Nucleic acid for coding CD8 alpha beta co-receptor, cell containing nucleic acid and pharmaceutical composition containing nucleic acid

Abstract

The present invention relates to the field of adoptive immune cell therapies, such as adoptive T cell therapies. It provides immune cells, such as gamma delta T cells, CD4 helper T cells, mucosa-related constant cells, innate lymphocytes, NK cells, macrophages, or other types of immune cells, or combinations thereof, engineered to express CD8 co-receptors, and it provides advantageous recombinant configurations of nucleic acids encoding CD8 alpha beta co-receptors and alpha beta TCR constructs, wherein the nucleic acid comprises a construct capable of mediating the expression of CD8 [alpha] and CD8 [beta] chains and of a TCR [alpha] chain construct and of a TCR [beta] chain construct under the control of a promoter. This improves adoptive immune cell therapy, e.g., it may improve the function and persistence of engineered immune cells. The immune cells can express an MHC I restricted alpha beta TCR construct as well as a CD8 co-receptor. Pharmaceutical compositions comprising the cells or nucleic acids are also provided. These are useful for the treatment of cancer or infectious diseases.

Description

[0001] This invention relates to the field of adoptive immunocellular therapy, such as adoptive T-cell therapy. It provides immune cells, such as γδ T cells, CD4 helper T cells, mucosa-associated homeostatic cells, innate lymphocytes, NK cells, macrophages, or other types of immune cells or combinations thereof, engineered to express a CD8 co-receptor, and provides a favorable rearrangement configuration of nucleic acids encoding a CD8αβ co-receptor and an αβ TCR construct, wherein the nucleic acid contains a construct capable of mediating the expression of CD8α and CD8β chains and TCRα and TCRβ chain constructs under the control of a single promoter. This improves adoptive immunocellular therapy; for example, it can improve the function and persistence of engineered immune cells. The immune cells can express an MHC I-restricted α-β TCR construct and a CD8 co-receptor. Pharmaceutical compositions comprising said cells or nucleic acids are also provided. These can be used to treat cancer or infectious diseases.

[0002] Some members of the MAGE cancer testis antigen family (CTA) are promising targets for immunotherapy because they offer safe expression profiles and are shared by many patients. CTA melanoma-associated antigen 4 (MAGE-A4) is highly expressed in many different types of solid tumors, such as gastric cancer, head and neck cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, and esophageal cancer, and can be targeted by T cells. 1-4 Adoptive transfer of T cells is currently the most effective mechanism of action in oncology. Therefore, it is used to treat MAGE-A4 positive (MAGE-A4) cells. + T-cell therapy (TCR-T cell therapy) with engineered T-cell receptors for solid tumors offers promising opportunities for highly effective cancer treatment with broad applicability.

[0003] CTC127 is an HLA-A receptor targeting the MAGE-A4 immunodominant epitope. 01:01 Restrictive TCR, previously provided by the inventors, and showing CD8 expressing CTC127. + Cytotoxic T cells slowed tumor growth and provided significant survival benefits. Although CD8... + Cytotoxic T cells (Tc) are considered the main cytolytic cells in the context of antitumor immunity, but CD4 + Helper T cells (Th) possess several properties beneficial for cancer immunotherapy because they function as helper T cells, supporting the engraftment and survival of cytotoxic T cells. Furthermore, they are capable of directly killing cancer cells. Indeed, preclinical and clinical reports in the literature support the ability of incorporating both tumor-specific cytotoxic and helper T cell populations into products to improve clinical response and survival. 5-8 ).

[0004] Co-receptors CD8 or CD4 stabilize the interaction between T cells and target cells by binding to the invariant portions of MHC (HLA) class I or II, respectively, and are therefore essential for the effective activation of T cells (a phenomenon known as co-receptor dependence). 9-12 ).

[0005] CTC127 is a CD8-dependent TCR, therefore in CD4 + It is expressed but not functional in helper T cells, while CD4 is expressed but not functional. + Helper T cells are born without CD8 co-receptors.

[0006] CD8 co-receptor constructs have been described in the past and are in clinical development. (See WO 2020 / 049496 A1) 13 The CD8α (CD8a) homodimer is disclosed in WO 2019 / 204662 A1. 14 The CD8αβ (CD8ab) heterodimer has been disclosed. CD8a homodimers or CD8ab heterodimer constructs comprising covalently linked α and β chains have been described in cases of autoreactive T cell depletion (WO 2020 / 208346 A1). 15 US 2022 / 202862 A1, WO2020 / 243134 A1, and CA 3201767 A1 disclose constructs encoding TCR α and β chains and CD8α and CD8β chains, which can be used in CD4 + It may be expressed in γδT cells.

[0007] According to the prior art, the inventors have solved the problem of providing advantageous constructs that can improve adoptive cell therapy using immune cells (e.g., T cells) expressing MHCI-restricted co-receptor-dependent TCRs.

[0008] The present invention, particularly the subject matter of the claims, solves this problem. CD8 ba co-receptor

[0009] This invention provides a nucleic acid encoding a CD8αβ co-receptor, wherein the nucleic acid contains an operon capable of mediating the expression of the CD8α and CD8β chains under the control of a single promoter from RNA, wherein the nucleic acid fragment encoding the CD8β chain is closer to the promoter than the nucleic acid fragment encoding the CD8α chain.

[0010] Preferably, at least one nucleic acid fragment encoding the CD8β chain or the CD8α chain is located between a nucleic acid fragment encoding one of the TCRα chain constructs or the TCRβ chain constructs and a nucleic acid fragment encoding the other of the TCRα chain constructs and the TCRβ chain constructs.

[0011] RNA is a single RNA. As described below, it can be considered a polycistronic mRNA.

[0012] As demonstrated in the experimental section, in in vitro co-culture assays (peptide titration, tumor cell recognition, and killing), co-engineering of T cells (e.g., CTC127 T cells) with the novel heterodimeric CD8 co-receptor construct of this invention resulted in the gain of helper T cell function compared to T cells without CD8 co-receptors, and produced more rapid and sustained tumor rejection in the NSG mouse model. T cell function was also improved compared to prior art CD8 constructs. The use of this toolkit technique has the potential to significantly enhance the efficacy of TCR-engineered T cells by leveraging the powerful functions of helper T cells.

[0013] As explained in more detail below, the inventors also surprisingly discovered that the expression of the nucleic acid of the present invention provides cytotoxic functions for other immune cells, such as γ-δT cells expressing α-βTCR, thus allowing for the engineering of cell types of great interest in allogeneic adoptive T-cell therapy.

[0014] The nucleic acid of this invention encodes CD8α and CD8β chains. Upon expression, CD8α and β chains are generated and present on the cell surface of cells containing the nucleic acid of this invention. The CD8α and CD8β chains are not covalently linked in the form of proteins in the CD8α-β co-receptor of this invention. When present on the cell surface, the TCRα or β chain constructs are also not covalently linked in the form of proteins; that is, when present on the cell surface, the CD8α chain, CD8β chain, TCRα chain construct, and TCRβ chain construct are not covalently linked to each other.

[0015] With WO 2019 / 204662 A1 ( 14 Compared to existing heterodimer CD8 constructs disclosed in [the present invention], this invention provides a CD8βα (CD8 ba) heterodimer in which the order of subunits in the operon is reversed. The inventors were able to demonstrate that this alteration is beneficial to Th cell function.

[0016] The nucleic acid of this invention comprises an operon capable of mediating the expression of the CD8α and CD8β chains under the control of a single promoter of a single mRNA. In the context of this invention, an operon is a functional unit of DNA containing a gene cluster under the control of a single promoter. Therefore, an operon contains a nucleic acid segment comprising both the CD8α and CD8β chains under the control of a single promoter. These genes are transcribed together into a single mRNA molecule. The single RNA molecule can be considered polycistronic because it contains a nucleic acid segment encoding at least two proteins expressed from the same promoter. Transcription may or may not be further regulated by repressors or inducers.

[0017] The promoter is operatively linked to the first gene of the operon. This can be, for example, a nucleic acid segment encoding the CD8β chain. It could also be another gene, for example, as described below. However, it is not a nucleic acid segment encoding the CD8α chain, because the inventors have found it advantageous if, on a single mRNA transcribed from the operon, the segment encoding the CD8α chain is 3' downstream of the segment encoding the CD8β chain. Without being bound by theory, this could be due to the reduced translation of the downstream CD8α chain compared to the upstream CD8β chain.

[0018] The promoter mediates the transcription of mRNA. It may be a constitutive or inducible promoter. Preferably, it is active in human T cells. It may be active in CD4 and CD8 T cells, or primarily or only in CD4 T cells. It may also be active only or primarily in γδ T cells. Preferably, the promoter is active in immune cells, such as in CD8 T cells and CD4 T cells, as well as γδ T cells and / or NK cells and / or NKT cells, and most preferably, it is active in all immune cells. For example, the promoter may be human EF1α, PGK, β-actin, IF4A1, GAPDH, GRP78, HSP70, β-kinin, ubiquitin B, CD45, MP71, or CMV promoters. CD45 is an exemplary promoter with specific activity in immune cells. Preferably, the promoter is EF1α.

[0019] The operon may also contain a transcription terminator, preferably downstream of the nucleic acid fragment encoding the CD8α chain.

[0020] CD8α and β chains typically originate from the same species. For example, they can be from humans, mice, rats, rabbits, chickens, camels, monkeys, apes, goats, sheep, pigs, or cattle. Often, especially in cases involving the treatment of human patients, they are human CD8α and β chains. They can also be genetically engineered forms of the CD8 co-receptor.

[0021] Both CD8α and β chains are members of the immunoglobulin superfamily. They can form heterodimers containing intracellular regions, preferably including a cytoplasmic tail, a transmembrane region, and an extracellular immunoglobulin variable (IgV)-like extracellular domain. The extracellular IgV-like domain of CD8α can interact with the α3 portion of class I MHC molecules. This allows the TCR, which also interacts with class I MHC, to approach CD8 when binding to target cells containing MHC I molecules that present TCR-corresponding antigens. The cytoplasmic tail of the CD8 co-receptor interacts with tyrosine kinase Lck (a lymphocyte-specific protein tyrosine kinase), which phosphorylates the ITAM motif of the CD3 and ζ chains of the TCR, thereby initiating a phosphorylation cascade that ultimately activates transcription factors such as NFAT, NFKB, and AP-1.

[0022] In the context of this invention, the CD8α chain can be any isoform of human CD8α, namely 1, 2 (secretory isoform) or 3, optionally, as shown in Examples 1 and 2, it is CD8α1 having SEQ ID NO:2.

[0023] In the context of this invention, the CD8β chain can be any isoform of human CD8β, namely 1, 2, 3 (secretory isoform), 4, 5, 6 (secretory isoform), 7, or 8 (secretory isoform). Optionally, as shown in Examples 1 and 2, it is CD8β5 having SEQ ID NO: 1. The inventors further demonstrated in experiments described below that the CD8β5 and CD8β1 isoforms are expressed particularly well in γδT cells compared to other isoforms.

[0024] CD8 α1β5 is also effective. To avoid the formation of soluble CD8αβ heterodimers, preferably, at least one of the α or β chains is not a secretory isomer lacking a transmembrane domain. The CD8α and / or CD8β transmembrane domains can be transmembrane domains derived from other proteins (e.g., from CD4); however, they are preferably not fusion proteins. Sequences of usable wild-type human CD8α and β chains are disclosed herein.

[0025] Alternatively, modified sequences can be used, such as variant CD8α or CD8β chains containing one or more mutations, which increase the binding affinity to the MHC class I α3 moiety compared to wild-type CD8α or CD8β chains. For example, using Wang et al. 2011 ( 16 High-affinity mutants of CD8α were generated and characterized using in vitro evolution methods. The modified sequence or variant preferably has at least 95% amino acid identity with the wild-type isoform of the CD8α or β chain, for example, optionally, as described herein, with at least 95% amino acid identity with the sequence of SEQ ID NO: 1 or 2, respectively.

[0026] Polycistronic RNAs comprise nucleic acid fragments encoding at least two proteins expressed from the same promoter. In the context of this invention, a nucleic acid fragment is part of a continuous nucleic acid sequence containing other nucleic acid fragments. It is generally defined as encoding a specific function. For example, a nucleic acid fragment may contain a promoter, a terminator, a functional element (e.g., a cleavage site), or encode a CD8α or CD8β strand, a TCRα strand construct, or a TCRβ strand construct.

