ ID NO:5:

[0157] >CTX2026_VH

[0158] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTKYAQKFQGRVTMT R DTS I STAYM E LS R LRS D DTAVYYCAR RH S D M I G YYYG M DVWGQGTTVTVSS

[0159] SEQ ID NO:6:

[0160] >103.2_VL

[0161] DIVLTQSPVTLSVTPGDSVSLSCRASQSISNNLHWYRQKSHESPRLLIKYASQSIFGIPSRFSGSGSGTEFTLSINSVETE

[0162] DFGIYFCQQSNSWPHTFGTGTKLELK

[0163] SEQ ID NO:7:

[0164] >103.2_VH

[0165] QVQLQQSGAEVVRPGTSVKVSCKASGYAFTSYLIHWIKQRPGQGLEWIGVINPRSGDSHYNEKFKDRTTLTADQSSS

[0166] TAYMQLSSLTSDDSAVYFCARSDYGAYWGQGTLVTVSA Nucleotide sequences

[0167] SEQ ID NO:8:

[0168] >P2A-CTX2026(VL-VH)-41BB (nucleotide)

[0169] GAGGGCTCTGGAGCCACAAACTTTTCCCTTCTGAAGCAGGCCGGGGATGTCGAGGAGAAC

[0170] CCCGGACCAATGGCTCTGCTGCTGCTGCTGCCCCTGCTGTGGGCCGGGGCCCTGGCTATG

[0171] GATATCCAGATGACACAGAGCCCCTCCAGCGTGAGCGCCAGCGTGGGGGATAGGGTGACC

[0172] ATTACCTGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCCTGGTACCAGCAGAAGCCC

[0173] GGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGC

[0174] AGATTCAGCGGCAGCGGCTCCGGCACCGATTTCACACTGACAATCAGCAGCCTGCAGCCC

[0175] GAGGACTTCGCCACCTACTACTGCCAGCAGGCCACCGACTTTCCCCCCACATTCGGGGGC

[0176] GGGACAAAGGTGGAGATCAAGGGGGGCGGGGGCAGCGGCGGCGGGGGCAGCGGGGGAGGG

[0177] GGGAGCCAGGTGCAGCTGGTGCAGTCCGGGGCCGAGGTGAAAAAGCCCGGCGCCTCCGTG

[0178] AAGGTCAGCTGCAAGGCCAGCGGGTACACCTTCACAGGCTACTACATGCACTGGGTGCGG

[0179] CAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATTAACCCCAACAGCGGGGGGACC

[0180] AAGTACGCCCAGAAGTTCCAGGGGCGGGTCACCATGACCAGGGACACAAGCATCTCCACA

[0181] GCCTACATGGAGCTGTCCAGGCTGCGGTCCGATGACACCGCCGTGTATTACTGCGCCAGG

[0182] AGGCACAGCGATATGATCGGGTACTACTACGGCATGGATGTGTGGGGGCAGGGCACCACA

[0183] GTGACAGTGAGCTCCGGGAAGCACCTGTGCCCCAGCCCCCTCTTCCCCGGCCCCAGCAAG

[0184] CCCTTCTGGGTGCTGGTGGTGGTGGGGGGGGTGCTGGCCTGCTACAGCCTGCTGGTCACA

[0185] GTGGCCTTCATCATCTTCTGGGTCAAGCGGGGGAGGAAGAAACTCCTGTACATCTTTAAG

[0186] CAGCCCTTCATGAGGCCAGTCCAGACCACCCAGGAGGAGGACGGCTGTAGCTGTAGGTTC CCAGAGGAGGAGGAAGGAGGCTGTGAGCTGTGA

[0187] SEQ ID NO:9:

[0188] >P2A-103.2(VL-VH)-41BB (nucleotide)

[0189] GAGGGCTCTGGAGCCACAAACTTTTCCCTTCTGAAGCAGGCCGGGGATGTCGAGGAGAAC

[0190] CCCGGACCAATGGCTCTGCTGCTGCTGCTGCCCCTGCTGTGGGCCGGGGCCCTGGCTATG

[0191] GACATTGTGCTGACACAGTCTCCCGTGACTTTGTCAGTCACCCCTGGCGACTCAGTCAGT

[0192] CTCTCCTGTAGAGCCAGCCAGTCCATTTCCAACAACCTGCACTGGTACCGGCAGAAGAGC

[0193] CACGAGAGCCCCAGGCTGCTGATCAAGTACGCCAGCCAGTCCATCTTTGGCATCCCCTCC

[0194] CGGTTCAGCGGGAGCGGCAGCGGGACAGAGTTCACCCTGTCCATCAACAGCGTGGAGACC

[0195] GAGGACTTCGGGATCTACTTCTGCCAGCAGTCCAACAGCTGGCCCCACACATTCGGGACC

[0196] GGCACCAAGCTGGAGCTGAAGGGGGGCGGGGGCAGCGGCGGCGGGGGCAGCGGGGGAGGG

[0197] GGGAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGGTGCGGCCCGGCACAAGCGTG

[0198] AAGGTGAGCTGCAAGGCCAGCGGGTACGCCTTCACAAGCTACCTGATCCACTGGATCAAG

[0199] CAGAGGCCCGGCCAGGGGCTGGAGTGGATCGGCGTGATCAACCCCAGGAGCGGGGATTCC

[0200] CACTACAACGAGAAGTTCAAGGATCGGACAACACTGACCGCCGACCAGTCCAGCAGCACC

[0201] GCCTACATGCAGCTGAGCAGCCTGACAAGCGACGATAGCGCCGTGTACTTCTGCGCCAGG

[0202] TCCGATTACGGCGCCTACTGGGGCCAGGGCACACTGGTGACCGTGAGCGCCGGGAAGCAC

[0203] CTGTGCCCCAGCCCCCTCTTCCCCGGCCCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTG

[0204] GGGGGGGTGCTGGCCTGCTACAGCCTGCTGGTCACAGTGGCCTTCATCATCTTCTGGGTC

[0205] AAGCGGGGGAGGAAGAAACTCCTGTACATCTTTAAGCAGCCCTTCATGAGGCCAGTCCAG

[0206] ACCACCCAGGAGGAGGACGGCTGTAGCTGTAGGTTCCCAGAGGAGGAGGAAGGAGGCTGT GAGCTGTGA

[0207] SEQ ID NQ:10:

[0208] >P2A-103.2(VL-VH)-CD28 (nucleotide)

[0209] GAGGGCTCTGGAGCCACAAACTTTTCCCTTCTGAAGCAGGCCGGGGATGTCGAGGAGAAC CCCGGACCAATGGCTCTGCTGCTGCTGCTGCCCCTGCTGTGGGCCGGGGCCCTGGCTATG

[0210] GACATTGTGCTGACACAGTCTCCCGTGACTTTGTCAGTCACCCCTGGCGACTCAGTCAGT

[0211] CTCTCCTGTAGAGCCAGCCAGTCCATTTCCAACAACCTGCACTGGTACCGGCAGAAGAGC

[0212] CACGAGAGCCCCAGGCTGCTGATCAAGTACGCCAGCCAGTCCATCTTTGGCATCCCCTCC CGGTTCAGCGGGAGCGGCAGCGGGACAGAGTTCACCCTGTCCATCAACAGCGTGGAGACC GAGGACTTCGGGATCTACTTCTGCCAGCAGTCCAACAGCTGGCCCCACACATTCGGGACC

[0213] GGCACCAAGCTGGAGCTGAAGGGGGGCGGGGGCAGCGGCGGCGGGGGCAGCGGGGGAGGG

[0214] GGGAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGGTGCGGCCCGGCACAAGCGTG AAGGTGAGCTGCAAGGCCAGCGGGTACGCCTTCACAAGCTACCTGATCCACTGGATCAAG CAGAGGCCCGGCCAGGGGCTGGAGTGGATCGGCGTGATCAACCCCAGGAGCGGGGATTCC CACTACAACGAGAAGTTCAAGGATCGGACAACACTGACCGCCGACCAGTCCAGCAGCACC GCCTACATGCAGCTGAGCAGCCTGACAAGCGACGATAGCGCCGTGTACTTCTGCGCCAGG TCCGATTACGGCGCCTACTGGGGCCAGGGCACACTGGTGACCGTGAGCGCCGGGAAGCAC CTGTGCCCCAGCCCCCTCTTCCCCGGCCCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTG GGGGGGGTGCTGGCCTGCTACAGCCTGCTGGTCACAGTGGCCTTCATCATCTTCTGGGTC AGAAGCAAGAGGAGCCGGCTGCTCCACAGCGACTACATGAACATGACACCCAGAAGGCCC GGCCCCACACGGAAGCACTACCAGCCCTACGCCCCCCCACGGGACTTTGCCGCCTACAGG TCCTGA

[0215] SEQ ID N0:11:

[0216] >CI5 (CI5gammaT2ACI5deltaP2A; nucleotide)

[0217] ATGGTGTCCCTGCTGCACGCCTCTACACTGGCTGTTCTGGGAGCCCTGTGTGTGTATGGC

[0218] GCCGGACATCTGGAACAGCCCCAGATCAGCAGCACCAAGACACTGAGCAAGACCGCCAGA

[0219] CTGGAATGCGTGGTGTCCGGCATCACAATCAGCGCCACAAGCGTGTACTGGTACAGAGAA

[0220] AGACCCGGCGAAGTGATCCAGTTCCTGGTGTCCATCAGCTACGATGGCACCGTGCGGAAA

[0221] GAGAGCGGCATCCCTTCTGGCAAGTTCGAGGTGGACAGAATCCCCGAGACAAGCACCAGC ACACTGACCATCCACAACGTGGAAAAGCAGGATATCGCCACCTACTACTGCGCCCTGTGG GAGATCCAAGAGCTGGGCAAGAAAATCAAGGTGTTCGGCCCTGGCACCAAGCTGATCATC

[0222] ACCGACAAACAGCTGGACGCCGACGTGTCCCCTAAGCCTACCATCTTCCTGCCTTCTATC GCCGAGACAAAGCTGCAGAAGGCCGGCACCTATCTGTGCCTGCTGGAAAAGTTCTTCCCA GACGTGATCAAGATCCACTGGGAAGAGAAGAAGTCCAACACCATCCTGGGCAGCCAAGAG

[0223] GGCAACACCATGAAGACCAACGACACCTACATGAAGTTCAGCTGGCTGACCGTGCCTGAG AAGTCCCTGGACAAAGAACACCGGTGCATCGTGCGGCACGAGAACAACAAGAACGGCGTG GACCAAGAGATCATCTTCCCACCTATCAAGACCGACGTCATCACAATGGACCCCAAGGAC AACTGCAGCAAGGACGCCAACGATACCCTGCTGCTGCAGCTGACAAACACCAGCGCCTAC TACATGTATTTGCTGCTGTTGCTGAAGTCCGTGGTGTACTTCGCCATCATCACCTGTTGC CTGCTGCGGAGAACCGCCTTCTGCTGCAATGGCGAGAAAAGCGTCGACAGCGGCTCTGGC

[0224] AGATCTGGCTCTGGCGAAGGCAGAGGCTCTCTGCTGACATGTGGCGACGTGGAAGAGAAC CCCGGACCTCGCTTAATTAACATGGAACGGATCAGCTCCCTGATCCACCTGAGCCTGTTT TGGGCTGGCGTGATGAGCGCCATTGAGCTGGTGCCTGAACACCAGACCGTGCCAGTGTCT

[0225] ATTGGCGTGCCAGCCACACTGCGGTGTAGCATGAAGGGCGAAGCCATCGGCAACTACTAC ATCAACTGGTATCGCAAGACCCAGGGAAATACCATGACCTTCATCTACCGCGAGAAGGAC ATCTACGGCCCAGGCTTCAAGGACAATTTCCAGGGCGACATCGACATTGCCAAGAACCTG

[0226] GCCGTGCTGAAGATTCTGGCCCCTAGCGAGAGAGATGAGGGCAGCTACTACTGTGCTTGC GACGCCCTGAAGAGGACCGACACCGATAAGCTGATCTTCGGCAAGGGCACCAGAGTGACC GTGGAACCTAGAAGCCAGCCTCACACCAAGCCTAGCGTGTTCGTGATGAAGAACGGCACC