[0027] The inventors further demonstrate that it is advantageous if at least one nucleic acid fragment encoding the CD8β chain or the CD8α chain is located between a nucleic acid fragment encoding one of the TCRα chain constructs or the TCRβ chain constructs and a nucleic acid fragment encoding the other of the TCRα chain constructs and the TCRβ chain constructs.

[0028] Preferably, at least one nucleic acid fragment encoding the CD8β chain or the CD8α chain is located between the nucleic acid fragment encoding the TCRβ chain construct and the nucleic acid fragment encoding the TCRα chain construct. This order means that the nucleic acid fragment encoding the TCRβ chain construct is closer to the promoter than the nucleic acid fragment encoding the TCRα chain construct, or more specifically, the nucleic acid fragment encoding the TCRβ chain construct is closer to the promoter than the nucleic acid fragment encoding the CD8β chain or the CD8α chain, and the nucleic acid fragment encoding the CD8β chain or the CD8α chain is closer to the promoter than the nucleic acid fragment encoding the TCRα chain construct. Preferably, only one nucleic acid fragment encoding the CD8β chain or the CD8α chain is located between the nucleic acid fragment encoding the TCRβ chain construct and the nucleic acid fragment encoding the TCRα chain construct. Optionally, the only nucleic acid fragment may be a nucleic acid fragment encoding the CD8β chain.

[0029] Optionally, at least one nucleic acid fragment encoding the CD8β or CD8α chain is located between the nucleic acid fragment encoding the TCRα chain construct and the nucleic acid fragment encoding the TCRβ chain construct. This means that the nucleic acid fragment encoding the TCRα chain construct is closer to the promoter than the nucleic acid fragment encoding the CD8β or CD8α chain, and the nucleic acid fragment encoding the CD8β or CD8α chain is closer to the promoter than the nucleic acid fragment encoding the TCRβ chain construct. In one embodiment, only one nucleic acid fragment encoding the CD8β or CD8α chain is located between the nucleic acid fragment encoding the TCRα chain construct and the nucleic acid fragment encoding the TCRβ chain construct; optionally, said only nucleic acid fragment is a nucleic acid fragment encoding the CD8α chain. Optionally, said only nucleic acid fragment may be a nucleic acid fragment encoding the CD8β chain.

[0030] In the nucleic acid of the present invention, preferably, the nucleic acid fragment encoding at least the CD8α chain is located between the nucleic acid fragment encoding one of the TCRα chain construct or the TCRβ chain construct and the nucleic acid fragment encoding the other of the TCRα chain construct and the TCRβ chain construct, and more preferably between the nucleic acid fragment encoding the TCRβ chain construct and the nucleic acid fragment encoding the TCRα chain construct.

[0031] Optionally, in the nucleic acid of the present invention, at least the nucleic acid fragment encoding the CD8β chain is located between the nucleic acid fragment encoding the TCRα chain or the TCRβ chain and the nucleic acid fragment encoding the other of the TCRα chain and the TCRβ chain, preferably between the nucleic acid fragment encoding the TCRβ chain and the nucleic acid fragment encoding the TCRα chain.

[0032] In another embodiment, the nucleic acid fragments encoding the CD8β and CD8α chains are located between a nucleic acid fragment encoding one of the TCRα or TCRβ constructs and a nucleic acid fragment encoding the other of the TCRα or TCRβ constructs, preferably between a nucleic acid fragment encoding the TCRβ construct and a nucleic acid fragment encoding the TCRα construct. The inventors have demonstrated in experiments shown herein that this arrangement of nucleic acids also produces very good results. Alternatively, the nucleic acid fragments encoding the CD8β and CD8α chains may also be located between a nucleic acid fragment encoding the TCRα construct and a nucleic acid fragment encoding the TCRβ construct. In all embodiments of the invention, the CD8 and TCR chains are rearranged, i.e., at least one of the CD8a or CD8b chains is between the TCR chains. Preferably, only one of the CD8a and CD8b chains is between the TCR chains, and the other is closer to the promoter than the first TCR chain or less close to the promoter than the second TCR chain. This is the opposite of a configuration where the two TCR chains are directly interconnected and the two CD8 chains are directly interconnected. In this context, "direct" may include adapters and / or cleavage sites, but not other nucleic acid fragments encoding proteins.

[0033] In a preferred embodiment, the nucleic acid fragment encoding the CD8β chain is linked to the nucleic acid fragment encoding the CD8α chain via a nucleic acid fragment encoding a cleavage site. Optionally, cleavage sites are also present between other proteins encoded by the operon, wherein preferably, all cleavage sites may be identical. Optionally, each cleavage site may be independently selected from any cleavage site described herein.

[0034] The cleavage site can be any sequence that separates two (or more) polypeptides, for example, such as WO 2020208346A1 ( 15 As disclosed in ( ).

[0035] The term cleavage is used here for convenience, but cleavage sites can lead to the separation of peptides into individual entities through mechanisms other than classical cleavage. For example, for the self-cleaving peptide of foot-and-mouth disease virus (FMDV) 2A (see below), various models have been proposed to explain cleavage activity: proteolysis by host cell proteases, self-proteolysis, or translational effects (Donnelly et al. (2001)). 17 While the exact molecular mechanism of 2A peptide-mediated cleavage is not fully understood, it is believed to involve a ribosome "jump" in the formation of a glycyl-prolyl peptide bond, rather than a true proteolytic cleavage. The exact mechanism of this cleavage is not important to the purposes of this invention, as long as the cleavage site is located between nucleic acid sequences encoding the protein, allowing the protein to be expressed as a separate entity.

[0036] The cleavage site is preferably a self-cleaving peptide. This term refers to a peptide that functions such that when a polypeptide comprising a protein and a self-cleaving peptide is generated, it is immediately cleaved or separated into distinct and discrete first and second polypeptides without any external cleavage activity.

[0037] The self-cleaving peptide is preferably a 2A element, such as a P2A element, T2A element, E2A element, or F2A element. These elements may originate from apthoviruses or cardiviruses. The major 2A / 2B cleavage of apthoviruses and cardiviruses is mediated by a 2A cleavage at their own C-terminus. In apthoviruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short segment of about 18 amino acids that, together with the N-terminal residues (conserved proline residues) of the protein 2B, represents an autonomous element capable of mediating its own C-terminal cleavage (Donnelly et al. (2001), as described above). 17 ).

[0038] In addition to repetitive sequences in oral thrush viruses or cardioviruses, piconemavirus-like insect viruses, type C rotaviruses, and trypanosomes, as well as bacterial sequences, 2A elements have also been found in piconemaviruses (Donnelly et al. (2001), as described above). 17 The 2A element peptide shares the core sequence motif DXEXNPGP (SEQ ID NO: 7).

[0039] Four members of the 2A peptide family are frequently used in life science research and are preferred 2A elements in this invention. These are P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A from equine rhinitis A virus; P2A from porcine chezinvirus-1 2A; and T2A from Thosea asigna virus 2A.

[0040] T2A EGRGSLL TCGDVEENPGP (SEQ ID NO: 3)

[0041] P2A ATNFSLLKQAGDVEENPGP (SEQ ID NO: 4)

[0042] E2A QCTNYALLKLAGDVESNPGP (SEQ ID NO: 5)

[0043] F2A VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6)

[0044] Adding an optional GSG (Gly-Ser-Gly) adapter, such as a self-cleaving peptide like peptide 2A, to the N-terminus of the cleavage site can improve efficiency and is therefore preferred for all cleavage sites (e.g., P2A elements). Adapters such as GSG adapters at the C-terminus of the cleavage site may also exist.

[0045] Therefore, the connection can be a direct connection or a connection via a linker, which may optionally contain another encoded protein. In addition to the TCRα and β chains and the CD8α and β chains, the operon may contain 0, 1, 2, 3, 4, 5, 6, 7, or 8 additional encoded proteins, preferably also separated from each other by cleavage sites.

[0046] Different 2A peptides exhibit varying self-cleavage efficiencies, with T2A and P2A being the most efficient and F2A the least. Therefore, up to 50% of F2A linkers can remain in the cell as fusion proteins, potentially leading to unpredictable outcomes, including gain-of-function. One study reported that the 2A site results in ribosome detachment at approximately 60% of the time, and the binding of P2A and T2A results in approximately 10% ribosome readthrough, leading to a reduction in downstream peptide expression of about 70%. However, the degree of reduction detected in this study varied depending on the exact structure used, with some structures showing almost no reduction; furthermore, in tricistronic transcripts, it reported higher levels of ribosome detachment after a single 2A sequence than after the merging of two 2A sequences, which is inconsistent with linear translation models.

[0047] In the context of this invention, the P2A element is preferred, for example, in combination with a GSG adapter directly at the N-terminus of the P2A element. Therefore, the nucleic acid of this invention preferably comprises a nucleic acid fragment encoding a CD8β chain, which is connected to a nucleic acid fragment encoding a CD8α chain via a nucleic acid fragment encoding a P2A element, wherein, optionally, between the nucleic acid fragment encoding the CD8β chain and the nucleic acid fragment encoding the P2A element, there is a nucleic acid fragment encoding a adapter (e.g., a GSG adapter) located directly upstream of the P2A element.

[0048] The cleavage site can be self-cleaving, so that when the polypeptide is generated, it is immediately cleaved into a single peptide without any external cleavage activity.

[0049] Alternatively, the cleavage site can be an enzyme cleavage site, such as the furin cleavage site. Frin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process potential precursor proteins into their biologically active products. Frin is a calcium-dependent serine intraprotease that efficiently cleaves precursor proteins at paired basic amino acid processing sites. Examples of furin substrates include parathyroid hormone, transforming growth factor β1 precursor, prealbumin, pre-β-secretase, membrane type 1 matrix metalloproteinases, the β subunit of pre-nerve growth factor, and von Willebrand factor. Frin cleaves proteins downstream of a basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg') and accumulates in the Golgi apparatus.

[0050] The cleavage site can also be a tobacco etch virus (TEV) cleavage site. TEV protease is a highly sequence-specific cysteine ​​protease, a chymotrypsin-like protease. It is highly specific to its target cleavage site and is therefore frequently used for controlled cleavage of fusion proteins in vitro and in vivo. The common TEV cleavage site is ENLYFQS (SEQ ID NO: 8), where the cleavage occurs between Q and S. Mammalian cells (e.g., human cells) do not express TEV protease. Therefore, in an embodiment where this nucleic acid construct includes a TEV cleavage site and is expressed in mammalian cells, the exogenous TEV protease must also be expressed in mammalian cells.

[0051] In an alternative implementation, the internal ribosome entry site (IRES) is located between the nucleic acid fragment encoding the CD8β chain and the nucleic acid fragment encoding the CD8α chain. An IRES is an RNA element that can initiate translation in a cap-independent manner as part of a broader protein synthesis process. In eukaryotic translation, initiation typically occurs at the 5' end of the mRNA molecule because 5' cap recognition is required to assemble the initiation complex. They are described as distinct regions of the RNA molecule capable of recruiting eukaryotic ribosomes into the mRNA. This process is also known as cap-independent translation. IRES elements have been shown to possess distinct secondary and even tertiary structures, but similar structural features at the primary or secondary structure level common to all IRES fragments have not yet been reported. For example, Hellen et al. (… 18 The IRES disclosed in () is used in the context of this invention. Preferably, the IRES is SEQ ID NO: 9 or SEQ ID NO: 15.

[0052] The nucleic acid of the present invention further encodes a TCRα strand construct and a TCRβ strand construct. The TCRα strand construct and the TCRβ strand construct together form a TCR construct, which is capable of recognizing epitopes in the context of MHC molecules, such as cancer epitopes, i.e., those that are MHC-restricted. Preferably, it is MHC class I restricted. In the context of the present invention, MHC class I is MHC class Ia, i.e., HLA-A, HLA-B, or HLA-C in human systems. The TCRα strand construct and the TCRβ strand construct are preferably both expressed by the same promoter, i.e., they form a single operon.

[0053] In a preferred embodiment, the operon comprises a nucleic acid fragment encoding the following components. CD8α chain and CD8β chain and TCRα chain constructs and TCRβ chain constructs or chimeric antigen receptors.