[0227] AACGTGGCCTGCCTGGTCAAAGAGTTCTACCCTAAGGACATCCGGATCAACCTGGTGTCT

[0228] AGCAAGAAGATCACCGAGTTCGACCCCGCCATCGTGATCAGCCCTAGCGGCAAGTACAAC GCCGTGAAGCTGGGGAAGTACGAGGACAGCAATAGCGTGACCTGCTCCGTGCAGCATGAT AACAAGACCGTGCACAGCACCGATTTCGAAGTGAAAACCGACTCCACCGACCACGTGAAG

[0229] CCCAAAGAGACAGAGAACACCAAGCAGCCCAGCAAGAGCTGCCACAAGCCTAAGGCCATC GTGCACACCGAGAAAGTGAACATGATGAGCCTGACAGTGCTGGGCCTGAGAATGCTGTTC GCCAAGACAGTGGCCGTGAACTTCCTGCTGACCGCCAAGCTGTTCTTCCTCGAGGGCTCTG

[0230] GAGCCACAAACTTTTCCCTTCTGAAGCAGGCCGGGGATGTCGAGGAGAACCCCGGACCA

[0231] Amino acid sequences (underlined the amino acid sequences after processing of the signal peptides)

[0232] SEQ ID NO:12:

[0233] >P2A-CTX2026(VL-VH)-41BB (amino acid)

[0234] EGSGATNFSLLKQAGDVEENPGPMALLLLLPLLWAGALAMDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQATDFPPTFGGGTKVEIKGGGGSG GGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTKYAQ KFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARRHSDMIGYYYGM DVWGQGTTVTVSSGKHLCPSPLFPGPSK PFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL-

[0235] SEQ ID NO:13:

[0236] >P2A-103.2(VL-VH)-CD28 (amino acid)

[0237] EGSGATNFSLLKQAGDVEENPGPMALLLLLPLLWAGALAMDIVLTQSPVTLSVTPGDSVSLSCRASQSISNNLHWYR QKSHESPRLLIKYASQSIFGIPSRFSGSGSGTEFTLSINSVETEDFGIYFCQQSNSWPHTFGTGTKLELKGGGGSGGGGS GGGGSQVQLQQSGAEVVRPGTSVKVSCKASGYAFTSYLIHWIKQRPGQGLEWIGVINPRSGDSHYNEKFKDRTTLT ADQSSSTAYMQLSSLTSDDSAVYFCARSDYGAYWGQGTLVTVSAGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL LVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL-

[0238] SEQ ID NO:14:

[0239] >P2A-103.2(VL-VH)-CD28 (amino acid)

[0240] EGSGATNFSLLKQAGDVEENPGPMALLLLLPLLWAGALAMDIVLTQSPVTLSVTPGDSVSLSCRASQSISNNLHWYR QKSHESPRLLIKYASQSIFGIPSRFSGSGSGTEFTLSINSVETEDFGIYFCQQSNSWPHTFGTGTKLELKGGGGSGGGGS GGGGSQVQLQQSGAEVVRPGTSVKVSCKASGYAFTSYLIHWIKQRPGQGLEWIGVINPRSGDSHYNEKFKDRTTLT ADQSSSTAYMQLSSLTSDDSAVYFCARSDYGAYWGQGTLVTVSAGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL LVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS-

[0241] SEQ ID NO:15:

[0242] >CI5 (CI5gammaT2ACI5deltaP2A; amino acid)

[0243] MVSLLHASTLAVLGALCVYGAGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLVSISYDGTVRK ESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCALWEIQELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAET KLQKAGTYLCLLEKFFPDVIKIHWEEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGV DQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKSVDSGSGR SGSGEGRGSLLTCGDVEENPGPRLI NMERISSLIH LS LF WAG VM S AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNY YINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDALKRTDTDKLIFGKGTR VTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSV

[0244] QHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAK LFFLEGSGATNFSLLKQAGDVEENPGP

[0245] General design for aCD277-Costim:

[0246] P2A - Signal peptide - VL (anti-CD277) - linker - VH (anti-CD277) - hinge - TM - IntracellularDomain

[0247] Nucleotide:

[0248] SEQ ID NO:16:

[0249] >P2A (same in all aCD277-Costim)

[0250] GAGGGCTCTGGAGCCACAAACTTTTCCCTTCTGAAGCAGGCCGGGGATGTCGAGGAGAAC

[0251] CCCGGACCA SEQ ID N0:17:

[0252] >CD33 signal peptide ((same in all aCD277-Costim) ATGGCTCTGCTGCTGCTGCTGCCCCTGCTGTGGGCCGGGGCCCTGGCTATG

[0253] SEQ ID N0:18:

[0254] >Linker_(G4S)3

[0255] GGGGGCGGGGGCAGCGGCGGCGGGGGCAGCGGGGGAGGGGGGAGC

[0256] SEQ ID N0:19:

[0257] >CD28-hinge (same in all aCD277-Costim) GGGAAGCACCTGTGCCCCAGCCCCCTCTTCCCCGGCCCCAGCAAGCCC

[0258] SEQ ID NQ:20:

[0259] >CD28-TM (same in all aCD277-Costim) TTCTGGGTGCTGGTGGTGGTGGGGGGGGTGCTGGCCTGCTACAGCCTGCTGGTCACAGTG GCCTTCATCATCTTCTGGGTC

[0260] SEQ ID N0:21:

[0261] >CD28-lntracellularDomain AGAAGCAAGAGGAGCCGGCTGCTCCACAGCGACTACATGAACATGACACCCAGAAGGCCC GGCCCCACACGGAAGCACTACCAGCCCTACGCCCCCCCACGGGACTTTGCCGCCTACAGG TCC

[0262] SEQ ID NO:22:

[0263] >41BB_lntracellularDomain AAGCGGGGGAGGAAGAAACTCCTGTACATCTTTAAGCAGCCCTTCATGAGGCCAGTCCAG ACCACCCAGGAGGAGGACGGCTGTAGCTGTAGGTTCCCAGAGGAGGAGGAAGGAGGCTGT GAGCTG

[0264] SEQ ID NO:23:

[0265] >CTX2026_VL

[0266] GATATCCAGATGACACAGAGCCCCTCCAGCGTGAGCGCCAGCGTGGGGGATAGGGTGACC ATTACCTGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCCTGGTACCAGCAGAAGCCC GGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGC AGATTCAGCGGCAGCGGCTCCGGCACCGATTTCACACTGACAATCAGCAGCCTGCAGCCC GAGGACTTCGCCACCTACTACTGCCAGCAGGCCACCGACTTTCCCCCCACATTCGGGGGC GGGACAAAGGTGGAGATCAAG

[0267] SEQ ID NO:24:

[0268] >CTX2026_VH

[0269] CAGGTGCAGCTGGTGCAGTCCGGGGCCGAGGTGAAAAAGCCCGGCGCCTCCGTGAAGGTC AGCTGCAAGGCCAGCGGGTACACCTTCACAGGCTACTACATGCACTGGGTGCGGCAGGCC CCCGGCCAGGGCCTGGAGTGGATGGGCTGGATTAACCCCAACAGCGGGGGGACCAAGTAC GCCCAGAAGTTCCAGGGGCGGGTCACCATGACCAGGGACACAAGCATCTCCACAGCCTAC ATGGAGCTGTCCAGGCTGCGGTCCGATGACACCGCCGTGTATTACTGCGCCAGGAGGCAC AGCGATATGATCGGGTACTACTACGGCATGGATGTGTGGGGGCAGGGCACCACAGTGACA

[0270] GTGAGCTCC SEQ ID NO:25:

[0271] >103.2_VL

[0272] GACATTGTGCTGACACAGTCTCCCGTGACTTTGTCAGTCACCCCTGGCGACTCAGTCAGT

[0273] CTCTCCTGTAGAGCCAGCCAGTCCATTTCCAACAACCTGCACTGGTACCGGCAGAAGAGC

[0274] CACGAGAGCCCCAGGCTGCTGATCAAGTACGCCAGCCAGTCCATCTTTGGCATCCCCTCC

[0275] CGGTTCAGCGGGAGCGGCAGCGGGACAGAGTTCACCCTGTCCATCAACAGCGTGGAGACC

[0276] GAGGACTTCGGGATCTACTTCTGCCAGCAGTCCAACAGCTGGCCCCACACATTCGGGACC

[0277] GGCACCAAGCTGGAGCTGAAG

[0278] SEQ ID NO:26:

[0279] >103.2_VH

[0280] CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGGTGCGGCCCGGCACAAGCGTGAAGGTG

[0281] AGCTGCAAGGCCAGCGGGTACGCCTTCACAAGCTACCTGATCCACTGGATCAAGCAGAGG

[0282] CCCGGCCAGGGGCTGGAGTGGATCGGCGTGATCAACCCCAGGAGCGGGGATTCCCACTAC

[0283] AACGAGAAGTTCAAGGATCGGACAACACTGACCGCCGACCAGTCCAGCAGCACCGCCTAC

[0284] ATGCAGCTGAGCAGCCTGACAAGCGACGATAGCGCCGTGTACTTCTGCGCCAGGTCCGAT

[0285] TACGGCGCCTACTGGGGCCAGGGCACACTGGTGACCGTGAGCGCC

[0286] Amino acid:

[0287] SEQ ID NO:27:

[0288] >P2A (same in all aCD277-Costim)

[0289] EGSGATNFSLLKQAGDVEENPGP

[0290] SEQ ID NO:28:

[0291] >CD33 signal peptide (same in all aCD277-Costim)

[0292] MALLLLLPLLWAGALAM

[0293] SEQ ID NO:29:

[0294] >linker_(G4S)3 (same in all aCD277-Costim)

[0295] GGGGSGGGGSGGGGS

[0296] SEQ ID NQ:30:

[0297] >CD28-hinge (same in all aCD277-Costim)

[0298] GKHLCPSPLFPGPSKP

[0299] SEQ ID N0:31:

[0300] >CD28-TM (same in all aCD277-Costim)

[0301] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0302] SEQ ID NO:32:

[0303] >CD28_lntracellularDomain (Used in 103.2-CD28)

[0304] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0305] SEQ ID NO:33:

[0306] >41BB_lntracellularDomain (Used in 103.2-41BB and CTX2026-41BB)

[0307] KRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCEL

[0308] SEQ ID N0:4: >CTX2026_VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQATDFPPTFGGGTKVEIK

[0309] SEQ ID N0:5:

[0310] >CTX2026_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTKYAQKFQGRVTMT R DTS I STAYM E LS R LRS D DTAVYYCAR RH S D M I G YYYG M DVWGQGTTVTVSS

[0311] SEQ ID N0:6:

[0312] >103.2_VL DIVLTQSPVTLSVTPGDSVSLSCRASQSISNNLHWYRQKSHESPRLLIKYASQSIFGIPSRFSGSGSGTEFTLSINSVETE DFGIYFCQQSNSWPHTFGTGTKLELK

[0313] SEQ ID N0:7:

[0314] >103.2_VH

[0315] QVQLQQSGAEWRPGTSVKVSCKASGYAFTSYLIHWIKQRPGQGLEWIGVINPRSGDSHYNEKFKDRTTLTADQSSS TAYMQLSSLTSDDSAVYFCARSDYGAYWGQGTLVTVSA

[0316] CD277 consist of 3 isoforms BTN3A1, BTN3A2, BTN3A3

[0317] CTX2026 and 103.2 bind to all 3 isoforms, hence we use these.

[0318] Below the sequences obtained from NCBI (nucleotide) and Uniprot (amino acid)

[0319] SEQ ID NO:34:

[0320] Nucleotide sequences

[0321] >cDNA_BTN3Al (based on mRNA sequence from NCBI Reference Sequence: XM_047418120.1) ATGAAAATGGCAAGTTTCCTGGCCTTCCTTCTGCTCAACTTTCGTGTCTGCCTCCTTTTGCTTCAGCTGCTCATGC CTCACTCAGCTCAGTTTTCTGTGCTTGGACCCTCTGGGCCCATCCTGGCCATGGTGGGTGAAGACGCTGATCTG CCCTGTCACCTGTTCCCGACCATGAGTGCAGAGACCATGGAGCTGAAGTGGGTGAGTTCCAGCCTAAGGCAGG TGGTGAACGTGTATGCAGATGGAAAGGAAGTGGAAGACAGGCAGAGTGCACCGTATCGAGGGAGAACTTCG ATTCTGCGGGATGGCATCACTGCAGGGAAGGCTGCTCTCCGAATACACAACGTCACAGCCTCTGACAGTGGAA AGTACTTGTGTTATTTCCAAGATGGTGACTTCTATGAAAAAGCCCTGGTGGAGCTGAAGGTTGCAGCACTGGGT TCTGATCTTCACGTTGATGTGAAGGGTTACAAGGATGGAGGGATCCATCTGGAGTGCAGGTCCACTGGCTGGT ACCCCCAACCCCAAATACAGTGGAGCAACAACAAGGGAGAGAACATCCCGACTGTGGAAGCACCTGTGGTTGC AGACGGAGTGGGCCTGTATGCAGTAGCAGCATCTGTGATCATGAGAGGCAGCTCTGGGGAGGGTGTATCCTG TACCATCAGAAGTTCCCTCCTCGGCCTGGAAAAGACAGCCAGCATTTCCATCGCAGACCCCTTCTTCAGGAGCG CCCAGAGGTGGATCGCCGCCCTGGCAGGGACCCTGCCTGTCTTGCTGCTGCTTCTTGGGGGAGCCGGTTACTTC CTGTGGCAACAGCAGGAGGAAAAAAAGACTCAGTTCAGAAAGAAAAAGAGAGAGCAAGAGTTGAGAGAAAT GGCATGGAGCACAATGAAGCAAGAACAAAGCACAAGAGTGAAGCTCCTGGAGGAACTCAGATGGAGAAGTAT CCAGTATGCATCTCGGGGAGAGAGACATTCAGCCTATAATGAATGGAAAAAGGCCCTCTTCAAGCCTGCGGAT GTGATTCTGGATCCAAAAACAGCAAACCCCATCCTCCTTGTTTCTGAGGACCAGAGGAGTGTGCAGCGTGCCAA GGAGCCCCAGGATCTGCCAGACAACCCTGAGAGATTTAATTGGCATTATTGTGTTCTCGGCTGTGAGAGCTTCA TATCAGGGAGACATTACTGGGAGGTGGAGGTAGGGGACAGGAAAGAGTGGCATATAGGGGTGTGCAGTAAG AATGTGCAGAGAAAAGGCTGGGTCAAAATGACACCTGAGAATGGATTCTGGACTATGGGGCTGACTGATGGG AATAAGTATCGGACTCTAACTGAGCCCAGAACCAACCTGAAACTTCCTAAGCCCCCTAAGAAAGTGGGGGTCTT CCTGGACTATGAGACTGGAGATATCTCATTCTACAATGCTGTGGATGGATCGCATATTCATACTTTCCTGGACGT CTCCTTCTCTGAGGCTCTATATCCTGTTTTCAGAATTTTGACCTTGGAGCCCACGGCCCTGACTATTTGTCCAGCG

[0322] SEQ ID NO:35:

[0323] >cDNA_BTN3A2 (based on mRNA sequence from NCBI Reference Sequence: NM_001197246.2) ATGAAAATGGCAAGTTCCCTGGCTTTCCTTCTGCTCAACTTTCATGTCTCCCTCCTCTTGGTCCAGCTGCTCACTC CTTGCTCAGCTCAGTTTTCTGTGCTTGGACCCTCTGGGCCCATCCTGGCCATGGTGGGTGAAGACGCTGATCTG CCCTGTCACCTGTTCCCGACCATGAGTGCAGAGACCATGGAGCTGAAGTGGGTAAGTTCCAGCCTAAGGCAGG TGGTGAACGTGTATGCAGATGGAAAGGAAGTGGAAGACAGGCAGAGTGCACCGTATCGAGGGAGAACTTCG ATTCTGCGGGATGGCATCACTGCAGGGAAGGCTGCTCTCCGAATACACAACGTCACAGCCTCTGACAGTGGAA AGTACTTGTGTTATTTCCAAGATGGTGACTTCTATGAAAAAGCCCTGGTGGAGCTGAAGGTTGCAGCACTGGGT TCTAATCTTCACGTCGAAGTGAAGGGTTATGAGGATGGAGGGATCCATCTGGAGTGCAGGTCCACCGGCTGGT ACCCCCAACCCCAAATACAGTGGAGCAACGCCAAGGGAGAGAACATCCCAGCTGTGGAAGCACCTGTGGTTGC

[0324] AGATGGAGTGGGCCTATATGAAGTAGCAGCATCTGTGATCATGAGAGGCGGCTCCGGGGAGGGTGTATCCTG CATCATCAGAAATTCCCTCCTCGGCCTGGAAAAGACAGCCAGCATTTCCATCGCAGACCCCTTCTTCAGGAGCG CCCAGCCCTGGATCGCAGCCCTGGCAGGGACCCTGCCTATCTTGCTGCTGCTTCTCGCCGGAGCCAGTTACTTCT TGTGGAGACAACAGAAGGAAATAACTGCTCTGTCCAGTGAGATAGAAAGTGAGCAAGAGATGAAAGAAATGG GATATGCTGCAACAGAGCGGGAAATAAGCCTAAGAGAGAGCCTCCAGGAGGAACTCAAGAGGAAAAAAATCC AGTACTTGACTCGTGGAGAGGAGTCTTCGTCCGATACCAATAAGTCAGCC

[0325] SEQ ID NO:36:

[0326] >cDNA_BTN3A2 (based on mRNA sequence from NCBI Reference Sequence: NM_001197246.2) ATGAAAATGGCAAGTTCCCTGGCTTTCCTTCTGCTCAACTTTCATGTCTCCCTCTTCTTGGTCCAGCTG CTCACTCCTTGCTCAGCTCAGTTTTCTGTGCTTGGACCCTCTGGGCCCATCCTGGCCATGGTGGGTGAAG ACGCTGATCTGCCCTGTCACCTGTTCCCGACCATGAGTGCAGAGACCATGGAGCTGAGGTGGGTGAGTTC CAGCCTAAGGCAGGTGGTGAACGTGTATGCAGATGGAAAGGAAGTGGAAGACAGGCAGAGTGCACCGTAT CGAGGGAGAACTTCGATTCTGCGGGATGGCATCACTGCAGGGAAGGCTGCTCTCCGAATACACAACGTCA CAGCCTCTGACAGTGGAAAGTACTTGTGTTATTTCCAAGATGGTGACTTCTACGAAAAAGCCCTGGTGGA GCTGAAGGTTGCAGCATTGGGTTCTGATCTTCACATTGAAGTGAAGGGTTATGAGGATGGAGGGATCCAT

[0327] CTGGAGTGCAGGTCCACTGGCTGGTACCCCCAACCCCAAATAAAGTGGAGCGACACCAAGGGAGAGAACA TCCCGGCTGTGGAAGCACCTGTGGTTGCAGATGGAGTGGGCCTGTATGCAGTAGCAGCATCTGTGATCAT GAGAGGCAGCTCTGGTGGGGGTGTATCCTGCATCATCAGAAATTCCCTCCTCGGCCTGGAAAAGACAGCC AGCATATCCATCGCAGACCCCTTCTTCAGGAGCGCCCAGCCCTGGATCGCGGCCCTGGCAGGGACCCTGC CTATCTCGTTGCTGCTTCTCGCAGGAGCCAGTTACTTCTTGTGGAGACAACAGAAGGAAAAAATTGCTCT GTCCAGGGAGACAGAAAGAGAGCGAGAGATGAAAGAAATGGGATACGCTGCAACAGAGCAAGAAATAAGC CTAAGAGAGAAGCTCCAGGAGGAACTCAAGTGGAGGAAAATCCAGTACATGGCTCGTGGAGAGAAGTCTT TGGCCTATCATGAATGGAAAATGGCCCTCTTCAAACCTGCGGATGTGATTCTGGATCCAGACACGGCAAA CGCCATCCTCCTTGTTTCTGAGGACCAGAGGAGTGTGCAGCGTGCTGAAGAGCCGCGGGATCTGCCAGAC AACCCTGAGAGATTTGAATGGCGTTACTGTGTCCTTGGCTGTGAAAACTTCACATCAGGGAGACATTACT GGGAGGTGGAAGTGGGGGACAGAAAAGAGTGGCATATTGGGGTATGTAGTAAGAACGTGGAGAGGAAAAA

[0328] AGGTTGGGTCAAAATGACACCGGAGAACGGATACTGGACTATGGGCCTGACTGATGGGAATAAGTATCGG GCTCTCACTGAGCCCAGAACCAACCTGAAACTTCCTGAGCCTCCTAGGAAAGTGGGGATCTTCCTGGACT ATGAGACTGGAGAGATCTCGTTCTATAATGCCACAGATGGATCTCATATCTACACCTTTCCGCACGCCTC TTTCTCTGAGCCTCTATATCCTGTTTTCAGAATTTTGACCTTGGAGCCCACTGCCCTGACCATTTGCCCA ATACCAAAAGAAGTAGAGAGTTCCCCCGATCCTGACCTAGTGCCTGATCATTCCCTGGAGACACCACTGA CCCCGGGCTTAGCTAATGAAAGTGGGGAGCCTCAGGCTGAAGTAACATCTCTGCTTCTCCCTGCCCACCC TGGAGCTGAGGTCTCCCCTTCTGCAACAACCAATCAGAACCATAAGCTACAGGCACGCACTGAAGCACTT TAC

[0329] SEQ ID NO:1:

[0330] Amino acid sequences CD277 isoforms without signal peptide

[0331] >sp 10004811 BT3A1_HUIV1AN Butyrophilin subfamily 3 member Al

[0332] Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA

[0333] ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSDL HVDVKGYKDG GIHLECRSTG WYPQPQIQWS NNKGENIPTV EAPVVADGVG LYAVAASVIM RGSSGEGVSC TIRSSLLGLE KTASISIADP FFRSAQRWIA ALAGTLPVLL LLLGGAGYFL WQQQEEKKTQ FRKKKREQEL REMAWSTMKQ EQSTRVKLLE ELRWRSIQYA SRGERHSAYN EWKKALFKPA DVILDPKTAN PILLVSEDQR SVQRAKEPQD LPDNPERFNW HYCVLGCESF ISGRHYWEVE VGDRKEWHIG VCSKNVQRKG WVKMTPENGF WTMGLTDGNK YRTLTEPRTN LKLPKPPKKV GVFLDYETGD ISFYNAVDGS HIHTFLDVSF SEALYPVFRI LTLEPTALTI CPA

[0334] SEQ ID N0:2:

[0335] >sp | P78410 | BT3A2_HUI\ / lAN Butyrophilin subfamily 3 member A2

[0336] Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA

[0337] ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSNL HVEVKGYEDG GIHLECRSTG WYPQPQIQWS NAKGENIPAV EAPVVADGVG LYEVAASVIM RGGSGEGVSC IIRNSLLGLE KTASISIADP FFRSAQPWIA ALAGTLPILL LLLAGASYFL WRQQKEITAL SSEIESEQEM KEMGYAATER EISLRESLQE ELKRKKIQYL TRGEESSSDT NKSA

[0338] SEQ ID NO:3:

[0339] >sp 10004781 BT3A3_HUMAN Butyrophilin subfamily 3 member A3

[0340] Q FSVLGPSGPI LAMVGEDADL PCHLFPTMSA

[0341] ETMELRWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSDL HIEVKGYEDG GIHLECRSTG WYPQPQIKWS DTKGENIPAV EAPVVADGVG LYAVAASVIM RGSSGGGVSC IIRNSLLGLE KTASISIADP FFRSAQPWIA ALAGTLPISL LLLAGASYFL WRQQKEKIAL SRETEREREM KEMGYAATEQ EISLREKLQE ELKWRKIQYM ARGEKSLAYH EWKMALFKPA DVILDPDTAN AILLVSEDQR SVQRAEEPRD LPDNPERFEW RYCVLGCENF TSGRHYWEVE VGDRKEWHIG VCSKNVERKK GWVKMTPENG YWTMGLTDGN KYRALTEPRT NLKLPEPPRK VGIFLDYETG EISFYNATDG SHIYTFPHAS FSEPLYPVFR ILTLEPTALT ICPIPKEVES SPDPDLVPDH SLETPLTPGL ANESGEPQAE VTSLLLPAHP GAEVSPSATT NQNHKLQART EALY