[0054] The operon encodes both the TCRα and TCRβ constructs. Therefore, both peptides can be expressed from the same promoter. This may be beneficial in ensuring that all cells expressing TCRs also express the CD8 co-receptor, and vice versa.

[0055] As long as the fragment encoding the CD8β chain is closer to the promoter than the fragment encoding the CD8α chain, and preferably, at least one nucleic acid fragment encoding the CD8β chain or the CD8α chain is located between the nucleic acid fragment encoding one of the TCRα chain constructs or the TCRβ chain constructs and the nucleic acid fragment encoding the other of the TCRα chain constructs and the TCRβ chain constructs, these fragments may have different configurations.

[0056] In one embodiment, the operon may contain the nucleic acid sequence of SEQ ID NO:10 (a nucleic acid fragment encoding the CD8β chain is linked to a nucleic acid fragment encoding the CD8α chain via a nucleic acid fragment encoding a cleavage site).

[0057] For example, in a preferred embodiment of the invention, the promoter is operatively linked to a nucleic acid fragment encoding the CD8β chain, which is linked via a nucleic acid fragment encoding a cleavage site to a nucleic acid fragment encoding either the TCRα chain construct or the TCRβ chain construct; the nucleic acid fragment encoding either the TCRα chain construct or the TCRβ chain construct is linked via a nucleic acid fragment encoding a cleavage site to a nucleic acid fragment encoding the CD8α chain; and the nucleic acid fragment encoding the CD8α chain is linked via a nucleic acid fragment encoding a cleavage site to another nucleic acid fragment encoding either the TCRα chain construct or the TCRβ chain construct.

[0058] Preferably, the promoter is operatively linked to a nucleic acid fragment encoding the CD8β chain, which in turn links to a nucleic acid fragment encoding a cleavage site, which links to a nucleic acid fragment encoding the TCRβ chain construct, which links to a nucleic acid fragment encoding the CD8α chain, which links to a nucleic acid fragment encoding the TCRα chain construct, which in turn links to a nucleic acid fragment encoding the CD8α chain, which links to a nucleic acid fragment encoding the TCRα chain construct, as illustrated in the example below in transfected γδT cells. This arrangement leads to optimal expression and T cell activation.

[0059] In another embodiment of the invention, the promoter is operatively linked to a nucleic acid fragment encoding either a TCRα or TCRβ construct, the nucleic acid fragment encoding either the TCRα or TCRβ construct being linked to a nucleic acid fragment encoding a CD8β strand via a cleavage site, the nucleic acid fragment encoding the CD8β strand being linked to a nucleic acid fragment encoding a CD8α strand via a cleavage site, and the nucleic acid fragment encoding the CD8α strand being linked to a nucleic acid fragment encoding the other of the TCRα or TCRβ constructs via a cleavage site. Preferably, in this embodiment, the TCRβ strand is also closer to the promoter than the TCRα strand. Optionally, in this embodiment, the TCRα strand is closer to the promoter than the TCRβ strand.

[0060] In another embodiment of the invention, the promoter is operatively linked to a nucleic acid fragment encoding either the TCRα or TCRβ construct, the nucleic acid fragment encoding either the TCRα or TCRβ construct being linked to a nucleic acid fragment encoding the CD8β construct via a cleavage site, the nucleic acid fragment encoding the CD8β construct being linked to a nucleic acid fragment encoding the other of the TCRα or TCRβ constructs via a cleavage site, and the nucleic acid fragment encoding the other of the TCRα or TCRβ constructs being linked to a nucleic acid fragment encoding the CD8α construct via a cleavage site.

[0061] The construct used in Example 1 below is a) Figures 1-3 (a) or (b) Figures 4-7 The construct of ), in which all cleavage sites are P2A elements.

[0062] In addition to the nucleic acid fragments encoding the TCRα and TCRβ constructs and the CD8α and β chains, the operon may also contain one or more other genetic elements, such as encoding another receptor, transmembrane protein, nucleoprotein, or cytoplasmic protein that enhances immune cell function, encoding cytokines that stimulate immune cells, and / or selection genes, such as DHFR for methotrexate resistance, or EGFR for antibody-mediated selection or consumption. Additional genetic elements may be located, for example, between the promoter and the next coding element of the operon, such as between the promoter and the nucleic acid fragment encoding the CD8β chain, the nucleic acid fragment encoding the CD8β chain being linked via a cleavage site to a nucleic acid fragment encoding one of the TCRα or TCRβ constructs, the nucleic acid fragment encoding one of the TCRα or TCRβ constructs being linked via a cleavage site to a nucleic acid fragment encoding the CD8α chain, and the nucleic acid fragment encoding the CD8α chain being linked via a cleavage site to a β chain construct encoding the other of the TCRα or TCRβ constructs.

[0063] Additional genetic elements may alternatively (or optionally, additionally) be located at the distal end of a genetic element that is furthest from the promoter listed in any of the above embodiments. It may optionally (or optionally, additionally) be located between genetic elements encoding the TCRα chain construct or the TCRβ chain construct or the CD8β chain or the CD8α chain in any of the above configurations.

[0064] Optionally, all cutting sites are identical; for example, all cutting sites can be 2A elements, such as P2A elements.

[0065] Alternatively, the cleavage sites can be different. Preferably, the nucleic acid fragments encoding the TCRα and β chains and the nucleic acid fragments encoding the CD8β and α chains can be linked by nucleic acid fragments encoding cleavage sites (e.g., 2A elements, especially P2A elements), and the nucleic acid fragments encoding the two TCR chains on one side and the nucleic acid fragments encoding the CD8β and α chains on the other side can be linked by nucleic acid fragments encoding the IRES site.

[0066] The TCRα and / or β chain construct may contain all the features or domains corresponding to its natural counterpart (i.e., the TCRα or β chain), but this is not required. Preferably, the TCRα and / or β chain construct contains at least one variable region or a variable region and a constant region, for example, the variable region and / or constant region having at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with human variable regions or constant regions.

[0067] The TCRα chain construct and / or TCRβ chain construct preferably include constant regions. For adoptive TCR-T cell therapy, the TCR construct preferably comprises full-length TCRα and β chains including variable and constant regions (including transmembrane regions). The constant region can be a human constant region, a mouse constant region, or a chimeric constant region, such as a minimal mouse constant region. The TCR construct can be substantially or entirely derived from humans to minimize immunogenicity. To prevent pairing with endogenous TCR chains, the constructs of the present invention may optionally contain one or more, for example 1-5, 1-10, or 1-20, amino acid exchanges, preferably 9, compared to human sequences. 19 ).

[0068] To prevent pairing with endogenous TCR chains, the constant regions of the TCRα and β chain constructs can be mouse constant regions ( 20 For example, it can have at least 80% or at least 90% sequence identity with the mouse constant region. TCRs with mouse constant regions generally have stronger expression than TCRs with human constant regions.

[0069] Additional cysteine ​​residues may be provided alternatively or separately to allow additional disulfide bonds to form between the TCR chains. 21 , 22 Furthermore, codon modifications to the TCR sequence can be used to enhance the functional expression of transgenic TCRs. 23 Since the α and β TCR constructs are preferably expressed by a single promoter, for example, linked via a cleavage site such as a 2A element (e.g., P2A), stoichiometric expression of both chains can be achieved. 24 This leads to enhanced functional expression of the transgenic TCR.

[0070] In order to specifically recognize epitopes in the context of MHC, particularly for therapeutic purposes in human patients, the present invention provides a TCR α and β chain construct encoding a TCR that can recognize epitopes in the context of human MHC, preferably in the context of human MHC I.

[0071] In a preferred embodiment, the TCR construct is capable of recognizing epitopes of tumor antigens complexed with human MHC I, such as antigens from solid tumors, such as MAGE antigens, such as MAGE-A4.

[0072] Typically, the nucleic acid sequences provided by this invention are codons optimized for expression in human cells.

[0073] In a preferred embodiment, the nucleic acid of the present invention comprises SEQ ID NO: 25 or a sequence having at least 80%, such as at least 90%, at least 95%, or at least 99%, sequence identity.

[0074] In another embodiment, the nucleic acid of the present invention comprises SEQ ID NO: 26 or a sequence having at least 80%, such as at least 90%, at least 95%, or at least 99% sequence identity.

[0075] In the comparative sequences, such as SEQ ID NO: 14, the two TCR strands encode adjacent to each other, and the two CD8 strands encode adjacent to each other, thus having a tandem configuration. For example, in SEQ ID NO: 14, the configuration is CD8β-P2A-CD8α-P2A-TCRβ-P2A-TCRα. It can be seen that, in contrast, in the nucleic acids of the present invention, such as SEQ ID NO: 25 and 26, the TCR and CD8 strands are rearranged. The inventors surprisingly found that expression from the rearranged sequences, particularly from SEQ ID NO: 25 and SEQ ID NO: 26, is superior to expression from the tandem constructs, such as those with the configuration CD8β-P2A-CD8α-P2A-TCRβ-P2A-TCRα (… Figure 14 ).

[0076] Of course, the TCR is not limited to the TCRs exemplified in the sequence list. Therefore, nucleic acid fragments encoding the TCRα and / or β chain constructs of the nucleic acids of the present invention, particularly SEQ ID NO:25 and 26, can replace different TCRα and / or β chain construct coding sequences. In one embodiment, the frame regions of the TCRα and β chain constructs are identical to those in SEQ ID NO:25 and 26. Optionally, in addition, the CDR sequence has at least 50%, preferably at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with the CDR of the TCR shown in SEQ ID NO:25 and 26. The CDR1 and CDR2 sequences may also be identical to those shown in SEQ ID NO:25 and 26, while the CDR3 sequence has at least 50%, preferably at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with the CDR3 of the TCR used in SEQ ID NO:25 and 26. Of course, the encoded TCR construct still needs to be able to specifically bind to the homologous peptide / MHC construct.

[0077] Furthermore, the CD8α and β chains encoded by the nucleic acids of this invention can be different. For example, as disclosed herein, different isoforms of the CD8α and / or β chains can be used. Variants of different isoforms, such as those used in SEQ ID NO:25 and 26, can also be used as long as they still promote the binding of the TCR to the peptide / MHC complex. Variants can carry substitutions, deletions, or insertions, preferably substitutions. For example, the nucleic acids of this invention can have at least 80%, for example, at least 90%, at least 95%, or at least 99% sequence identity with the sequences encoding CD8β and CD8α at positions 1000-2499 of SEQ ID NO:26 or preferably positions 1-729 and 1795-2499 of SEQ ID NO:25.

[0078] The nucleic acid of this invention can be a vector, such as an expression vector. It is typically DNA. The nucleic acid can be, for example, a minicircle DNA, a plasmid, doggybone DNA, or RNA.

[0079] Recently, DNA constructs using doggybone DNA (dbDNA) and synthesized closed linear DNA have been developed. The cell-free process relies on amplification, such as using Phi29 DNA polymerase, where the template is amplified followed by incubation with the protease TelN to complete a single closed linear DNA. The resulting dbDNA may contain, for example, the desired sequence (e.g., a CD8β chain encoded by a cleavage site linked to the CD8α chain, and optional TCRα and β chain constructs), a promoter, and a poly A tail, but lacks bacterial sequences, such as antibiotic resistance genes.

[0080] Microcircular DNA can be derived from plasmids in which bacterial sequences (such as origins of replication) have been excised. Microcircular DNA is a small (typically less than 5 kb, preferably about 4 kb) circular plasmid derivative that has been released from all prokaryotic vector moieties. They have been used as transgenic vectors for genetic modification in mammalian cells, with the advantage that, because they do not contain bacterial DNA sequences, they are less likely to be perceived as foreign and destroyed. The smaller size of microcircular DNA also expands their cloning capabilities and facilitates their delivery into cells, for example, using transposases. Suitable nucleic acids and protocols are specified, for example, in WO 2017 / 158019 A1 (… 25 It is published in ( ).

[0081] Microcircular DNAs lack origins of replication, so they typically do not replicate within target cells, and the encoded genes are lost with cell division. A new addition to this field is non-viral self-replicating microcircular DNA, attributed to the presence of the S / MAR element.

[0082] Plasmids (containing circular DNA at the origin of replication) can also be used in the context of this invention, but preferably with less DNA to be transferred; therefore, the nucleic acid is preferably doggybone DNA or microcircular DNA, with microcircular DNA being the most preferred.