[0342] Amino acid sequences CD277 isoforms including signal peptide

[0343] SEQ ID NO:37:

[0344] >BT3A1_HUMAN Butyrophilin subfamily 3 member Al

[0345] MKMASFLAFL LLNFRVCLLL LQLLMPHSAQ FSVLGPSGPI LAMVGEDADL PCHLFPTMSA ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSDL HVDVKGYKDG GIHLECRSTG WYPQPQIQWS NNKGENIPTV EAPVVADGVG LYAVAASVIM RGSSGEGVSC TIRSSLLGLE KTASISIADP FFRSAQRWIA ALAGTLPVLL LLLGGAGYFL WQQQEEKKTQ FRKKKREQEL REMAWSTMKQ EQSTRVKLLE ELRWRSIQYA SRGERHSAYN EWKKALFKPA DVILDPKTAN PILLVSEDQR SVQRAKEPQD LPDNPERFNW HYCVLGCESF ISGRHYWEVE VGDRKEWHIG VCSKNVQRKG WVKMTPENGF WTMGLTDGNK YRTLTEPRTN LKLPKPPKKV GVFLDYETGD ISFYNAVDGS HIHTFLDVSF SEALYPVFRI LTLEPTALTI CPA

[0346] SEQ ID NO:38:

[0347] >BT3A2_HUMAN Butyrophilin subfamily 3 member A2

[0348] MKMASSLAFL LLNFHVSLLL VQLLTPCSAQ FSVLGPSGPI LAMVGEDADL PCHLFPTMSA ETMELKWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSNL HVEVKGYEDG GIHLECRSTG WYPQPQIQWS NAKGENIPAV EAPVVADGVG LYEVAASVIM RGGSGEGVSC IIRNSLLGLE KTASISIADP FFRSAQPWIA ALAGTLPILL LLLAGASYFL WRQQKEITAL SSEIESEQEM KEMGYAATER EISLRESLQE ELKRKKIQYL TRGEESSSDT NKSA SEQ ID NO:39:

[0349] >BT3A3_HUMAN Butyrophilin subfamily 3 member A3

[0350] MKMASSLAFL LLNFHVSLFL VQLLTPCSAQ FSVLGPSGPI LAMVGEDADL PCHLFPTMSA ETMELRWVSS SLRQVVNVYA DGKEVEDRQS APYRGRTSIL RDGITAGKAA LRIHNVTASD SGKYLCYFQD GDFYEKALVE LKVAALGSDL HIEVKGYEDG GIHLECRSTG WYPQPQIKWS DTKGENIPAV EAPVVADGVG LYAVAASVIM RGSSGGGVSC IIRNSLLGLE KTASISIADP FFRSAQPWIA ALAGTLPISL LLLAGASYFL WRQQKEKIAL SRETEREREM KEMGYAATEQ. EISLREKLQE ELKWRKIQYM ARGEKSLAYH EWKMALFKPA DVILDPDTAN AILLVSEDQR SVQRAEEPRD LPDNPERFEW RYCVLGCENF TSGRHYWEVE VGDRKEWHIG VCSKNVERKK GWVKMTPENG YWTMGLTDGN KYRALTEPRT NLKLPEPPRK VGIFLDYETG EISFYNATDG SHIYTFPHAS FSEPLYPVFR ILTLEPTALT ICPIPKEVES SPDPDLVPDH SLETPLTPGL ANESGEPQAE VTSLLLPAHP GAEVSPSATT NQNHKLQART EALY

[0351] Sequences referred to (part 2):

[0352] General design for aCD277-Costim:

[0353] P2A - Signal peptide - VL (anti-CD277) - linker - VH (anti-CD277) - hinge - TM - IntracellularDomain

[0354] Nucleotide:

[0355] SEQ ID NO:40:

[0356] >BAFF-R-lntracellularDomain

[0357] AGCTGGCGCCGCCGCCAGCGCCGCCTGCGCGGCGCGAGCAGCGCGGAAGCGCCGGATGGC

[0358] GATAAAGATGCGCCGGAACCGCTGGATAAAGTGATTATTCTGAGCCCGGGCATTAGCGAT

[0359] GCGACCGCGCCGGCGTGGCCGCCGCCGGGCGAAGATCCGGGCACCACCCCGCCGGGCCAT

[0360] AGCGTGCCGGTGCCGGCGACCGAACTGGGCAGCACCGAACTGGTGACCACCAAAACCGCG GGCCCGGAACAGCAG

[0361] SEQ ID NO:41:

[0362] >CD40-lntracellularDomain

[0363] AAAAAAGTGGCGAAAAAACCGACCAACAAAGCGCCGCATCCGAAACAGGAACCGCAGGAA

[0364] ATTAACTTTCCGGATGATCTGCCGGGCAGCAACACCGCGGCGCCGGTGCAGGAAACCCTG

[0365] CATGGCTGCCAGCCGGTGACCCAGGAAGATGGCAAAGAAAGCCGCATTAGCGTGCAGGAA

[0366] CGCCAG

[0367] SEQ ID NO:42:

[0368] >OX40-lntracellularDomain

[0369] GCGCTGTATCTGCTGCGCCGCGATCAGCGCCTGCCGCCGGATGCGCATAAACCGCCGGGC

[0370] GGCGGCAGCTTTCGCACCCCGATTCAGGAAGAACAGGCGGATGCGCATAGCACCCTGGCG

[0371] AAAATT

[0372] SEQ ID NO:43:

[0373] >TACI-lntracellularDomain

[0374] AAAAAACGCGGCGATCCGTGCAGCTGCCAGCCGCGCAGCCGCCCGCGCCAGAGCCCGGCG

[0375] AAAAGCAGCCAGGATCATGCGATGGAAGCGGGCAGCCCGGTGAGCACCAGCCCGGAACCG

[0376] GTGGAAACCTGCAGCTTTTGCTTTCCGGAATGCCGCGCGCCGACCCAGGAAAGCGCGGTG

[0377] ACCCCGGGCACCCCGGATCCGACCTGCGCGGGCCGCTGGGGCTGCCATACCCGCACCACC

[0378] GTGCTGCAGCCGTGCCCGCATATTCCGGATAGCGGCCTGGGCATTGTGTGCGTGCCGGCG

[0379] CAGGAAGGCGGCCCGGGCGCG SEQ ID NO:44:

[0380] >CD8aHinge ACCACCACACCAGCTCCTAGACCTCCAACTCCTGCTCCTACAATCGCCAGCCAGCCACTG TCTCTGAGGCCAGAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTG GATTTCGCCTGCGAC

[0381] SEQ ID NO:45:

[0382] >CD8aTransmembrane ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGTTGCTGCTGAGCCTGGTCATC ACCCTGTACTGC

[0383] SEQ ID NO:46:

[0384] >lgG4Hinge GGCGGCGGTGGATCAGAGTCCAAGTACGGACCCCCTTGTCCCCCATGTCCA

[0385] SEQ ID NO:47:

[0386] >A3A_VL

[0387] GATATCGTAATGACTCAGAGTCACAAACTGATGTCTACATCAGTTGGCGACCGGGTGAGC ATCACTTGTAAAGCTAGCCAAGACGTGTCAAGCGCTGTAGCCTGGTATCAGCAGAAACCC GGTCAGTCTCCTAAACTTCTTATCAACAGCGCCTCATACAGGTATACAGGGGTTCCTGAA AGGTTCACGGGCAGCGGATCAGGGACAGACTTCACGTTCACCATTAGCAGCGTACAAGCG GAAGACCTGGCTGTCTACTACTGCCAGCAGCATTACCGGACACCCTTCACATTTGGGGCA GGGACGAAACTTGAGCTGAAG

[0388] SEQ ID NO:48:

[0389] >A3A_VH CAAGTTCAATTACAACAGTCTGGAGCGGAGCTCGTCCGTCCAGGTGCCAGCGTGACTCTC AGCTGCAAAGCCAGCGGCTACACCTTTACAGACTATGAAATGCACTGGGTAAAGCAGACT CCAGTACACGGACTTGAGTGGATTGGAGCCATCGACCCCGAGACAGGTGGCACAGCTTAT AATCAAAAGTTTAACGGCAAGGCGATTTTAACCGCTGACAAATCCTCTACCACCGCATAT

[0390] ATGGAGTTACGGAGCTTGACTTCTGAGGACTCAGCCGTGTACTACTGTAGCAGAGGCCCC TACT ACT ATGGCACCACCTATGGATGCTTCGACTTTTGGGGACAGGGCACAGCCCTCACG GTCTCCTCA

[0391] SEQ ID NO:49:

[0392] >4G2_VL GATATTGTTATGACTCAATCACAGAAATTTATGAGCACGTCCGTAGGCGACAGAGTCTCT GTCACATGTAAGGCTAGCCAAAATGTCGGGACCAACGTCGCTTGGTACCAACAAAAGCCT GGTCAATCCCCTAAGGCACTTATATATTCTGCTAGCTCCAGGTATTCTGGTGTCCCTGAT AGGTTTACAGGATCTGGGAGTGGCACTGATTTCACTCTCACCATCAGCAACGTCCAATCA GAGGATTTGGCCGAATATTTCTGTCAACAATACAATATCTATCCGCTGGCTTTTGGCGGC GGAACCAAACTGGAAATAAAA

[0393] SEQ ID NQ:50:

[0394] >4G2_VH

[0395] GAGGTCCAATTACAGCAAAGTGGAGCAGACTTAGTTCGGCCCGGTGCTTCCGTGAAATTA TCTTGTACAGCATCAGGATTTAATATTAAAGATGACTATATCCATTGGGTTAAACAACGC CCAGAACAAGGGTTGGAATGGATAGGATGGATCGATCCTGAGTCTGGCGATACAGAATAT GCAAGCAAATTTCAGGGGAAAGCTACTATCTCCGCTGACACTTCTTCCAACACCGCTTAT CTGCAACTGAGCTCATTAACTTCCGAAGACACAGCAGTCTATTACTGTACAACATGGAAT TGGTATGGCGGGTCTTCCGCGTGGTTCCCTTATTGGGGACAAGGTACTCTCGTTACCGTA

[0396] TCTGCC

[0397] SEQ ID N0:51:

[0398] Amino acid:

[0399] >BAFF-R-lntracellularDomain

[0400] SWRRRQRRLRGASSAEAPDGDKDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVP

[0401] VPATELGSTELVTTKTAGPEQQ

[0402] SEQ ID NO:52:

[0403] >CD40-lntracellularDomain

[0404] KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0405] SEQ ID NO:53:

[0406] >OX40-lntracellularDomain

[0407] ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0408] SEQ ID NO:54:

[0409] >TACI-lntracellularDomain

[0410] KKRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPG

[0411] TPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA

[0412] SEQ ID NO:55:

[0413] >CD8aHinge

[0414] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0415] SEQ ID NO:56:

[0416] > CD8aTransmembrane

[0417] IYIWAPLAGTCGVLLLSLVITLYC

[0418] SEQ ID NO:57:

[0419] >lgG4Hinge

[0420] GGGGSESKYGPPCPPCP

[0421] SEQ ID NO:58:

[0422] >A3A_VL

[0423] DIVMTQSHKLMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKLLINSASYRYTGVPERFT

[0424] GSGSGTDFTFTISSVQAEDLAVYYCQQHYRTPFTFGAGTKLELK

[0425] SEQ ID NO:59:

[0426] >A3A_VH

[0427] QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYN

[0428] QKFNGKAILTADKSSTTAYMELRSLTSEDSAVYYCSRGPYYYGTTYGCFDFWGQGTALTVSS

[0429] SEQ ID NQ:60:

[0430] >4G2_VL

[0431] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYS

[0432] ASSRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNIYPLAFGGG TKLEIK SEQ ID N0:61:

[0433] >4G2_VH

[0434] EVQLQQSGADLVRPGASVKLSCTASGFNIKDDYIHWVKQRPEQGLEWIGW IDPESGDTEYASKFQGKATISADTSSNTAYLQLSSLTSEDTAVYYCTTWNW YGGSSAWFPYWGQGTLVTVSA

[0435] The following Examples illustrate the different embodiments of the invention. Unless stated otherwise all recombinant DNA techniques are carried out according to standard protocols as described in e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA.

[0436] EXPERIMENTAL SECTION 1

[0437] The present inventors designed antiCD277-costimulatory receptors and tested them next to a tumor reactive immune receptor in engineered immune cells. The inventors establish that the specific selection of the epitope bound by the antiCD277-scFv can enhance the T cell function. In two different tumor xenograft models, the inventors show superior tumor control for engineered T cells co-expressing the tumor reactive immune receptor and the anti-CD277- costimulatory receptor, compared to engineered T cells expressing the tumor reactive immune receptor only.

[0438] Materials and Methods

[0439] Antibodies and flow cytometry analysis

[0440] The following antibodies were used: CD8a-PerCP-Cy5.5 (1 :100; clone RPA-T8; 301032), CD4-AF700 (1 :40; clone RPA-T4; 300526), apTCR-PE-Cy5 (1:80; clone IP26; 11-9986-42), from Biolegend. ybTCR-PE-Cy7 (1 :20; clone IMMU510; B10247) and Pan-ybTCR-PE (1:10; clone IMMU510; B49176) from Beckman Coulter. ybTCR-APC (1 :5; clone B1 ; 555718) from BD Biosciences. huCD45-PB (1 :30; clone HI30; 2120145) and mCD45-APC (1 :30; clone 30- F11S; 1115560)from Sony Biotechnology. apTCR-FITC (1 :10; clone IP26; 11-9986-42) from Invitrogen. CD4-PE-Cy7 (1 :100; clone RPA-T4; 25-0049-42) and Fixable Viability Dye eFluor506 from eBioscience. CD277ec-teramers were produced in house as described before

[0016] , All samples were analyzed on a BD LSRFortessa or BD Canto using FACS-Diva Software (BD Biosciences).

[0441] Cell lines and PBMCs RPMI-8226, SCC9, Fadu, and Phoenix-Ampho cells were obtained from ATCC. Phoenix- Ampho and SCC9 cells were cultured in DMEM supplemented with 1% Pen / Strep (Invitrogen) and 10% FCS (Bodinco, Alkmaar, The Netherlands). All other cell lines were cultured in RPMI with 1% Pen / Strep and 10% FCS. Primary fresh PBMCs were isolated by Ficoll-Paque (GE Healthcare, Eindhoven, The Netherlands) from buffy coats supplied by Sanquin Blood Bank (Amsterdam, The Netherlands).

[0442] Construction of antiCD277 costim receptors

[0443] Synthetic DNA for the antiCD277 co-receptors were ordered from BaseClear (Leiden, Netherlands). They were all subcloned into pMP71 already containing ybTCR-CI5, using Xhol and Hindll I . All enzymes were supplied by NEB (Massachusetts, USA).

[0444] Retroviral transduction of ap T cells and cell lines

[0445] Briefly, packaging cells (Phoenix-Ampho) were transfected with helper constructs gag-pol (pHIT60), env (pCOLT-GALV) and pMP71 or pBullet retroviral vectors containing genes codifying for the different proteins. In the case of human PBMCs, they were pre-activated with anti CD3 (30 ng / mL; Orthoclone OKT3; Janssen-Cilag) and IL-2 (50 lU / mL; Proleukin, Novartis). CD4 and CD8 T cells were transduced twice with a viral supernatant within 48 or 3 hours respectively, in the presence of 6 pg / mL polybrene (Sigma-Aldrich). For PMBCs 50 lU / mL of IL-2 was added. TCR-transduced T cells were expanded by stimulation with anti- CD3 / CD28 Dynabeads (500,000 beads / 106 cells; Life Technologies) and IL-2 (50 lU / mL). Thereafter, TCR-transduced T cells were depleted of the non-engineered a T cells.

[0446] Depletion of non-engineered T cells and selection of engineered T cells a T cells transduced with ybTCR either alone, or together with antiCD277 costim were incubated with a biotin-labeled anti-a TCR antibody (clone BW242 / 412; Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequently incubated with an anti-biotin antibody coupled to magnetic beads (anti-biotin MicroBeads; Miltenyi Biotec). Thereafter, the cell suspension was loaded onto an LD column and a TCR+ T cells were depleted by MACS cell separation, per the manufacturer’s protocol (Miltenyi Biotec). After depletion, TEGs were expanded, using a T cell rapid expansion protocol (REP)

[0017] , After a -depletion, T cells were selected using human CD4 or CD8 microbeads and MS columns (Miltenyi Biotec). The procedure was carried out according to the manufacturer's protocol.

[0447] In vitro functional T cell assays

[0448] For cytokine detection, 5 x 10A4 engineered T cells were co-cultured for 20 hours with different tumor cell lines a 1:1 effector-to-target ratio (E:T), in round-bottom 96-well plates, in absence or presence of pamidronate. After incubation, supernatants were collected, and either frozen or used directly to detect IFNy levels. ELISA was performed using IFN gamma Human Uncoated ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA).

[0449] For killing assays 5 x 10A3 RPMI 8226 or SCC9 cells expressing luciferase-GFP were cocultured with engineered T cells at 3:1, 1 :1, 0.3:1 effector: target ratio in absence or presence of pamidronate. After 20 hours luciferin was added at 12.5 ug / ml and luminescence signal was measured on a Softmax pro machine.

[0450] In order to assess proliferation, T cells were resuspended at 1 x 10A6 cells / ml using a 2 pM solution of CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, Massachusetts, USA) in PBS. The cell suspension was incubated for 20 min at 37°C. Cells were washed two times with complete RPMI medium, and resuspended in culture medium. For the proliferation assay with tumor cell lines, 2.5 x 10A5 labeled engineered T cells were co-cultured, together with 2.5 x 10A5 tumor cells in 48-well plates for 6 days, 100 pM pamidronate was added to indicated cultures to boost recognition. On Day 6, T cell proliferation was analyzed by flow cytometry.

[0451] 3D model

[0452] The 3D model was previously described in detail

[0015] , RPMI-8226 tumor cells were stained with Vybrant DiO (Thermo Fisher, United States) and multipotent mesenchymal stromal cells (MSCs) stained with Vybrant DiD (Thermo Fisher, United States). Both cell types seeded in Matrigel (Corning, United States) on the same day. After four days, the different engineered T cells were stained with Vybrant Dil (Thermo Fisher, United States) and administered to the model, together with and 10 pM PAM (Calbiochem, United States). Six days later, the Matrigel was dissolved using Dispase (Corning, United States) to retrieve the cells from the model. Tumor, T cells and stromal cells were quantified by FACS, using Flow count Fluorospheres (Beckman Coulter, United States).

[0453] Animal models

[0454] NOD.Cg-Prkdcscidll2rgtm1Wjl / SzJ (NSG) mice originally obtained from Jackson Laboratory (Bar Harbor, ME, USA) were purchased from Charles River Laboratories (AS, USA). Experiments were conducted under institutional guidelines, after obtaining permission from the Animal Welfare Body Utrecht, and in accordance with the current Dutch laws on Animal Experimentation. Mice were housed in sterile conditions, using an individually ventilated cage (IVC) system and fed with sterile food and water. Irradiated mice were given sterile water with the antibiotic Ciproxin for the duration of the experiment. Mice were randomized and divided into 10 mice / group. Adult mice (16-30 weeks old) received sublethal total body irradiation (1.75 Gy) on Day -1. On Day 0, depending on the model, NSG mice were injected either intravenously with 5 x 10A6 RPMI 8226-luciferase cells resuspended in PBS or subcutaneously with 0.5 x 10A6 SCC9-luciferase or 1 x 10A6 Fadu tumor cells resuspended in mixture of matrigel and PBS (1 :1) or PBS respectively. On Days 1 and 7, mice were injected intravenously with 1 x 10A7 of different engineered T cells. All mice received 0.6 x 10A6 III of IL-2 (Proleukin; Novartis) in 100 pl incomplete Freund’s adjuvant (IFA) subcutaneously, together with the first T cell injection, and every 3 weeks until Day 45. Pamidronate (10mg / kg body weight) was injected intravenously, together with the T cell injections, and every 3 weeks until the end of the experiment, in order to enhance activation of TEGs. Tumor growth was monitored weekly by bioluminescence for RPMI-8226 and SCC9. Furthermore, in the subcutaneous Fadu model, tumor volumes were measured twice per week, and calculated by using the formula tumor volume = 0.4x (length x width2). In the all models, mouse survival was assessed at least twice a week, by monitoring weight loss and symptoms of disease (sign of paralysis, weakness, and reduced motility).

[0455] Generation and screening of antibodies against CD277

[0456] Murine antibodies against the extracellular domain of CD277 were generated by the Utrecht Monoclonal Antibody Facility (UMAB) located at the UMC Utrecht (Netherlands) using their cellular immunization method. Supernatants of the hybridoma clones generated by UMAB were used for screening.

[0457] UMewt and UMecD277 were incubated with hybridoma supernatant, after washing the cells were stained with anti-mouse Ig-APC and analyzed by flow cytometry. The MFI on UMecD277 normalized to the MFI of the control cell line UMewt.

[0458] RPMI-8226 cells were incubated with hybridoma clone supernatant, after 15 minutes scFv- 103.2-HA was added. Cells were washed once, and incubated with anti-HA antibody to detect scFv103.2 binding. The cells were analyzed by FACS. The data is normalized to scFv103.2- HA binding alone.

[0459] 5 10A4 CL5-T cells were cocultured with FaDu cells 1 :1 E:T ratio and hybridoma clone supernatant in the presence of 30 pM pamidronate for 20 hours. IFNy production by CI5-T cells was determined using IFN gamma Human Uncoated ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA).

[0460] Results

[0461] Generation of T cells expressing a tumor reactive TCR and antiCD277-costimulatory receptor

[0462] For expressing the chimeric costimulatory receptors targeting CD277 in combination with a y952TCR, the inventors based the design on previously reported NKG2D chimeric costimulatory receptors [9], The present tri-cistronic construct contained the TCRy9 chain, TCR62 chain, and antiCD277-costimulatory receptor genes separated by T2A and P2A selfcleaving peptide sequences, respectively (Figure 1A). The mature antiCD277-costimulatory receptor contains an extracellular antiCD277 scFv linked to the CD28 hinge-transmembrane peptide and the intracellular signaling domain of 4-1 BB.

[0463] The inventors created two antiCD277-costimulatory receptors (anti-CD277-costim) using the scFv derived from mAb 103.2

[0010] and mAb CTX2026 [3], These two antibody clones recognize slightly overlapping epitopes on top of the IgV-like domain of CD277, but the relative orientation of the antibodies differ significantly (Figure 1B). mAb 103.2 recognizes a conformational epitope consisting of residues Ser30-Thr33, Tyr50-Lys54, and Arg72-Asp73 of the mature protein [8], CTX2026 also recognizes a conformational epitope containing residues Ser30, Thr33, Glu35-Lys37, Val46, Val49, Ala51-Gly53, Arg59, and Tyr98-Tyr105 [3], Despite the overlap in epitopes these two antibodies have distinct effects on y952T cells activation, where 103.2 is an inhibitory antibody and CTX2026 is an activating antibody [3, 10], 103.2 has also been shown to inhibit activation of abT cells engineered with a y952TCR

[0010] , However, antibody fragments of 103.2 are not able to inhibit y952T cells activation [8], and F(ab’)2 and Fab fragments are able to activate y952T cells (WO2020136218A1) like other agonistic antibodies 20.1 [8, 10] or CTX2026 [3],

[0464] Human donor derived a T cells were transduced with using retrovirus containing either the non-functional y952TCR LM1 or the tumor reactive y952TCR CI5 alone

[0011] , or y952TCR CI5 in combination with either of the antiCD277-costim, 103.2-41 BB or CTX2026-41BB. After transduction the successfully transduced cells were enriched by depleting a TCR expressing cells as described previously

[0012] , To confirm surface expression of both the y952TCR as the antiCD277-costim, the engineered T cells were stained with anti-ybTCR-PECy7 and CD277ec-streptavidinPE tetramers and analyzed by FACS. The engineered T cells with CI5 only stained for positive for ybTCR, while the engineered T cells expressing CI5 in combination with 103.2-41 BB or CTX2026-41 BB stained positive for both ybTCR and CD277ec-tetramer (Figure 1 C), confirming that both antiCD277-costim molecules were able to bind to CD277.