[0083] In one embodiment, the DNA is a transposon or contains a transposon. Therefore, the flanking structures of the operon can be inverted repeat sequences capable of being mobilized by transposases, preferably by Sleeping Beauty transposases such as SB100X. Suitable transposases are, for example, those described in WO 2009 / 003671 A2 (…). 26 Disclosed in WO 2017 / 158029 A1. Suitable transposons are, for example, found in WO 2017 / 158029 A1. 27 The information is disclosed in [the relevant document]. For example, the vector can be a p2 vector, or preferably a p4 or p5 vector.

[0084] The vector can also be a viral vector, such as a lentiviral vector, a gamma retroviral vector, adenoviral vector, or AAV. The vector can also be suitable for CRISPR / Cas homology-directed repair (HDR) genome engineering, binding to a guide RNA provided in the kit. For example, the guide RNA can be homologous to a portion of the endogenous TCR α, β, δ, or γ loci. Therefore, advantageously, particularly for αβT cells, the insertion of the nucleic acid of the present invention disfunctionalizes the endogenous TCR, thereby preventing mismatches and / or GVHD. In γδT cells, expression of the endogenous TCR, which often has antitumor activity, can also be expected, thus allowing for the selection of other loci for engineering.

[0085] The present invention also discloses a method for preparing cells containing the nucleic acids of the present invention. A vector, such as purified microcircular DNA, can be transferred to recipient cells by transfection, lipid transfection, jet injection, or preferably electroporation.

[0086] The present invention also provides a cell comprising the nucleic acid of the present invention, wherein the cell preferably also expresses CD8αβ co-receptor and TCR. The cell may be a bacterial cell, such as *Escherichia coli*, but it is preferably a eukaryotic cell, such as a mammalian cell. Typically, for research purposes, the cell is a mouse or human cell. Therapeutic cells are typically human cells, intended for use in treating human subjects.

[0087] For therapeutic purposes, the αβT cells engineered with the nucleic acids of this invention can be allogeneic, but they are preferably homologous, and usually autologous. This avoids immune rejection or the need for immunosuppression to avoid graft-versus-host disease (GvHD). αβT cells expressing the CD8 co-receptor of this invention are preferably CD4+ T cells, as CD8+ T cells already naturally express the co-receptor. However, a cell mixture containing both CD4+ and CD8+ cells can also be transfected with the co-receptor of this invention.

[0088] The cells of the present invention preferably further express a type I restricted T cell receptor construct.

[0089] The cells are preferably immune cells, such as T cells, NK cells, or NKT cells. The T cells can be αβT cells or γδT cells. As described, CD4αβT cells can be used, or more preferably a mixture of CD4 and CD8αβT cells and γδT cells.

[0090] However, one advantage of this invention is that, using the nucleic acids of this invention encoding the CD8α and β chains and the TCRα and β chain constructs, other immune cells, such as γδT cells, NK cells or NKT cells, monocytes or macrophages or B cells, can also be endowed with cytotoxic functions against target cells, particularly tumor cells. Immune cells expressing TCR and CD8 can be advantageously activated by target cells expressing homologous antigens and presented in an MHC-I-restricted manner. The inventors have demonstrated that the claimed nucleic acid conformation is optimal for expression in γδT cells. Therefore, the γδT cells of this invention are a preferred embodiment.

[0091] In one implementation, if the cells are CD4 T cells, they are derived from PBMCs (peripheral blood mononuclear cells). They can be obtained in large quantities from the subject, for example, from the subject's PBMCs, particularly by stimulating T cells. CD4 T cells can be isolated from other T cells before stimulation, but this is not necessary. In fact, it is advantageous if both CD4 and CD8 T cells are engineered with the nucleic acids of the present invention and therefore do not separate from each other. T cells can be isolated from other cells in the PBMCs, for example, by reducing the concentration of B cells. Suitable methods are, for example, known in the art.

[0092] For example, as is known in the art, T cells can be stimulated and expanded with anti-CD3 or anti-TCR (γ and / or δ chain) antibodies, which are optionally bound to a solid carrier, preferably in combination with an anti-CD28 antibody. They can also be stimulated by contacting target cells expressing their homologous antigens (after engineered cells to express transgenic TCRs) and / or by adding cytokines such as IL-1β, IL-2, IL-4, IL-5, IL-7, IL-15, IL-21, IFNγ and / or by adding bisphosphonates (e.g., zoledronic acid, risedronate, ibandronate, alendronate, opaldronate, neriddronate, pamidronate, tiludronate, clodronate, and etidronate).

[0093] In a preferred embodiment, the present invention provides a cellular composition comprising the γδT cells of the present invention. The composition may be purified, or optionally further comprise CD8+ and CD4+ T cells and / or NK cells and / or NKT cells. Advantageously, the γδT cells (and optionally, other enumerated cells) comprise the nucleic acids of the present invention and express the CD8αβ co-receptor. In one embodiment, the composition comprising the cells of the present invention can be obtained by transfecting PBMCs or cells derived therefrom of a subject (e.g., a subject to be treated or an unrelated donor) with the nucleic acids of the present invention. Immune cells, especially γδT cells

[0094] γδT cells are a fascinating subset of T cells that do not express α-βTCRs but do express γδTCRs. The ligands for γδTCRs remain largely unknown. Based on the γδTCR-specific data identified to date, it is generally believed that the receptors bind to their targets in a peptide-independent MHC presentation manner. They can bind conformational epitopes, i.e., specific surface structures of proteins and possibly other molecules. For example, some γδTCRs are known to bind to phosphate antigens and aminobisphosphonates, which may be induced by metabolic changes in cells such as tumor cells. The targets of the Vγ9Vδ2 TCR are known to be the binding spatial and conformational changes of CD277 on the cell membrane of cancer cells.

[0095] Therefore, γδT cells possess a naturally high tumor cell killing potential. They have also been found to naturally infiltrate solid tumors, and tumors rich in γδT cells have a significantly better prognosis.

[0096] Furthermore, when γδT cells are engineered to express α-βTCR, the exogenous and endogenous α and βTCR chains do not pair with those of engineered αβT cells. The γ and δTCR chains do not pair with the αβTCR chains. Therefore, the autoimmune risk arising from this pairing is eliminated (US 2003219463 A1). 33 ), van der Veeken et al., 2006 ( 34 ), van der Veeken et al., 2009 ( 35 )).

[0097] Another feature that makes adoptive T cell therapy with γδT cells particularly interesting is that allogeneic γδT cells do not induce GvHD. Because they do not carry the classic endogenous αβTCR that recognizes epitopes in the MHC context, they do not recognize “foreign” MHC.

[0098] Using allogeneic T cells for adoptive T-cell therapy is of great interest because they can be used as off-the-shelf therapeutic agents, eliminating the need for costly and time-intensive engineering of the patient's own T cells. The cost per dose would be reduced by approximately 10-fold due to scalability. Furthermore, patients with disease progression can receive treatment immediately, and the T cells are well-adapted (typically the opposite of the patient's T cells, which may have been affected by the patient's disease and / or previous treatments). Patient-specific manufacturing failures can also be avoided, and cells can be more easily standardized.

[0099] Because they do not induce GvHD, γδ cells would be an ideal choice for allogeneic adoptive T-cell therapy. This characteristic is shared with other immune cells such as NK cells, but γδ cells have a longer lifespan and can generate high numbers of cells, for example, from PBMCs from healthy donors.

[0100] Allogeneic CARγδT cells have been successfully used in clinical trials targeting CD20 lymphoma. 28 They were found to have a good safety profile, with an overall response rate of 75% and a complete remission rate of 69% across all dose levels. Circulating CAR γδ T cells were detectable in the blood at the highest dose level on day 28. However, it is known in the art that CAR T cells can lead to tumor resistance problems because tumor cells can downregulate the surface expression of CAR recognition-dependent antigens and select for these cells through treatment.

[0101] In view of this, the inventors have solved the problem of providing advantageous cells for allogeneic γδT cell therapy in this invention. Specifically, in one embodiment, the invention provides an immune cell expressing the following components: a) TCRα and TCRβ chain constructs of MHC class I restricted TCR constructs, and b) A CD8 co-receptor containing both CD8α and CD8β chains.

[0102] The immune cells are selected from γδT cells, NK cells, NKT cells, mucosa-associated invariant T (MAIT) cells, innate lymphocytes (ILC), CD4+ T helper cells, monocytes, macrophages and B cells.

[0103] The co-receptor is CD8 containing both CD8α and CD8β chains, i.e., it is not CD8a. As shown herein, CD8 with both α and β chains provides better co-stimulation than CD8a. Preferably, if immune cells express CD8, the nucleic acid encoding CD8 is a nucleic acid of the present invention as described herein.

[0104] However, nucleic acids can also encode CD8αβ constructs, meaning they can be nucleic acids that encode CD8αβ co-receptors, where the nucleic acid contains an operon capable of mediating the expression of both the CD8α and CD8β chains under the control of a single promoter from a polycistronic mRNA, wherein the nucleic acid segment encoding the CD8α chain is closer to the promoter than the nucleic acid segment encoding the CD8β chain.

[0105] The CD8α chain can be any isoform of human CD8α, i.e., 1, 2 (secretory isoform) or 3, optionally, as shown in Examples 1 and 2, it is CD8α1 having SEQ ID NO:2.

[0106] In the context of this invention, the CD8β chain can be any isoform of human CD8β, namely 1, 2, 3 (secretory isoform), 4, 5, 6 (secretory isoform), 7, or 8 (secretory isoform). Optionally, as shown in Examples 1 and 2, it is CD8β5 having SEQ ID NO: 1. The inventors have demonstrated that the CD8β5 and CD8β1 isoforms are expressed particularly well in γδT cells compared to other isoforms.

[0107] The α and β isomers can combine with each other; for example, CD8 can be CD8α1β1, CD8α1β2, CD8α1β3, CD8α1β4, CD8α1β5, CD8α1β6, CD8α1β7, CD8α1β8, CD8α2β1, CD8α2β2, CD8α2β3, CD8α2β4, CD8α2β5, CD8α2β6, CD8α2β7, CD8α2β8, CD8α3β1, CD8α3β2, CD8α3β3, CD8α3β4, CD8α3β5, CD8α3β6, CD8α3β7, or CD8α3β8. Preferably, it is CD8α1β5. CD8α1β1 is also a good combination. To avoid the formation of soluble CD8αβ heterodimers, preferably, at least one of the α or β chains is not a secretory isomer lacking a transmembrane domain. The CD8α and / or CD8β transmembrane domains can be transmembrane domains derived from other proteins (e.g., from CD4); however, they are preferably not fusion proteins. This document discloses sequences of usable wild-type human CD8α and β chains.

[0108] Alternatively, modified sequences can be used, such as variant CD8α or CD8β chains containing one or more mutations, which increase the binding affinity to the MHC class I α3 moiety compared to wild-type CD8α or CD8β chains. For example, using Wang et al. 2011 ( 16High-affinity mutants of CD8α were generated and characterized using in vitro evolution methods. The modified sequence or variant preferably has at least 95% amino acid identity with the wild-type isoform of the CD8α or β chain, for example, optionally, as described herein, with at least 95% amino acid identity with the sequences of SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0109] The TCR construct is MHC class I restricted, meaning it is only valid if it can recognize an epitope in the MHC class I context. The co-receptor CD8 can interact with the complex.

[0110] Immune cells can be, for example, immune cells that do not naturally express αβT cell receptors, preferably γδT cells.

[0111] γδT cells can be derived, for example, from PBMCs or T cells isolated therefrom. They can be enriched by antibodies against γ and / or δTCR chains and / or by consuming αβT cells and / or by bisphosphonate stimulation and / or by stimulation with antiCD3 antibodies. 29 ).

[0112] The inventors can demonstrate that 100-fold expansion of TCR-engineered γδT cells (e.g., PBMCs from healthy donors) is possible. γδT cell expansion protocols are known in the art, such as those described by Tan et al. (…). 29 ), US 2003219463 A1 ( 33 ), van der Veeken et al., 2006 ( 34 ), van der Veeken et al., 2009 ( 35 ) or WO2019 / 104269A1 ( 36 The γδT cells of this invention carrying the αβTCR construct can be stimulated with anti-CD3 antibodies that stimulate all subsets of γδT cells. Optionally, the Vg9Vd2 subset can be specifically stimulated with isopentenyl pyrophosphate (IPP), bromoethanol pyrophosphate (BrHPP), or bisphosphonates (e.g., zoledronic acid). γδT cells can also be generated from iPSCs, such as human iPSCs. Optionally, engineered γδT cells expressing co-receptors and MHC class I restricted TCRs can specifically amplify the antigen using cells expressing the target antigen and / or cells coated with homologous peptides.

[0113] The inventors have also demonstrated that co-expression of the CD8 co-receptor, particularly the CD8αβ co-receptor, and the MHC class I restricted TCR construct confers functions typically seen in CD8 cytotoxic T cells, such as IFN-γ production when co-cultured with tumor cells expressing the associated epitope and MHC class I, as well as cytotoxicity. Conversely, γδT cells expressing only a TCR lacking the CD8 co-receptor fail to elicit a full-scale γδT cell response against targeted solid tumor cells. Therefore, the γδT cells of this invention, co-expressing the CD8 co-receptor and the MHC class I restricted TCR construct, particularly γδT cells derived from the nucleic acids of this invention as described herein, can be advantageously used in adoptive T-cell therapy, such as allogeneic adoptive T-cell therapy.

[0114] Unlike CAR T cells, T cells expressing MHC class I restricted TCRs can target intracellular antigens, such as tumor antigens. This also applies to intracellular solid tumor antigens, such as MAGE, for example, MAGE-A4. Therefore, downregulation of cell surface expression is insufficient to evade the action of these T cells.

[0115] The inventors also demonstrated that γδT cells can naturally sense the loss of HLA (human leukocyte antigen / MHC class I) in tumor cells. For example, the HLA-negative cell line K562 may be recognized by unengineered γδT cells through innate immune function. This could provide an additional mode of action if tumor cells attempt to escape by downregulating HLA.

[0116] γδT cells can also be derived from patients who will receive cell therapy after being engineered. However, the donor for γδT cell source is preferably a healthy donor. Therefore, the donor is not diagnosed with cancer and generally is also not diagnosed with infectious diseases and / or other illnesses. The donor may be a relative, such as a sibling of the patient, but this is not necessary due to the lack of GvHD. Preferably, the donor shares MHC I, the donor shares MHC I restricted by the TCR with which the γδT cells are engineered, and most preferably, the donor is haploidentical to that MHC. For example, the donor may have two HLA-A receptors. The 01:01 allele is the second most common HLA allele in Europe. It is also the CTC127 (a TCR that recognizes the MAGA-A4 epitope) (WO2022 / 243514 A1). 30 HLA is restricted by )).

[0117] Along with the fact that γδT cells do not express high levels of MHC II molecules, this has significantly reduced the risk of host resistance to graft-versus-graft reactions. Optionally, other HLA molecules can be downregulated, for example, by repressive RNA or by knockout methods such as CRISPR / Cas engineering. Donors who are also haploidentical for one or preferably two other HLA molecules can be selected. Furthermore, a library of γδT cells with the most common HLA types (preferably haploidentical forms) can be provided so that γδT cells suitable for a specific patient can be selected.

[0118] Immune cells can be CD4+ T helper cells. Therefore, this invention provides a CD4+CD8αβ+ T helper cell that further expresses an MHC class I restricted TCR construct. The TCR construct is preferably a heterologous TCR construct, i.e., the α and β chains are preferably expressed under the control of a heterologous promoter. The inventors have demonstrated that the co-expression of an MHC class I restricted TCR construct and CD8 containing both α and β chains provides a functional T cell capable of responding to a target antigen of the TCR (presented by a suitable MHC). This response may include killing target cells and / or secreting cytokines.

[0119] Immune cells can be innate lymphocytes (ILCs). ILCs originate from common lymphoprogenitor cells (CLPs). In response to pathogenic tissue damage, ILCs promote immunity by secreting signaling molecules and regulating innate and adaptive immune cells. ILCs are primarily tissue-resident cells, found in both lymphoid (immune-associated) and non-lymphoid tissues, and rarely in the blood. They are particularly abundant on mucosal surfaces and play a crucial role in mucosal immunity and homeostasis. Characteristics that allow them to differentiate from other immune cells include regular lymphoid morphology, the absence of rearranged antigen receptors on T cells and B cells (due to the lack of the RAG gene), and phenotypic markers typically found on bone marrow or dendritic cells. Based on differences in developmental pathways, phenotypes, and signaling molecules produced, ILCs can be classified into five groups: NK cells, ILC1s, ILC2s, ILC3s, and lymphotissue inducer (LTi) cells (Wikipedia). ILCs do not naturally express CD4 or CD8 co-receptors, but in the context of this invention, they can be engineered to express the CD8 co-receptor as defined herein. In a preferred embodiment, the ILCs of the present invention express the CD8 ba co-receptor and MHCI-restricted TCRs of the present invention. Preferred ILCs are NK cells.

[0120] Immune cells can be NK (natural killer) cells, NKT cells, monocytes, macrophages, or B cells that express the TCRα chain construct and the TCRβ chain construct of the MHCI-restricted TCR.

[0121] If immune cells do not naturally express CD3, such as macrophages, B cells, or NK cells, the expression of the TCR and CD8 co-receptor allows immune cells to target environments that express antigens targeted by the TCR. Therefore, for example, if the TCR targets epitopes that form tumor antigens, immune cells can target tumors.

[0122] Furthermore, optionally, immune cells can be further engineered to express CD3, thereby also allowing activation of cells via TCR binding, which can lead to cytotoxic killing of target cells expressing homologous antigens, cytokine secretion, and / or phagocytosis (particularly in the case of macrophages). If the immune cells are B cells, activation may also lead to antibody secretion. This can be helpful, for example, if B cells further express antibodies, e.g., through genetic engineering, which can target B-cell epitopes of antigens such as tumor antigens. The TCR can target the same or related antigens. In one embodiment, the CD3 signaling domain can be expressed as a fusion protein having TCRα and / or β chains, e.g., as Sebestyén et al. ( 31 ) and Govers et al. 32 As disclosed in ( ).

[0123] The immune cells of the present invention advantageously comprise a nucleic acid encoding a co-receptor and a TCR. The nucleic acid fragment of the present invention encoding the co-receptor and TCR chain is operatively linked to a heterologous promoter, such as the promoters disclosed herein.

[0124] The CD8 co-receptor expressed by immune cells (e.g., the γδT cells of the present invention) is a heterodimeric CD8αβ co-receptor because this results in a significantly higher level of co-stimulation than the expression of the CD8α homodimeric co-receptor. Due to the advantageous effects shown by the inventors, the CD8 co-receptor is a CD8 ba co-receptor, i.e., as described herein, the γδT cells preferably contain and express the nucleic acid of the present invention. Pharmaceutical Composition

[0125] The present invention also provides a pharmaceutical composition comprising the nucleic acid of the present invention or the cells of the present invention. The pharmaceutical composition optionally further comprises a suitable buffer and / or excipient.

[0126] The pharmaceutical compositions of the present invention are typically intended for intravenous administration. They may contain pharmaceutically acceptable carriers, such as buffers, like physiological saline or PBS. They may contain excipients, such as stabilizers, such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins, such as albumin or casein, or protein-containing reagents, such as bovine serum or skim milk. If the composition contains nucleic acids instead of cells, it may be a dried composition, such as a lyophilized composition. Such compositions typically contain fillers, such as non-reducing sugars, such as trehalose. They may also include buffers, for example, as described herein.

[0127] T cells or other immune cells typically occur at a rate of 1 × 10⁻⁶ per kilogram of body weight. 5 -5×10 9 A concentration of approximately 1 × 10⁻⁶ cells is administered to the subject or patient. 6 –5 × 10 11 Cells, for example, 1×10 8 –2×10 10 Individual cells can be administered as a single-dose intravenous injection to human subjects. These parameters can be adjusted by healthcare professionals based on factors such as the patient's age, sex, weight, and medical condition.

[0128] In one embodiment, the pharmaceutical composition of the present invention comprises autologous T cells of a subject, such as autologous CD4 and CD8 T cells, engineered in vitro to express the nucleic acid of the present invention. Optionally, the nucleic acid of the present invention, particularly an expression vector, may also be administered to the subject for in vivo transduction of the T cells.

[0129] In another embodiment, the pharmaceutical composition comprises immune cells, such as the γδT cells of the present invention, co-expressing a) the TCRα and TCRβ strand constructs of an MHC class I restricted TCR construct, and b) a CD8 co-receptor. Preferably, these cells contain and express the nucleic acids of the present invention. Optionally, they may also co-express a) the TCRα and TCRβ strand constructs of an MHC class II restricted TCR construct, and b) a CD4 co-receptor.

[0130] This invention also provides pharmaceutical compositions of the invention for treating subjects in need, particularly subjects suffering from cancer or infectious diseases, preferably cancers such as solid tumors. Subjects suffering from diseases (e.g., cancer or infectious diseases) are also designated herein as patients. Preferably, the pharmaceutical composition is used for adoptive cell therapy, such as adoptive T-cell therapy for cancer.

[0131] It can be an adoptive cell therapy, such as adoptive T-cell therapy, or a therapy targeting HLA-A. T-cell receptor (TCR) gene therapy for the epitope of cancer testis antigen MAGEA4 presented on 01, such as TCR CTC127 (as disclosed in WO 2022 / 243514 A1 (30)) or a variant thereof, having at least 90% sequence identity in the CDR1, CDR2 and CDR3 regions, and preferably having at least 98% or at least 99% sequence identity with CTC127 on the complete sequence of the variable region.

[0132] In one embodiment, the cells used for adoptive T-cell therapy are allogeneic γδT cells of the present invention, preferably expressing a) the TCRα chain construct and TCRβ chain construct of an MHC class I restricted TCR construct, and b) a CD8 co-receptor, wherein the cells contain and express the CD8 ba nucleic acid of the present invention.

[0133] Of course, other antigens or epitopes can also be targeted. Optionally, the TCR construct targets antigens expressed by solid tumors, such as MAGE antigens, such as MAGEA4. As described herein, the TCR construct can be, for example, CTC127 or a variant thereof. The TCR can be expressed in CD4 and CD8 T cells or γδ T cells, preferably in γδ T cells, as described herein.

[0134] A method for treating subjects in need is also disclosed, for example, those suffering from cancer or infectious diseases, preferably cancer, such as solid carcinoma or lymphoma. This therapy typically involves intravenous administration of the pharmaceutical composition of the invention, preferably by infusion. Typically, prior to T-cell administration, for example on days 5, 4, and 3 prior to T-cell administration, it is administered by fludarabine and cyclophosphamide (e.g., infusion of 30 mg / m²). 2 Fludarabine and 500 mg / m 2 Cyclophosphamide is used for pretreatment of patients.

[0135] The subjects are typically mammalian subjects, such as mice or humans. Preferably, they are human patients.

[0136] Infectious diseases may be, for example, bacterial infections caused by multidrug-resistant bacteria, viral infections, or fungal infections. Viral infections are well-suited for adoptive cell therapy using cells with MHC I-restricted TCRs, such as T cells. Viral diseases may be coronavirus-mediated diseases, such as SARS-CoV-1, MERS, or SARS-CoV-2. Influenza virus infection is also possible.

[0137] Solid tumors can include, for example, melanoma, gastric cancer, head and neck cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, and esophageal cancer. Cancer-testis antigen melanoma-associated antigen 4 (MAGEA4) is highly expressed in all these solid tumors. Cancer can also be lymphoma. For example, adoptive T-cell therapy targeting CD19-mediated B-cell lymphoma has shown great potential. Combination with the CD8 βα co-receptor construct of this invention can further improve this therapy.

[0138] However, without being limited thereto, the invention is further illustrated in the following examples with reference to the accompanying drawings and sequences. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety. Attached Figure Description

[0139] Figure 1 CTC127 is CD8-dependent. Tc and Th cells were transduced with CTC127 and co-cultured with peptide-loaded K562-A0101. After overnight culture, IFNγ was measured in the supernatant by ELISA. Experiments were repeated using two T cell donors. Data were pooled. Mean ± SEM values ​​are shown. Tc, CD8 + Cytotoxic T cells; Th, CD4 + Helper T cells.