[0465] AntiCD277-costim 103.2-41 BB enhanced the tumor reactivity of engineered T cells better than AntiCD277-costim CTX2026-41BB

[0466] Having established that the antiCD277-costim molecules are expressed and able to bind their ligand, the next step was to determine whether antiCD277-costim could also enhance the killing capacity of the engineered T cells. For this, the inventors used two tumor cell lines that are targeted by y952TCRs, the multiple myeloma cell line RPMI-8226 and the head and neck cancer cell line SCC9. These cell lines were both transduced with luciferase, so tumor cell viability could be measured by adding luciferin to the cultures. To be able to observe differences in tumor killing capacity of the engineered T cells the inventors used (i) three different effector-to-target ratio’s and (ii) co-culture conditions without and with pamidronate to increase recognition via the y952TCR.

[0467] The negative control LM1 T cells were not able to kill the tumor cells in any of the conditions, while CI5 T cells were able to kill RPMI-8226 cells in absence and presence of pamidronate and SCC9 cells only in the presence of pamidronate (Figure 2). As expected, tumor cells were killed more efficiently by CI5 T cells at higher effector-to-target ratio’s (Figure 2). CISIOS.2-41 BB T cells exhibited better tumor killing compared to CI5 T cells, especially at lower effector-to-target ratio’s. Moreover, CI5-103.2-41 BB T cells were able to kill SCC9 cells in the absence of pamidronate, albeit less efficient compared to the condition with pamidronate (Figure 2). In contrast, CI5- CTX2026-41 BB T cells killed RPMI-8226 cells to comparable levels as CI5 T cells and were even slightly worse in killing SCC9 cell in the presence pamidronate (Figure 2). This difference in efficacy between the two chimeric receptors bearing different anti-CD277 scFv showed that not all anti-CD277 scFv are equally effective in a functional chimeric costimulatory receptor. The relative binding orientation of the anti- CD277 scFv might be important to create a functional costimulatory receptor with increased efficacy.

[0468] Proliferation capacity of T cells engineered with antiCD277-costim was enhanced in both CD4+ and CD8+ T cells

[0469] Increased proliferative capacity of tumor specific T cells is important to sustain prolonged tumor control. To address if the co-expression of antiCD277-costim indeed results in improved proliferation, the engineered T cells were co-cultured for six days with RPMI-8226 or SCC9 cells, in the absence of presence of pamidronate. In line with the killing assay, the inventors observed increased proliferation of CI5-103.2-41 BB T cells in all conditions, for both CD4+ and CD8+ T cells, compared to CI5 T cells (Figure 3). The addition of pamidronate, in order to increase the intracellular phosphoantigen levels, led to an increase in proliferation for all engineered T cells. In contrast to results of the killing assay, there were no differences between CI5 and CI5-CTX2026-41 BB T cells, increased proliferation was observed for CI5- CTX2026-41 BB T cells, compared to CI5 T cells, when cultured with the two tumor cell lines (Figure 3), indicating that this construct can deliver additional costimulatory signals to the engineered T cells.

[0470] The intracellular 4-1 BB signaling domain results in the most potent antiCD277-costim In chimeric receptors for T cell therapy intracellular signaling domains derived from different co-stimulatory proteins lead to different efficacies of engineered T cells

[0013] , Using the 4-1 BB signaling domain in general leads to improved T cell fitness and long persistence, while the CD28 signaling domain induced fast initial expansion and improved cytotoxicity. To test the effect of the different costimulatory domains in anti-CD277-costim, CD4 T cells were engineered with CI5, CI5-103.2-41 BB, or CI5-103.2-CD28 (or the negative control TCR LM1 + / - 103.2-41 BB). After expansion the T cell activation potential and T cell induced cytotoxicity was assessed in co-culture assays with tumor cell lines. First the inventors addressed the T cell activation potential using IFNy secretion as a read out for T cell activation. The cocultures were done in absence of presence of 10 and 100 pM pamidronate. In line with the previous experiments, CI5-103.2-41 BB T cells were able to secrete IFNy in absence of pamidronate when co-cultured with tumor cells, either SCC9 or Fadu, and IFNy secretion was further enhanced in the presence of pamidronate (Figure 4A). Both CI5 and CI5-103.2-CD28 T cells benefit from increased phosphoantigen levels mediated by pamidronate to secrete IFNy in cocultures with tumor cells, moreover in the co-culture with SCC9 cells and 10 pM pamidronate CI5 T cells outperformed CI5-103.2-CD28 T cells. In conditions with 100 pM pamidronate no differences between CI5 and CI5-costim were observed due to the strong recognition of tumor cells of CI5 T cells. The lack of activation in all conditions for LM1 and LM 1-103.2-41 BB T cells showed the importance of TCR signaling for enhanced T cell activity, as seen for CI5- 103.2-41 BB T cells.

[0471] Next the inventors assessed if the difference in T cell activity between costimulatory signaling domains also resulted in a difference in the cytotoxic potency of the engineered T cells. Engineered CD4 T cells were cocultured with RPMI-8226-lucGFP cells in different effector target ratio’s in the presence of 10 pM pamidronate. Again CI5-103.2-41 BB T cells were more potent in killing RPMI-8226-lucGFP cells compared to CI5 T cells, especially at lower effector target ratio’s, 1 :1 and 0.3:1 (Figure 4B), while CI5-103.2-CD28 T cells were less effective in killing tumor cells compared to CI5 T cells.

[0472] Both assays imply that choice of intracellular signaling domain in antiCD277-costim is important for the potency of engineered T cells, and that the use of 41 BB signaling domain is preferred over the CD28 signaling domain.

[0473] Both CD4+ and CD8+ engineered T cells were enhanced by 103.2-41 BB

[0474] Tumor cell killing by engineered T cells can be mediated by both CD4+ and CD8+ T cells and many studies have shown that it is important to enhance both CD4+ and CD8+ T cells (reviewed in

[0014] ). The T cell proliferation experiment already showed that both subsets are improved by the co-expression of 103.2-41 BB, but the inventors did not formally show that the killing capacity of both subsets was enhanced as well. To address this, the inventors made used of our 3D bone marrow niche model

[0015] , in which the multiple myeloma cell line RPMI-8226 was cultured together with mesenchymal stromal cells (MSG) in Matrigel, T cells were added on top of a trans-well membrane to introduce the requirement of active migration. The engineered T cells were combined in a 1 :1 CD4+ and CD8+ ratio, either as CI5-T cells or CI5-costim T cells or as a combination of CI5-T cells with CI5-costim-T cells. After six days of coculture, the Matrigel was dissolved and the number and type of cells present in the wells was analyzed by FACS.

[0475] For CD4-CI5:CD8-CI5 T cell condition 50% of the tumor cells were killed over the course of the experiment, while combining one the CI5 T cell subsets with the other subset of CISIOS.2-41 BB T cells led to a significant increase in tumor cell killing (Figure 5A). There was a further increase in tumor cell killing to >80% when both CD4+ and CD8+ T cells were engineered with CI5-103.2-41 BB, indicating the added benefit of enhancing both T cell subsets.

[0476] The added benefit of the 3D bone marrow model is the presence of non-malignant MSCs, that can be used to monitor toxicity. In all conditions a similar number of MSC was present after six days of co-culture (Figure 5B), while large significant differences in tumor killing were observed between the different engineered T cell combinations. Based on this observation the inventors conclude that there is no aberrant bystander toxicity in any of the formats.

[0477] T cells co-expressing CI5 and 103.2-41 BB had superior tumor control in a multiple myeloma xenograft model

[0478] Next, the inventors tested whether the increased capacity to kill tumor cells by T cells coexpressing CI5 and 103.2-41 BB also translates to better tumor killing in vivo. For this the inventors used a multiple myeloma, RPMI-8226, xenograft model in NSG mice to test three engineered T cell products; LM1 , CI5, and CI5-103.2-41 BB. Tumor burden was monitored using bioluminescence imaging (BLI) once a week. Rapid tumor outgrowth was observed in the group treated with LM1 T cells, while in the group treated with CI5 T cells a delayed tumor outgrowth was observed, followed by a similar tumor growth curve as seen for the LM1 T cell group (Figure 6A). The group treated with CI5-103.2-41 BB T cells showed still low tumor burden on day 55. Only 3 mice in this group had detectable tumor signal on day 55 (Figure 6B). Mouse survival reflected the tumor outgrowth observed in BLI (Figure 6C). Mice treated with LM1 T cells had a median survival of 31 days, while mice treated with CI5 T cells had a significant longer median survival of 51 days. All mice treated with CI5-103.2-41 BB T cells were survived for over 75 days.

[0479] As 7 out of 10 mice in the CI5-103.2-41 BB T cell group had undetectable or very low BLI signal at day 55, 4 out of 7 tumor free mice were rechallenged with an intravenous injection of 5 106RPMI-8226 cells at day 59. The other 3 tumor free mice were used a non-rechallenged control group.

[0480] In both the non-rechallenged group as well as in the rechallenged group one mouse developed a solid tumor, while all other mice remained tumor free up to day 97 (Figure 7A). The mouse that developed a solid tumor in the non-rechallenged group needed to be sacrificed at day 93. At day 100 the remaining mice (n=2 non-rechallenged; n=4 rechallenged) were sacrificed, and the number of engineered T cells in the bone marrow and blood was determined using FACS. In both the non-rechallenged group as the rechallenged group similar numbers of T cells were present in the bone marrow, however no persisting T cells were detected in blood, which might be an explanation for the development of a solid tumor in some of these mice (Figure 7B).

[0481] T cells co-expressing CI5 and 103.2-41BB have superior solid tumor control in xenograft models

[0482] T cells expressing y952TCRs can also target solid tumors and in the in vitro assays a strong enhancement in tumor killing was observed for CI5-103.2-41 BB T cells. To determine if this effect is also observed in vivo, subcutaneous solid tumor models, using SCC9-lucGFP cells, in NGS mice was done. As in the MM model, the mice were divided in three groups that received a different engineered T cell product; LM1 , CI5, and CI5-103.2-41 BB. The engineered T cells were injected one and seven days after tumor injection in presence of pamidronate, additional pamidronate injections was given on day 21 and 42. Once a week tumor size was measured using BLI. Despite large differences in the in vitro killing assay, there was no significant difference in tumor control between the groups treated with LM1 T cells and CI5 T cells. The group treated with CI5-103.2-41 BB T cells completely prevented outgrowth of SCC9 tumor cells (Figure 8A).

[0483] Previously the inventors noticed that not all tumor cells were efficiently targeted by CI5 T cells at low pamidronate concentration, e.g. SCC9 was efficiently recognized by CI5 T cells at 10 pM PAM but Fadu was not. (Figure 4A). The inventors were interested to see whether CISIOS.2-41 BB T cells could also prevent the outgrowth of Fadu cells in a s.c. xenograft model. Again the mice were divided in three treatment groups; LM1 , CI5, and CI5-103.2-41 BB T cells. One and seven days after tumor injection the engineered T cells were injected in presence of pamidronate, an additional pamidronate injection was given on day 21. Twice a week tumor size was measured using a caliper. Again no significant difference in tumor size could be observed between the groups treated with LM1 and CI5 T cells (Figure 8B). In this Fadu model there was no complete tumor control for the group treated with CI5-103.2-41 BB T cells, but tumor outgrowth was significantly reduced.

[0484] At day 37 the mice were sacrificed and infiltration of engineered T cells in the tumors was determined in 4 mice of the CI5 T cell and CI5-103.2-41 BB T cell groups. In line with the reduced tumor outgrowth in the group treated with CI5-103.2-41 BB T cells, a significant higher infiltration of engineered T cells, a mean 1000 fold increase, was observed compared to CI5 T cells (Figure 8C). In bone marrow, there was also a significant higher number of CI5-103.2-41 BB T cells present compared to CI5 T cells, but in this compartment there was only a mean 10 fold increase (Figure 8D). The inventors conclude that, compared to CI5 T cells, CI5-103.2-41 BB T cells could not only persist longer in mice but also home to the tumor side to reduce tumor outgrowth.

[0485] Generation of novel antiCD277 antibodies resulted in antibodies with distinct characteristics

[0486] Murine UMe cells transduced a CD277 construct were used to immunize mice. The splenocytes of the mice were fused with murine myeloma cells to generate hybridomas. Clonal populations of hybridomas were generated using two rounds of limiting dilution. 29 subclones were used for in-depth characterization. Ideally antibodies with similar characteristics as 103.2 would be most preferred. The supernatants of all hybridoma subclones were able to stain the UMecD277 cell line, albeit differences in staining intensity were observed (Figure 9A), e.g. hybridoma subclone supernatant 4G5-H8 showed relatively weak staining, 16-fold increase in MFI over UMewt staining. Next, the inventors assessed whether the antibodies in the hybridoma subclone supernatants were able to inhibit binding of 103.2-scFv to RPMI-8226 cells. Here, the inventors observed distinct differences between the hybridoma subclones. One group strongly inhibited the binding of 103.2-scFv to RPMI-8226 cells indicating an overlapping epitope, while a second group was not able to inhibit 103.2- scFv binding and probably has a non-overlapping epitope (Figure 9B and Table 1). A third group, partially inhibited the binding of 103.2-scFv to RPMI-8226 cells, which could be caused by antibody concentration or lower affinity for CD277 of these subclones. Antibody 103.2 is a potent antagonist for y952TCR mediated activation. To assess whether the novel antibodies were also able to block y952TCR mediated activation, CI5-T cells were co-cultured with FaDu cells and 30 pM pamidronate in presence of the hybridoma subclone supernatants. The majority of the hybridoma subclone supernatant, inhibited the activation of CI5-T cells (Figure 9C), including the hybridoma subclone supernatants that did not inhibit 103.2-scFv binding. There were few hybridoma subclone supernatants that did not inhibit CI5-T cell activation (sub-clones; 4G5-H8, 6D12-D10, 6D12-H11, and 7E5A7), and again a group of subclones that partially inhibit CI5-T cell activation. This initial screening resulted in at least 3 different groups of antibodies with distinct characteristics; the first group are the 103.2 like antibodies that strongly inhibit 103.2-scFv binding as well as CI5-T cell activation, Group 2 consist of antibodies that did not inhibit 103.2 binding but did inhibit CI5-T cell activation, and Group 3 consist of antibodies that (partially) inhibited 103.2-scFv binding, bit did not inhibit CI5-T cell activation (Table 1). Table 1 anti-CD277 subclone characteristics

[0487] Conclusion

[0488] Here the inventors present a novel chimeric costimulatory receptor targeting CD277, a protein that is expressed on a broad range of tumor cells, for improving the efficacy, fitness, and persistence of engineered T cells. As an example the inventors combined this antiCD277- costim with a tumor reactive Y962TCR, and showed that the co-expression of CI5 and 103.2- 41 BB on T cells led to a significantly enhanced tumor control in vitro and in vivo, which was mediated by both CD4+ and CD8+ engineered T cells. However, the present data also shows that not all antiCD277 antibody fragments are equally effective in a functional chimeric coreceptor, as 103.2 was more effective that CTX2026.

[0489] References

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[0507] EXPERIMENTAL SECTION 2 AntiCD277-costim 103.2-41 BB has superior tumor control compared to NKG2D-41BB in in vivo models

[0508] Hereinbefore, it was shown that engineered T cells CI5-103.2-41 BB showed improved tumor control significantly compared to CI5 T cells in both hematological and solid tumor xenograft models (Figures 6 and 8), but no comparative data was provided on a previously developed chimeric costimulatory receptor NKG2D-41BB, that consist of the extracellular domain of NKG2D linked to CD28-hinge-TM connected to the intracellular signaling domain of 41BB. NKG2D recognizes stress antigens, MICA / B and ULBP1-6, that are expressed on many tumor cells [1, 2],

[0509] When comparing the tumor control in the multiple myeloma, RMPI-8226, xenograft model for engineered T cells expressing LM1, CI5, CI5-NKG2D-41 BB, and CI5-103.2-41 BB, a clear difference in tumor control between the mock Y952TCR-LM1 and the other engineered T cells was observed (figure 10A). However, despite a difference in tumor control in mice treated with CI5 and CI5-NKG2D-41BB T cells, clear tumor outgrowth was observed after 35 days, which was not observed for mice treated with CI5-103.2-41 BB T cells (Figure 10A).

[0510] More pronounced differences were observed in a xenograft model using SCC9-lucGFP cells, where no significant difference in tumor growth was observed between treatments with LM1 and CL5 T cells and a small, but significant, difference in tumor growth was observed between LM1 and CI5-NKG2D-41 BB. In this model tumor outgrowth was fully controlled in mice treated with CI5-103.2-41 BB T cells (Figure 10B).

[0511] This comparative data clearly shows the superior efficacy of engineered T cells expressing CI5-103.2-41 BB over engineered T cells expressing CI5-NKG2D-41BB, underpinning the importance for antigen selection in development of chimeric costimulatory receptors.

[0512] Altered antiCD277-costim designs resulted in similar efficacy enhancement as 103.2- 41BB

[0513] The intracellular signaling domains of costimulatory receptors are widely used to in CAR and COR designs to enhance the potency or persistence of the engineered immune cells. Here, we tested, next to 4-1 BB and CD28 intracellular domains, also the intracellular domains of BAFF-R, CD40, 0X40 (CD134), and TACI (CD267) in the antiCD277-costim construct (Figure 11A). Healthy donor (ds272) T cells were transduced with the constructs using retroviral particles containing genes encoding y952TCR A3 and one of the antiCD277-costim constructs.

[0514] To assess the efficacy of the altered antiCD277-costim constructs, the transduced T cells were co-cultured with SCC9 cells for 24h, and the degranulation of T cells, measured by CD107 surface expression on CD8+ T cells, was used to determine the level of T cell activation. In the absence of pamidronate no activation was expected for T cells expressing a Y962TCR alone, as observed before (Figure 3 and Figure 4A), in line with these observations, there was a low percentage, 7.2%, CD107+ CD8+ T cells for Y952TCR transduced T cells (Figure 11 B). The percentage of CD107+ CD8+ T cells was significantly increased for antiCD277-costim constructs, with mean percentage CD107+CD8+ T cells on average over 4-fold higher compared to Y952TCR A3T cells; 103.2-41 BB 39.9%, 103.2-CD28 30.2%, 103.2-BAFF-R 35.1 %, 103.2-CD40 47.8%, 103.2-0X40 28.0%, and 103.2-TACI 41.6% (Figure 11 B). Surprisingly, in these experiments 103.2-CD28 did enhance efficacy of the transduced T cells in contrast to the experiments reported above (Figure 4). Underlying reasons for this difference are hard to determine in general, but variations in donor reactivity are described in the context of CARs [3], For this reason, T cells of a third donor (ds243) were transduced with Y952TCR, Y952TCR-103.2-41 BB, and Y952TCR-103.2-CD28, and expanded. These engineered T cells were also assessed for their tumor reactivity using SCC9-lucGFP cells as targets in absence pf pamidronate. Both antiCD277-costim 103.2- 41 BB and 103.2-CD28 expressing Y952TCR T cells outperformed Y952TCR T cells in killing SCC9-lucGFP cells, as measured by total luciferase activity in the culture (Figure 11C).

[0515] In conclusion, an antiCD277-costim consisting of scFv 103.2, the hinge and transmembrane sequence of CD28, and a intracellular signaling domain of a costimulatory molecule, like 41 BB, BAFF-R, CD40, 0X40 (CD134), TACI (CD267), or CD28, enhance the efficacy of engineered T cells. Of note, some intracellular signaling domains, like e.g. the one of CD28, might be more affected by variation in donor T cells, while others, like 41 BB, work in all tested donors.

[0516] Next to the intracellular signaling domains other parts of a chimeric coreceptor can be varied as well, like the hinge and transmembrane sequences. To assess whether introducing different hinge or transmembrane sequences in the 103.2-41 BB construct affected the potency of the engineered T cells, several additional constructs were made containing scFv 103.2 and 41 BB intracellular signaling domain connected by one of the following hinge (H) - transmembrane (TM) combinations; CD8aHTM, CD8aH -CD28TM, lgG4H-CD28TM, or lgG4H-CD8aTM (figure 12A). After transduction of healthy donor T cells (ds272), with the different 103.2-H-TM-41 BB constructs and the only Y952TCR control, the T cells were cocultured with SCC9 cells for 24 hours. As a measure of T cell activation, surface expression of CD107 on CD8+ T cells was determined using FACS. Compared to T cells expressing Y952TCR alone, which had 6.6% CD107+CD8+ T cells after 24 hours of coculture wit SCC9 cells, all T cells co-expressing a antiCD277-costim, regardless of the HTM combination significantly enhanced the percentage of degranulated CD8+ T cells after coculture with SCC9 cells (CD28HTM 31.3%, CD8aHTM 47.9%, CD8aH -CD28TM 40.7%, lgG4H-CD28TM 54.0%, or lgG4H-CD8aTM 54.6%) (Figure 12B). Concluding that, next to variation in costimulatory intracellular signaling domains, the HTM sequences can also be varied to obtain a functional antiCD277 chimeric costimulatory receptor that significantly enhance y952TCR expressing T cells.

[0517] Unique binding epitope of antiCD277 scFv 103.2 is preferred for the enhancement of T cell activity

[0518] Hereinbefore it was found that not every antiCD277-scFv enhanced the potency y952TCR expressing T cells to the same extent, as the CTX2026 derived scFv hardly enhances the potency when used in an antiCD277-costim, despite its ability to bind to CD277 (Figure 1-3). From the novel CD277 antibodies, two antibodies were picked to incorporate in the chimeric costimulatory receptor, one from the “103.2-like” group (clone 3A3) and a second from “group 2” (clone 4G2) (Table 1). The scFv of 3A3 and 4G2 were combined with the CD28HTM-41 BB sequence to create novel antiCD277-costim molecules, now referred to as 3A3-41 BB and 4G2-41BB respectively. Healthy donor T cells were transduced with y952TCR, y952TCR- 103.2-41 BB, Y952TCR-CTX2026-41BB, y952TCR-3A3-41 BB, or y952TCR-4G2-41BB, and expanded. Before functional characterization of these engineered T cells, a FACS analysis was done to confirm y952TCR expression and CD277 binding or the antiCD277-costim molecules. T cells engineered with y952TCR alone were only ybTCR positive, while all T cells engineered with a Y952TCR-antiCD277costim were positive for YGTCR expression and were able to bind soluble CD277 tetramers, confirming properly folded anti-CD277scFv expression on the T cell surface (Figure 13A).