[0140] Figure 2 CD8 co-receptor confers function on CTC127 Th cells. Tc and Th cells were transduced with CTC127 and one of three CD8 co-receptor constructs (a, ab, or ba), respectively. They were then co-cultured with peptide-loaded K562-A0101. T cell numbers were adjusted according to individual transduction rates to ensure an equal number of TCR-expressing cells in each group. After overnight culture, IFNγ was measured in the supernatant by ELISA. IFNγ concentrations were normalized to a percentage of the maximum value to plot dose-response curves. Experiments were repeated with two T cell donors. Data from both donors were pooled. Mean ± SEM is shown. A) Dose-response curves for Th cells expressing CTC127 and different co-receptor constructs. B) Dose-response curves for Tc cells expressing CTC127 and different co-receptor constructs.

[0141] Figure 3Th cells expressing CTC127 and the heterodimeric CD8 ba co-receptor recognized cancer cells. Tc and Th cells were transduced with CTC127 and one of three CD8 co-receptor constructs (a, ab, or ba), respectively. They were co-cultured with cancer cell lines A375 and H1703 expressing MAGE-A4. Engineered K562-A0101-MAGE-A4 and K562-A0101 were used as positive and negative controls, respectively. The number of T cells was adjusted according to individual transduction rates to ensure that each group had the same number of TCR-expressing cells. IFNγ was measured in the supernatant by ELISA after overnight culture. A) Co-culture with Th cells. B) Co-culture with Tc cells. Experiments were repeated using two T cell donors. Data were pooled. Mean ± SEM is shown.

[0142] Figure 4 CTC127c-Th cells secrete a variety of cytokines that support Tc cells and also possess cytotoxic functions. Th cells were transduced with CTC127 or CTC127c and then co-cultured with A375 cancer cells or activated with beads coated with anti-CD3 / anti-CD28 (positive control). Cytokine concentrations in the supernatant were measured by multiplex flow cytometry microsphere array (CBA) after 24 hours. Experiments were repeated using two T cell donors. Data were pooled. Mean ± SEM is shown.

[0143] Figure 5 CTC127c-Th cells express the helper molecule CD154. Tc and Th cells were transduced with CTC127 or CTC127c, respectively, and then co-cultured with A375 cancer cells. 10 µg / ml anti-CD40 antibody was added to the culture to block CD154-CD40 interaction and subsequently downregulate CD154. CD154 was detected on the surface of T cells by flow cytometry after 24 hours. The experiment was repeated using two T cell donors. A) Flow cytometry image showing CD154 in one donor. + T cell frequencies. Mouse constant β-chain (mCb) stained with TCR CTC127 is shown on the x-axis. B) Summary of CD154 frequencies. Mean ± SEM values ​​from both T cell donors.

[0144] Figure 6CTC127c-Th cells exhibited cytotoxicity, albeit to a lesser degree than Tc cells. Th and Tc cells were transduced with CTC127 or CTC127c, respectively, and used for killing assays of luciferase-expressing K562-A0101-MAGE-A4 cells (A and B), or co-cultured with A375 cells (C, D). For the killing assay, a fixed number of target K562-A0101-MAGE-A4 cells were co-cultured with a titrated number of (A) Th or (B) Tc cells. The effector cell to target cell (E:T) ratio ranged from 64:1 to 0.125:1. After 24 hours, the luciferase activity of live target cells was quantified, and the lysis frequency was calculated using the following formula: 100 – [(signal with T cells) / (signal without T cells)].

[100] C, D) Cytokines were measured after co-culturing with A375 cancer cells at a 1:1 ratio for 24 hours. Pooled mean ± SEM of duplicates from two T cell donors.

[0145] Figure 7 CTC127xc-T cells induce early response and complete rejection in A375 tumors. (5x10) 6 A375 cancer cells were subcutaneously injected into NSG mice. Eight days later, when the average tumor size reached 50 mm... 3 At that time, intravenous transfer 1x10 7 Engineered Th and Tc cells (expressing CTC127xc or CTC127x) or a mixture of unengineered Th and Tc cells (without TCR) were used. Tumor growth was measured using calipers for 35 days. Individual mice reached the humane endpoint (tumor >1500 mm). 3 A) Mice were euthanized after tumor growth curves were obtained. B) Mean tumor growth curves for each group (mean ± SEM). Data are from two independent experiments. Nd, undetectable.

[0146] Figure 8 TCR-engineered γδT cells can be rapidly expanded and harvested with high purity γδT cells on day 14. Engineered γδT cells were stabilized using different TCR constructs and / or co-receptor constructs. Engineering and expansion using two different donors are shown. B. αβT cell contamination was less than 0.25% after harvesting on day 14. Therapeutic TCR constructs and co-receptors were strongly expressed in γδT cells.

[0147] Figure 9CD8 co-receptor-engineered αβTCR T cells strongly recognized different tumor cell lines during co-culture. γδT cells were either unengineered or engineered to express the TCR CTC127, or they were CD8 co-receptors engineered to express CTC127xcs. The K562 tumor cell line was engineered to express HLA-A0101 (K562-A1) or HLA-A01. 01. Add MAGE-A4 antigen (K562-A1-MA4). A375 and H1703 tumor cell lines are naturally HLA-A01. 01 and MAGE-A4 positive. Combined data from γδT cells from two different donors are shown.

[0148] Figure 10 γδT cells expressing MHC-I-restricted TCR and CD8 co-receptors exhibited cytotoxicity. γδT cells were transduced with CTC127 or CTC127xcs and used in a killing assay of K562-A0101-MAGE-A4 cells expressing luciferase. For the killing assay, a fixed number of target K562-A0101-MAGE-A4 cells were co-cultured with a titrated number of γδT cells. The effector cell to target cell (E:T) ratio ranged from 16:1 to 0.125:1. After 24 hours, the luciferase activity of live target cells was quantified, and the lysis frequency was calculated using the following formula: 100 – [(signal with T cells) / (signal without T cells)].

[100] . The pooled mean ± SEM of two T cell donor replicates is shown.

[0149] Figure 11 γδT cells can sense HLA loss. Co-culture of CTC127xcs γδT cells with HLA-negative and HLA-positive K562 cell lines showed that IFN-γ release was induced by HLA-negative cells. IFN-γ release in the supernatant was measured by ELISA. Representative data from one of the two donors are shown.

[0150] Figure 12Effects of CD8b isoforms on transgenic TCR and transgenic CD8 co-receptor expression in the CD8b-CD8a-TCRb-TCRa operon. γδ T cells (A, C, E, and G) or CD4+ Th cells (B, D, F, and H) were transduced with CTC127xc box retroviruses, with CD8b being one of eight isoforms (B1-B8). Flow cytometry analysis was performed after 9 to 14 days. A and B show the frequency of TCR+ cells (TCR was measured by expression of the transgenic mouse TCRβ constant chain (mCb)). C and D show the mCb expression level in positive cells by median fluorescence intensity (MFI). E and F show the frequency of CD8b+ cells. G and H show the CD8b expression level in positive cells by median fluorescence intensity (MFI). Pooled mean ± SEM of duplicates from two T cell donors is shown.

[0151] Figure 13 The effect of CD8b isoforms on the function of transduced T cells. γδ T cells (A) or CD4+ Th cells (B) were transduced using CTC127xc box retroviruses, where CD8b was one of eight isoforms (B1-B8). These were compared with MAGE-A4. + and HLA-A 01:01 + A375 target cancer cells (10,000) were co-cultured. The number of T cells was adjusted according to individual transduction rates to ensure that each group had the same number of TCR-expressing cells (10,000). After overnight culture, IFNγ in the supernatant was measured by ELISA. The pooled mean ± SEM of the two T cell donor replicates is shown.

[0152] Figure 14 The effect of transgene sequence on the expression of transgenes abTCR and CD8ab co-receptors on γδT cells. γδT cells were transduced with different retroviral constructs with different transgene sequences: “CXCX” (CD8b-TCRb-CD8a-TCRA), “XCCX” (TCRb-CD8b-CD8a-TCRa), “CTC127xc” (CD8b-CD8a-TCRb-TCRa), “CTC127x” (TCRb-TCRa, without co-receptor), or untransduced (“unengineered”). Transgene expression was analyzed by flow cytometry after 14 days, and functional analysis was performed by co-culturing with target cancer cells. abTCR +A. Cell frequency (measured by mouse TCRβ constant chain (mCb)). B. mCb expression level in mCb+ cells. C. CD8a+ cell frequency. D. CD8a expression level in CD8a+ cells. E. CD8b+ cell frequency. F. CD8b expression level in CD8b+ cells. G. Expression of homologous antigens (MAGE-A4) and HLA (A... IFNγ secretion from K562 cancer cells co-cultured overnight (01:01). 10,000 mCb+gdT cells were cultured from 10,000 target cells. The pooled mean ± SEM of duplicates from two T cell donors is shown.

[0153] Figure 15 Possible configurations of the operon. Configurations are shown in 5' to 3' orientations. The promoter is located upstream of the 5' end, before the first nucleotide sequence encoding the first element. CS, cleavage site.

[0154] Sequence

[0155] Amino acid sequence of human CD8β chain isomer 5 (SEQ ID NO: 1)

[0156] Amino acid sequence of human CD8α chain isomer 1 (SEQ ID NO: 2)

[0157] SEQ ID NO: 3 T2AEGRGSLLTCGDVEENPGP

[0158] SEQ ID NO: 4 P2A ATNFSLLKQAGDVEENPGP

[0159] SEQ ID NO: 5 E2A QCTNYALLKLAGDVESNPGP

[0160] SEQ ID NO: 6 F2A VKQTLNFDLLKLAGDVESNPGP

[0161] SEQ ID NO: 7 2A element peptide core sequence motif DXEXNPGP

[0162] SEQ ID NO: 8 contains the TEV cleavage site ENLYFQS

[0163] SEQ ID NO: 9 Preferred IRES

[0164] SEQ ID NO: 10 is a nucleic acid fragment encoding the CD8β chain, which is codon-optimized by linking a nucleic acid fragment encoding the P2A cleavage site to a nucleic acid fragment encoding the CD8α chain.

[0165] SEQ ID NO: 11 Comparative nucleic acid (operon with CTC127 in the configuration TCRβ-P2A-TCRα-P2A-CD8β-P2A-CD8α)

[0166] Comparative nucleic acids with the configuration TCRβ-P2A-TCRα-P2A-CD8α-P2A-CD8β (TCR127) SEQ ID NO: 12

[0167] SEQ ID NO: 13 The contrast nucleic acid with the configuration TCRβ-P2A-TCRα-P2A-CD8α (TCR127)

[0168] SEQ ID NO: 14 The contrast nucleic acid with the configuration CD8β-P2A-CD8α-P2A-TCRβ-P2A-TCRα (TCR127).

[0169] SEQ ID NO: 15 IRES

[0170] Amino acid sequence of human CD8β chain isomer 16 (SEQ ID NO: 16)

[0171] Amino acid sequence of human CD8β chain isomer 2 (SEQ ID NO: 17)

[0172] Amino acid sequence of human CD8β chain isomer 3 (SEQ ID NO: 18)

[0173] Amino acid sequence of human CD8β chain isomer 4 (SEQ ID NO: 19)

[0174] Amino acid sequence of human CD8β chain isomer 6 (SEQ ID NO: 20)

[0175] Amino acid sequence of human CD8β chain isomer 7 (SEQ ID NO: 21)

[0176] Amino acid sequence of human CD8β chain isomer 8 (SEQ ID NO: 22)

[0177] Amino acid sequence of human CD8α chain isomer 2 (SEQ ID NO: 23)

[0178] Amino acid sequence of human CD8α chain isomer 3 (SEQ ID NO: 24)

[0179] SEQ ID NO: 25 25 nucleic acids of the present invention (operon with CTC127 in configuration CD8β-P2A-TCRβ-P2A-CD8α-P2A-TCRα, with codon optimization). Figure 15The “1a” in the text.

[0180] SEQ ID NO: 26 The nucleic acid of the present invention (an operon with CTC127 of the configuration TCRβ-P2A-CD8β-P2A-CD8α-P2A-TCRα, with codon optimization). Figure 15 The “2a” in the text.

[0181] Example

[0182] Example 1 - CD8 co-receptor constructs

[0183] Materials and methods

[0184] To generate transduced T cells, PBMCs were isolated from fresh blood by gradient centrifugation, and the T cells contained therein (mixed CD8 and CD4 populations or single CD8 and / or CD4 populations separated by magnetic bead sorting) were stimulated with bead-fixed anti-CD3 and anti-CD28 antibodies and cultured in RPMI medium supplemented with 10% FBS, 1% penicillin / streptomycin, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 400 IU / mL human recombinant IL-2. On days 2 and 3 post-stimulation, the cells were transduced with transgenic retroviruses (SEQ ID NO: 11-14). The retroviruses were generated by transfecting GALV cells with transgenic plasmid DNA using the Lipofectamine transfection kit (Invitrogen) according to the manufacturer's instructions. The transduced T cells were further cultured for 9 to 12 days and used for assays. The expression of the transgenic TCR and co-receptor was assessed by flow cytometry using antibodies against the mouse constant TCR β chain and human CD8a, respectively.

[0185] Transduced γδT cells were generated from PBMCs by culturing with 1 µg / ml zoledronic acid. On days 4 and 5 post-stimulation, cells were transduced with a retrovirus containing the transgene (SEQ ID NO: 11-14). The retrovirus was generated by transfecting GALV cells with the transgene plasmid DNA. The transduced γδT cells were further cultured for 14 days and used for assays. The expression of the transgene TCR and co-receptor was assessed by flow cytometry using antibodies against the mouse constant TCR β chain and human CD8a, respectively.

[0186] result

[0187] Generation and functional testing of candidate CD8 co-receptor constructs

[0188] CTC127 identifies HLA-A The MAGE-A4 epitope presented at 01:01. Here, we show the CD8 expression for CTC127. +Tc cells effectively recognized peptide-loaded K562-A0101 target cells and secreted IFNγ, a cytokine associated with T cell activation and function, in a peptide titration assay. Conversely, CD4 cells expressing CTC127... + Th cells failed to recognize target cells carrying peptides. Figure 1 Furthermore, it does not secrete IFNγ. This is due to the lack of a CD8 co-receptor. Delivering the CD8 co-receptor molecule along with CTC127 to Th cells can enable them to target this HLA class I restricted antigen.

[0189] The CD8 coreceptor consists of two subunits (CD8a and CD8b), most commonly found as the CD8ab heterodimer on the surface of cytotoxic T cells. Some cells express the CD8a homodimer; however, this is rare. We designed three candidate constructs using wild-type sequences of the subunits: CD8a alone (used by Adaptimmune), CD8a followed by CD8b linked via a P2A element (used by Hans Stauss et al.). 17 (Suggested and used by Immatics), CD8b connected via P2A element followed by CD8a (constructed by the inventors). To test the functionality of the constructs, CTC127 and each candidate were co-transduced to a separate CD4. + Th or CD8 + In Tc cells, peptide titration was performed to test the function of two T cell subtypes. We observed that all three CD8 co-receptor constructs conferred function on Th cells, enabling them to secrete IFNγ in response to K562-A0101 carrying the CTC127 homodimeric peptide. However, the heterodimeric constructs (CD8ab and CD8ba) were superior to the CD8a homodimeric construct, exhibiting higher functional affinity (EC50). 50 () Figure 2 A). EC of CD8ab and CD8ba 50 Quite significantly. Importantly, adding different CD8 co-receptor constructs to Tc cells did not alter peptide sensitivity (ECG). 50 () Figure 2 B). Therefore, delivering the TCR and co-receptor simultaneously to all T cells on a single construct, without the need to separate them into Th and Tc cells, enables an efficient single-engineering process.

[0190] Next, we tested whether Th cells expressing the CTC127 and CD8 co-receptors could recognize MAGE-A4. + Tumor cells. For this experiment, we selected cells naturally expressing MAGE-A4 and HLA-A4. Cancer cell lines A375 and H1703 at 01:01. Except for A... In addition to 01:01, we also include the expression of HLA-A. Engineered K562 cells with a 01:01 ratio and K562 cells stably expressing the full-length MAGE-A4 gene were compared. We observed that Th cells expressing the homodimer CD8a construct could not recognize cancer cell lines A375 and H1703, but did recognize K562-A0101-MAGE-A4. These data suggest that CD8a homodimers only confer function on Th cells when tumor cells highly express MAGE-A4, and this is achieved only in engineered cell lines, not in cancer cell lines naturally expressing MAGE-A4. Interestingly, the literature... 18 The study has already described that the CD8a homodimer cannot confer function in the absence of CD8b. Both heterodimeric CD8 constructs allow Th cells to recognize all three cell lines, with the CD8ba construct resulting in the highest IFNγ secretion. Figure 3 A). Compared to A375, H1703 showed a lower recognition rate, which can be explained by differences in MAGE-A4 expression levels (1626.7 rpkm and 4268.5 rpkm, respectively). Importantly, the addition of the CD8 co-receptor did not lead to nonspecific recognition by antigen-negative K562-A0101 cells. We also performed the same experiment using Tc cells and observed that recognition in all cell lines was unaffected by the addition of the CD8 co-receptor, supporting our previous peptide titration data that overexpression of CD8 in Tc cells does not affect antigen recognition. Figure 3 Figure 3 B).

[0191] Based on these findings, we selected the CD8ba construct as the co-receptor, and all further experiments were performed using the TCR CTC127 and CD8ba co-receptor constructs, which are linked together by the P2A element on the same transgenic cassette, and are subsequently referred to as CTC127c.

[0192] Functional analysis of helper T cells expressing CTC127c

[0193] It was observed that CTC127c Th cells can transmit the class I allele HLA-A. 01:01 By specifically recognizing MAGE-A4, we set out to conduct a more in-depth functional analysis of these engineered T cells. Th cells have different functions compared to Tc cells. They are often referred to as “helper” T cells because they activate dendritic cells, macrophages, and B cells through cytokine and CD154-CD40 interactions, and support Tc cell function by secreting cytokines such as IL-2. Furthermore, a subset of Th cells are also capable of cell lysis. Through functional analysis, we aimed to determine whether Th cells expressing CTC127c maintain typical helper T cell function upon activation via HLA class I antigens and participation in the addition of CD8 co-receptors.

[0194] We first evaluated the comparison with MAGE-A4 + Cytokine secretion in co-cultures of A375 cancer cells (activating T cells via CTC127c) or anti-CD3 / anti-CD28 beads as positive controls (independent of TCR and co-receptor activation of T cells). This setup allowed us to observe whether the cytokine profile of CTC127c Th cells varied depending on the activation pathway. After 24 hours, we measured six cytokines in the supernatant using a multiplex flow cytometry microsphere array (CBA). We selected typical cytokines associated with helper cell and cytotoxic T cell function (IL-2, IL-4, IL-5, IL-13, IFNγ, and TNFα). IL-2 is a potent stimulant for both Th and Tc cells and supports their survival in vivo. IFNγ and TNFα are cytotoxic mediators that promote antigen presentation in tumor cells by inducing proteasome activity and MHC expression, thereby facilitating T cell detection. They also activate innate immune cells and have direct tumor-killing effects. IL-4, IL-5, and IL-13 are classic helper type 2 cytokines with pleiotropic immune functions, such as activation of B cells, dendritic cells, and other innate cell types, which can enhance Tc cell-induced antitumor function.

[0195] After co-culturing with A375, only Th cells expressing CTC127c were activated and secreted cytokines exceeding basal levels. Figure 4 By comparing CTC127c-Th cells activated by A375 with TCR-independent positive controls, we observed similar levels of most cytokines, indicating that the presence of the CD8 co-receptor did not significantly alter the qualitative characteristics of helper T cells.

[0196] Next, we evaluated the surface expression of CD154 (CD40L) on Th and Tc cells. CD154 is a major surface molecule expressed by mature Th cells and is essential for activating B cells (germinal center formation, antibody class switching, and affinity maturation) and dendritic cells (maturation and antigen presentation to Tc cells). In the context of antitumor immunity, CD154 plays a role in the activation of intratumoral dendritic cells and macrophages and can promote enhanced T cell responses in lymph nodes. We measured CD154 expression after overnight co-culture with A375 cancer cells. As expected, unengineered Th and Tc cells, as well as CTC127-Th cells, showed only low expression of CD154, which can be explained by basal levels. Conversely, 55% of CTC127c-Th cells responded to A375 cancer cells by expressing CD154, indicating antigen-specific activation of helper T cells. 20% of CTC127 and CTC127c-Tc cells expressed CD154 (CD40L). Figure 5 The significant difference in CD154 expression between Th and Tc CTC127c-T cells illustrates the distinct functions of these T cell subsets.

[0197] Finally, we measured the cytolytic capacity of two T cell subsets to determine whether CTC127c-Th cells could also perform this function, which is typically more pronounced in Tc cells. For the killing assay, we used a fixed amount of HLA-α-expressing cells. K562 cells were prepared with 01:01, MAGE-A4, and luciferase, and T cells were added at different effector cell to target cell (E:T) ratios. After 24 hours, we measured the luciferase activity of the remaining viable K562 cells and calculated the degree of T cell lysis. As expected, we observed that CTC127-Th cells did not kill cancer cells above the background level (as background, we considered unengineered T cells that did not express engineered TCRs, hence referred to as "unengineered"). However, CTC127c-Th cells showed specific killing ability up to an E:T ratio of 2:1. Figure 6 A). Conversely, CTC127 and CTC127c-Tc cells were highly effective at killing target cancer cells until the E:T ratio was approximately 0.5:1 ( Figure 6 B). The difference in cytolysis between CTC127c-Tc and CTC127c-Th cells may be due to the higher ability of Tc cells to secrete IFNγ and granzyme B. Figure 6 (C, D). IFNγ is associated with the expression of other cytolytic proteins and can itself trigger apoptosis in cancer cells. Granzyme B is a major protease involved in cell lysis.

[0198] Using this set of functional assays, we characterized CTC127c-Th cells and observed that they maintained typical "helper" functions of cytokine secretion and CD154 expression, and also acquired direct cell lysis capacity, although to a lesser extent than CD8. + T cells. Using two populations of T cells in a therapeutic product can leverage the unique capabilities of each subset and provide beneficial synergistic effects.

[0199] In vivo assessment of CTC127c

[0200] Our next step is to evaluate the efficacy of CTC127c-T cells against A375 tumors in vivo. Our previous in vivo experiments have shown that CTC127-Tc cells provide a significant survival benefit in mice. The following experiments were performed using a CTC127 variant (CTC127x), which resulted in a complete response in vivo. To assess the effect of CD8 co-receptor expression on tumor rejection, we compared CTC127x with CTC127xc (TCRs without and with the co-receptor).

[0201] We subcutaneously inoculated NSG mice with A375 tumors and waited 8 days until the average tumor size was 50 mm. 3 Mice were then intravenously transferred with mixed populations of Tc and Th cells (each mixture was divided into three groups: CTC127xc, CTC127x, and no TCR). Tumor growth in the mice was then tracked for a total of 35 days. Mice receiving unengineered T cells (no TCR) failed to control A375 tumors and reached the humane endpoint approximately 20 days after tumor cell injection. In contrast, both groups receiving CTC127 engineered T cells completely rejected the tumor and remained tumor-free until the end of the observation period. Figure 7 Compared to the CTC127x group (10 days and 15 days, respectively), the CTC127xc group showed a 33% faster tumor rejection response, demonstrating the significant benefit of using a co-receptor that allows for synergistic Tc and Th function in vivo. Mice in the CTC127xc group remained tumor-free until the end of the experiment (35 days).

[0202] These experiments demonstrate that the use of co-receptors in CTC127xc enables synergistic function of Tc and Th cells, thereby achieving faster tumor clearance in 100% of mice.

[0203] in conclusion

[0204] In this report, we showcase the development of toolkit technology that has the potential to significantly improve the clinical efficacy of TCR-engineered T cells.

[0205] We constructed a CD8 co-receptor construct containing β and α subunits in reverse order and benchmarked it against two other constructs: 1) the CD8a homodimer used by Adaptimmune and 2) the CD8ab heterodimer used by Immatics. Our CD8ba construct exhibited superior functional affinity compared to Adaptimmune's CD8a construct and better tumor cell recognition compared to Immatics' CD8ab construct.