[0519] To assess whether the new antiCD277-costim molecules, 3A3-41 BB and 4G2-41 BB, were able enhance the potency of Y952TCR expressing T cells, like 103.2-41 BB, the engineered T cells were cocultured with SCC9 cells in the absence of presence of 10 pM pamidronate for 20 hours. To assess the magnitude of T cell activation, the concentration of INFY was determined using ELISA. In line with earlier observations, in absence of pamidronate the functional Y952TCR expressing T cells hardly secrete any INFY, while Y952TCR-103.2-41 BB T cells did secrete INFY. The three other antiCD277costim molecules, CTX2026-41 BB, SAS- 41 BB and 4G2-41BB hardly induces any INFY secretion in the engineered T cells (Figure 13B). However, in presence of 10 pM pamidronate INFY secretion was included for T cells engineered with Y952TCR, Y952TCR-103.2-41 BB, Y952TCR-3A3-41 BB, or Y952TCR-4G2- 41 BB, indicating that for 3A3-41 BB and 4G2-41BB elevated levels of pAg were important to become activated, as was the case for the Y952TCR alone, and thus no large additive effect for these two antiCD277-costim molecules was observed. For the mock Y952TCR and Y952TCR-CTX2026-41 BB engineered T cells no IFNY secretion was observed in presence of 10 pM pamidronate (Figure 13B), again indicating that CTX2026-41 BB inhibits Y952TCR activity (Figure 2). To further establish that both 3A3-41BB and 4G2-41BB are hardly enhancing the potency of y952TCR engineered T cells, the proliferation capacity of the engineered T cells was assessed when cocultured with SCC9 cells for six days, in absence and presence of 10 pM pamidronate. Proliferation was assessed by measuring the Cell Trace Violet (CTV) intensity of the prelabeled T cells using FACS. In the conditions without pamidronate only y952TCR- 103.2-41 BB T cells had reduced CTV intensity, indicating that these cells proliferated upon coculture with SSC9, while all the other engineered T cells, expressing mock-y952TCR, y952TCR, Y952TCR-CTX-2026-41BB, Y952TCR-3A3-41 BB, or Y952TCR-4G2-41 BB did hardly proliferate in this condition (Figure 13C). Increasing the intracellular phosphoantigens levels by the addition of 10 pM pamidronate, had, as expected, no effect on mock-Y952TCR, but did induce proliferation of Y952TCR T cells. Again, Y952TCR-103.2-41 BB T cells showed enhanced proliferation compared to Y952TCR T cells. The three other antiCD277-costim molecules, CTX-2026-41BB, 3A3-41 BB, and 4G2-41BB co-expressed in Y952TCR T cells, resulted in diminished proliferation capacity compared to Y952TCR T cells (Figure 13C). These combined observations, strongly point in the direction that the epitope of 103.2 on CD277 is unique with respect to the other antiCD277-scFv tested here. The epitopes of 103.2 and CTX2026 were described above based on the published crystal structure of these antibody fragment in complex with CD277. The initial screening of novel CD277 mAbs, hinted that 3A3 could mimic 103.2, but 4G2 most likely bound a distinct epitope (Figure 9; Table 1). To further elucidate the epitope of mAb 3A3 and mAb 4G2, 31 single alanine substitutions were introduced in the CD277 construct used for immunization, containing the extracellular domains of BTN3A1 linked to the TM and intracellular sequence of BTN3A2 to facilitate surface expression. These single mutants were transiently expressed in HEK293T-CD277KO cells, 48 hours after transfection the cells were incubated with diluted hybridoma supernatant of mAb clone 3A3 or mAb clone 4G2 and stained using anti-mouse Ig-APC and analyzed using FACS. Perturbations in staining intensity between the different alanine substitutions revealed that mAb 3A3 and mAb 4G2 have highly overlapping epitopes, but some BTN3A1 alanine substitutions had distinct perturbation unique for 3A3 or 4G2, BTN3A1-F26A and BTN3A1-P27A for mAb 3A3 and BTN3A1-R44A for mAb 4G2 (Figure 14A). In order to better visualize the epitopes of mAb 3A3 and mAb 4G2, the alanine substitutions causing decreased MFI were plotted on the crystal structure of BTN3A1 (pdb code: 4f9p), all substitution with a normalized MFI between 0.7 and 0.5 were colored gray and the substitutions resulting in stronger decrease, below 0.5, were colored black (Figure 14B). The alanine substitutions leading to the largest decrease in MFI, thus more important for mAb binding, between mAb 3A3 and mAb 4G2 are located at distinct sides of the shared epitope, leading to different mAb orientation. To compare the epitopes of 3A3 and 4G2 with 103.2 and CTX-2026, the epitopes of these to mAbs were also mapped on the BTN3A1 structure, revealing that the epitopes of 3A3 and 4G2 were overlapping in part with CTX-2026, residues Tyr-98, Gln-100, Asp-101 , Asp-103, Phe-104, Tyr-105. The epitope of 3A3 had only one shared residue with 103.2, residue Glu- 32 which resulted in a moderate decrease in MFI, explaining the blocking of 103.2.

[0520] Recently, several studies mapped residues on CD277 important for y952TCR mediated T cell activation to residues Lys-37, Val-39, Arg-44, Lys-94, Leu-96, Tyr-98, Phe-104, Tyr-105, Lys- 107 [4, 5], These residues were plotted on the BTN3A1 structure, clearly displaying that the three mAbs that did not enhance y952TCR T cell activation, 3A3, 4G2, and CTX-2026, had overlapping epitopes with the residues important for y952TCR binding, while the epitope of 103.2 had no overlap with the y952TCR binding site and importantly is located at the opposite side of CD277 (Figure 14B).

[0521] To conclude, antiCD277-costim molecules in combination with y952TCR engineered T cells enhance T cells when the mAb epitope has no overlap with the y952TCR binding site, and ideally is located at the opposite side of CD277. Residues comprising the most preferred epitope are within this collection: Leu-5, Ser-8, Asp-20, Pro-22, His-24, Phe-26, Pro-27, Thr- 28, Met-29, Ser-30, Glu-32, Thr-33, Tyr-50, Ala-51 , Asp-52, Gly-53, Lys-54, Glu-55, Leu-71 , Arg-72, Asp-73, Thr-76, Ala-77, Lys-79, Ala-81 , and Arg-83, but the epitope preferably should not contain any of the following residues: Lys-37, Val-39, Arg-44, Lys-94, Leu-96, Tyr-98, Phe-104, Tyr-105, Lys-107.

[0522] Combination of antiCD277-costim with a tumor reactive a TCR has no restriction on antiCD277-scFv

[0523] Following the hypothesis that the unique enhancement of efficacy by 103.2-41 BB in combination with y952TCR was driven by non-overlapping binding sides for 103.2 and y952TCR, it is to be expected that both 103.2-41 BB and CTX2026-41 BB will be able to enhance the efficacy of T cells engineered with a different tumor antigen receptor. This was tested by transducing healthy donor T cells with a tumor reactive apTCR WT 1 [6], that recognizes WT1126-134 peptide (RMFPNAPYL) HLA-A*02:01 , alone, or in combination with 103.2-41 BB and CTX2026-41 BB. To assess differences in tumor reactivity of the engineered CD8+ T cells, apTCR-WT1 , apTCR-WT1 -103.2-41 BB, and apTCR-WT1-CTX2026-41 BB, were cocultured with SCC9 cells loaded with 0.05 pg / ml WT1126-134 peptide and after 24h the percentage of degranulated T cells were determined using FACS. At this peptide concentration, a substantial percentage of apTCR-WT1 T cells degranulated, however, this percentage differed between repetitive assays (Figure 15A). In all assays T cells expressing apTCR-WTI-103.2-41 BB or apTCR-WT1-CTX2026-41 BB had higher percentage of CD107+ T cells compared to apTCR-WT1 T cells. Next, the effect of the antiCD277-costim molecules was assessed in light of antigen density, to this end the WT1126-134 peptide was titrated in a coculture assay containing SCC9 cells and engineered T cells and after 20 hours the IFNy concentration was determined using ELISA. Following the differences observed in the degranulation assay, both T cells expressing apTCR-WTI-103.2-41 BB or apTCR-WT1-CTX2026-41 BB secreted INFy at higher levels compared to apTCR-WT1 T cells. At a WT1126-134 peptide concentration where no INFy could be detected for apTCR-WT1 T cells, 0.0005 pg / ml, antiCD277-costim apTCR-WT1 T cells still secreted detectible INFy levels (Figure 15B).

[0524] These data show that in absence of a competing binding on CD277, CTX2026-41 BB is a potent antiCD277-costim. Leading to the conclusion that all antiCD277-costim molecules presented here, could be widely applied together with antigen receptors that do not bind to CD277, but when combined with a y952TCR as “signal one” preferably antiCD277 mAbs that bind to the above specific epitope can be used to create a functional antiCD277-costim.

[0525] References

[0526] 1. Hernandez-Lopez, P., et al., Enhancing cancer targeting of y962TCR through modified NKG2D co-stimulation. bioRxiv, 2021: p. 2021.01.06.424553.

[0527] 2. Hernandez-Lopez, P., et al., Dual targeting of cancer metabolome and stress antigens affects transcriptomic heterogeneity and efficacy of engineered T cells. Nat Immunol, 2024. 25(1): p. 88-101.

[0528] 3. Song, H.W., et al., CAR-T cell expansion platforms yield distinct T cell differentiation states. Cytotherapy, 2024. 26(7): p. 757-768.

[0529] 4. Willcox, C.R., et al., Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1. Cell Rep, 2023. 42(4): p. 112321.

[0530] 5. Fulford, T.S., et al., Vy9V52 T cells recognize butyrophilin 2A1 and 3A1 heteromers. Nature Immunology, 2024.

[0531] 6. Najima, Y., et al., Induction of WTl-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD / SCID / IL2rgKO mice. Blood, 2016. 127(6): p. 722-34.

Claims

Claims1. Immune cell expressing- a primary immune receptor comprising a target cell binding domain; and- a costimulatory immune receptor comprising a CD277 binding domain, wherein the CD277 binding domain binds CD277 at an epitope that does not overlap with amino acid positions corresponding to 37, 39, 44,, 94, 96, 98, 104, 105 and 107 in any one of SEQ ID NO:1-3.

2. Immune cell according to claim 1, the costimulatory immune receptor comprising i) an extracellular domain that binds CD277; ii) a hinge and / or transmembrane domain; and iii) an intracellular signalling domain.

3. Immune cell according to claim 2, wherein- the extracellular domain that binds CD277 is an extracellular domain that binds CD277;- the hinge and / or transmembrane domain is or comprises one of CD8aHTM, CD8aH - CD28TM, lgG4H-CD28TM, and lgG4H-CD8aTM, preferably a domain of CD28; and / or- the intracellular signalling domain is or comprises one of 4-1 BB, BAFF-R, CD40, 0X40 (CD134), TACI (CD267), and CD28, preferably 4-1 BB.

4. Immune cell according to any one of claims 2-3, wherein the extracellular domain that binds CD277 is an extracellular domain that binds CD277 at amino acid positions corresponding to 5, 8, 20, 22, 24, 26, 27, 28, 29, 30, 32, 33, 50, 51, 52, 53, 54, 55, 71 , 72, 73, 76, 77, 79, 81, and / or 83 of any one of SEQ ID NO:1-3.

5. Immune cell according to any one of claims 2-4, wherein the extracellular domain that binds CD277 comprises- an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4 and / or SEQ ID NO:5; and / or- an amino acid sequence having at least 70% sequence identity with SEQ ID NO:6 and / or SEQ ID NO:7.

6. Immune cell according to any one of the previous claims, wherein the primary immune receptor comprising a target cell binding domain is or comprises a op T-cell receptor or extracellular domain thereof, a Chimeric Antigen Receptor (CAR) or extracellular domainthereof, a y<5 T-cell receptor or extracellular domain thereof, more preferably a y952 T-cell receptor or extracellular domain thereof.

7. Immune cell according to any one of the previous claims, wherein the target cell binding domain is- a cancer cell binding domain;- an infected cell binding domain; and / or- auto-antibody producing B cell binding domain, autoreactive T cell binding domain, or autoantigen presenting cell binding domain.

8. Immune cell according to any one of the previous claims, wherein the immune cell is a human immune cell, more preferably a human T cell, human Natural Killer (NK) cell, human Antigen Presenting Cell (APC), more preferably an op T-cell or a y<5 T-cell,9. Immune cell according to any one of the previous claims, wherein the immune cell is comprised in a pharmaceutical composition.

10. Immune cell according to any one of the previous claims, wherein the immune cell is for the prevention or treatment of cancer, infectious disease and / or autoimmune disease.

11. Immune cell according to claim 10, wherein the cancer is chosen from leukemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, non-Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumor, stomach cancer, esophageal cancer; wherein the infectious disease is chosen from bacterial infection, fungal infection, viral infection, COVID-19, Hanta virus infection, sepsis, pneumonia, meningitis, acute respiratory distress syndrome, necrotizing fasciitis; and / or wherein the auto-immune disease is chosen from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, psoriasis, Hashimoto’s thyroiditis, Sjogren’s syndrome, autoimmune hepatitis, pemphigus vulgaris and graft versus host disease after allogeneic stem cell transplantation or rejection of a transplant.

12. Method for producing the immune cell according to any one of claims 1-8, the method comprising- introducing, in an immune cell, a nucleic acid or nucleic acid combination encoding a primary immune receptor comprising a target cell binding domain and a costimulatory immune receptor comprising a CD277 binding domain as defined in any one of claims 1-8; or- introducing, in an immune cell having a primary immune receptor comprising a target cell binding domain, a nucleic acid or nucleic acid combination encoding a costimulatory immune receptor comprising a CD277 binding domain as defined in any one of claims 1-8.

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