[0206] The CD8ba co-receptor confers full function to Th cells expressing HLA class I restricted TCRs without harming Tc cells. We found that CTC127c-Th cells inhibit MAGE-A4 + Cancer cells perform full helper functions, including cytotoxicity, helper cytokine secretion, and CD154 expression. Furthermore, they are capable of directly killing cancer cells. In vivo, CTC127xc T cells induce MAGE-A4... + Complete and durable rejection of A375 tumors was achieved 33% faster than that of CTC127x T cells (which do not have co-receptors).

[0207] The data in this report were primarily generated in conjunction with CTC127. However, in general, the CD8ba co-receptor technology is independent of the TCR and can be used with any other TCR. Importantly, the TCR and co-receptor can be housed in the same construct and delivered simultaneously to T cells, eliminating the need for additional engineering steps during manufacturing and eliminating the generation of T cells expressing only one of the constructs. Furthermore, the TCR and CD8ba co-receptor can be used for cell types, such as γδ T cells or NK cells that do not express the TCR co-receptor, providing them with anti-tumor function. This is particularly useful for allogeneic approaches using γδ T or NK cells engineered to express class I restricted TCRs.

[0208] Our developed CD8ba co-receptor technology enables us to fully leverage the potential of TCR-T cell therapy by utilizing Tc and Th cell subsets with unique and synergistic functions, which is crucial for successfully combating solid tumors. Furthermore, it opens up possibilities for the use of γδ T and NK cells in allogeneic approaches.

[0209] Example 2: gd T cells

[0210] γδT cells were generated from healthy donor PBMCs by activation with 1 µg / mL zoledronic acid and transduced with a retrovirus containing a TCR and / or the co-receptor construct described herein. The retrovirus was generated by transfecting GALV cells with transgenic plasmid DNA using the Lipofectamine transfection kit (Invitrogen) according to the manufacturer's instructions. T cells were cultured for 14 to 28 days (fed every 2–3 days) in a medium containing recombinant human IL-7 and IL-15 prior to experimental use. Regardless of the engineered construct used, γδT cells were able to expand approximately 100-fold within 14 days of culture. Figure 8 Importantly, the contamination of αβT cells in the final cell population was less than 0.25%. Figure 8 Wild-type γδT cells do not express the CD8 co-receptor; therefore, they cannot function fully if engineered using only MHC class I restricted TCRs (e.g., CTC127). Only when co-engineered with the CD8 co-receptor (CTC127xcs) can γδT cells cross HLA-A receptors. 01:01 Identify cancer cells expressing MAGE-A4 ( Figure 9 In these experiments, γδT cells and tumor cells were co-cultured for 24 hours, and then IFNg in the supernatant was measured by ELISA as a surrogate for tumor cell recognition and γδT cell activation. To obtain the cytotoxic ability of γδT cells expressing CTC127 and a co-receptor construct, a fixed number of HLA-A-expressing cells were used. K562 cells with 01:01, MAGE-A4, and luciferase were cultured with γδT cells at different effector-to-target (E:T) ratios. After 24 hours, the luciferase activity of the remaining viable K562 cells was measured, and the degree of γδT cell lysis was calculated. Compared with CTC127 and unengineered γδT cells, CTC127xcs γδT cells showed superior cell lysis ability. Figure 10 Therefore, the addition of the CD8 co-receptor is crucial for unleashing the full potential of TCR-engineered γδT cells against cancer. γδT cells can additionally sense HLA loss in target cells, a trait that can provide an additional mode of action if tumor cells attempt to escape by downregulating HLA. This was demonstrated by recognizing the HLA-negative cell line K562, rather than its HLA-transduced counterpart (K562-A0101), after 24 hours of co-culture. Figure 11 ).

[0211] Example 3: Comparison of CD8b isoforms in gd T and CD4+ Th cells.

[0213] To assess the effects of CD8b isoforms on the expression and function of the TCR construct in γδT and CD4+Th cells, these cells were transduced with the CTC127xc construct (CD8b-CD8a-TCRb-TCRa), where the CD8b isoform was one of eight CD8b isoforms (B1-B8). TCR expression was assessed by flow cytometry. Figure 12 AD) and CD8b Figure 12 Transduction rate and expression level of EH transgene.

[0214] In γδT and CD4+Th cells, constructs with isoforms B1, B3, B5, or B8 showed the highest TCR transduction rate. Figure 12 AB) and expression level ( Figure 12 CD (measured for mouse TCRb constant chain: mCb).

[0215] The surface expression of the CD8 co-receptor was measured using the specific monoclonal antibody clone S21011A, which detects the CD8b chain independently of the CD8a chain. Only cells transduced with constructs containing CD8b isoforms B1, B2, and B5 showed high frequency (…). Figure 12 EF) and CD8b expression levels Figure 12 GH).

[0216] Next, peptide titration was performed to determine the functional affinity of TCR127x with combinations of CD8 co-receptors containing different CD8b isoforms.

[0217]

[0218] Isomers 8, 5, and 1 showed the lowest EC in γδT cells. 50 The values ​​are therefore the highest functional affinity. Isomers 5, 1, and 2 showed the lowest EC values ​​in CD4+Th cells. 50 value.

[0219] Next, using the expression HLA-A Cancer cells expressing the 01:01 and CTC127 homologous antigens were used for tumor cell recognition assays. γδT cells expressing CD8b isoforms 1 and 5 exhibited the strongest cancer cell recognition, as measured by IFNγ secretion. Figure 13 A). Consistent with these findings, CD4+ Th cells transduced with isoforms 1 and 5 exhibited strong cancer cell recognition, while Th cells expressing isoforms 2 and 3 also secreted similar amounts of IFNγ (A). Figure 13 B).

[0220] In summary, these results indicate that CD8b isoforms 1 or 5 are preferred isoforms for γδT or CD4+Th cells, as well as for cancers targeting TCR.

[0221] Example 4: Optimization of transgene cassette containing CD8ab co-receptor and ab TCR.

[0223] To optimize transduction efficiency and expression levels, the inventors generated a cassette variant containing two subunits (α and β) of the CD8 co-receptor and two subunits (α and β) of the TCR in a "rearranged" order, and linked them via a P2A element:

[0224] - "CXCX": CD8b-TCRb-CD8a-TCRa

[0225] - "XCCX": TCRb-CD8b-CD8a-TCRa

[0226] The “CTC127xc” construct was tested, which has the following transgene sequence: CD8b-CD8a-TCRb-TCRa.

[0227] γδT cells from two independent donors were transduced using each construct, and the frequency and expression levels of the transgene were measured by flow cytometry. Surprisingly, higher transduction rates were observed in both rearranged constructs compared to CTC127xc and CTC127x. Figure 14 A, C, E). Besides a higher frequency of transduced cells, the expression levels of transgenes (TCRb, CD8a, and CD8b) in the CXCX construct were higher than those in the CTC127xc construct (…). Figure 14 B, D, F). Both rearranged constructs also conferred higher function on γδT cells, as demonstrated by IFNγ secretion after co-culturing with K562-A0101-MAGEA4 target cells. Figure 14 G). Therefore, the "rearranged" construct, especially CXCX, is superior to the "tandem" construct (CTC127xc) to achieve higher transduction, expression levels, and function.

[0228] References

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Claims

1. A nucleic acid encoding a CD8 alpha beta co-receptor, wherein the alpha and beta chains are not covalently linked, and a TCR alpha chain construct and a TCR beta chain construct, wherein the nucleic acid comprises an operon, under the control of one promoter, which is capable of mediating expression of the CD8 alpha and CD8 beta chains and the TCR alpha chain construct and the TCR beta chain construct, wherein the nucleic acid fragment encoding the CD8 beta chain is closer to the promoter than the nucleic acid fragment encoding the CD8 alpha chain, and wherein at least one nucleic acid fragment encoding a CD8 alpha chain or a CD8 beta chain is located between the nucleic acid fragment encoding one of the TCR alpha chain construct and the TCR beta chain construct and the nucleic acid fragment encoding the other of the TCR alpha chain construct and the TCR beta chain construct.

2. The nucleic acid according to claim 1, wherein at least one nucleic acid fragment encoding a CD8 alpha chain or a CD8 beta chain is located between the nucleic acid fragment encoding the TCR beta chain construct and the nucleic acid fragment encoding the TCR alpha chain construct, preferably wherein only one nucleic acid fragment is located between the nucleic acid fragment encoding the TCR beta chain construct and the nucleic acid fragment encoding the TCR alpha chain construct, said only one nucleic acid fragment encoding a CD8 alpha chain or a CD8 beta chain, optionally wherein said only one nucleic acid fragment is the nucleic acid fragment encoding the CD8 alpha chain.

3. The nucleic acid according to any one of the preceding claims, wherein at least the nucleic acid fragment encoding the CD8 alpha chain is located between the nucleic acid fragment encoding one of the TCR alpha chain construct or the TCR beta chain construct and the nucleic acid fragment encoding the other of the TCR alpha chain construct and the TCR beta chain construct, preferably between the nucleic acid fragment encoding the TCR beta chain construct and the nucleic acid fragment encoding the TCR alpha chain construct.

4. The nucleic acid according to any one of the preceding claims, wherein the promoter is operably linked to the nucleic acid fragment encoding the CD8 beta chain, which is linked to the nucleic acid fragment encoding one of the TCR alpha chain construct or the TCR beta chain construct by a nucleic acid fragment encoding a cleavage site, which is linked to the nucleic acid fragment encoding the CD8 alpha chain by a nucleic acid fragment encoding a cleavage site, which is linked to the nucleic acid fragment encoding the other of the TCR alpha chain construct or the TCR beta chain construct by a nucleic acid fragment encoding a cleavage site.

5. The nucleic acid according to claim 4, wherein the promoter is operably linked to the nucleic acid fragment encoding the CD8 beta chain, which is linked to the nucleic acid fragment encoding the TCR beta chain construct by a nucleic acid fragment encoding a cleavage site, which is linked to the nucleic acid fragment encoding the CD8 alpha chain by a nucleic acid fragment encoding a cleavage site, which is linked to the nucleic acid fragment encoding the TCR alpha chain construct by a nucleic acid fragment encoding a cleavage site.

6. The nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid fragment encoding the CD8 beta chain and the CD8 alpha chain is located between the nucleic acid fragment encoding one of the TCR alpha chain construct or the TCR beta chain construct and the nucleic acid fragment encoding the other of the TCR alpha chain construct and the TCR beta chain construct, preferably between the nucleic acid fragment encoding the TCR beta chain construct and the nucleic acid fragment encoding the TCR alpha chain construct.

7. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid fragment encoding the CD8 beta chain is linked to the nucleic acid fragment encoding the CD8 alpha chain by a nucleic acid fragment encoding a cleavage site.

8. The nucleic acid according to claim 7, wherein the cleavage site is a self-cleaving peptide selected from the group comprising a P2A element, a T2A element, an E2A element and an F2A element, preferably a P2A element.

9. The nucleic acid according to any one of claims 1 to 6, wherein an internal ribosome entry site (IRES) is located between the nucleic acid fragment encoding the CD8 beta chain and the nucleic acid fragment encoding the CD8 alpha chain.

10. The nucleic acid according to any one of claims 1 to 9, wherein the operon is flanked by inverted repeat sequences which are mobilizable by a transposase, preferably by a Sleeping Beauty transposase such as SB100X.

11. The nucleic acid according to any one of claims 1 to 10, which is a minicircle, a plasmid, a doggybone DNA or an RNA, preferably a minicircle.

12. A cell comprising the nucleic acid according to any one of claims 1 to 11, preferably wherein the cell further expresses a Class I restricted alpha beta T cell receptor construct, wherein the cell is selected from the group comprising an immune cell, such as a CD4 T cell and / or a CD8 T cell, a gamma delta T cell, an NK cell, an NKT cell, a mucosa-associated invariant T (MAIT) cell, an innate lymphoid cell (ILC), a monocyte, a macrophage and a B cell.

13. The immune cell according to claim 12, which is a gamma delta T cell.

14. The immune cell according to claim 12, which is a CD4+ T helper cell.

15. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 11 or the cell according to any one of claims 12 to 14, and optionally a suitable buffer and / or excipient.

16. The pharmaceutical composition according to claim 15 for use in the treatment of a cancer or an infectious disease, preferably for adoptive T cell therapy in cancer, wherein, Optionally, the cell for use in adoptive T cell therapy is an allogeneic gamma delta T cell according to claim 13.

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