Inducible T cell receptor and its use

Abstract

The present invention relates to (nucleic acid encoded) inducible T cell receptors (iTCR), compositions and kits, vectors and host cells comprising such (nucleic acid encoded) inducible T cell receptors, their use in the preparation of inducible T cell receptors and host cells comprising such T cell receptors, methods for preparing such inducible T cell receptors and for dimerizing T cell receptors, and medical uses of such compounds or pharmaceutical compositions comprising these compounds, in particular for the treatment of cancer. The present invention relates to a combination comprising one or more nucleic acid molecules, wherein the one or more nucleic acid molecules comprise a nucleic acid sequence A encoding a TCR alpha chain connected to a dimerization domain and a nucleic acid sequence B encoding a TCR beta chain connected to a dimerization domain, as well as proteins encoded by such nucleic acid molecules and corresponding uses and methods.

Description

Field of the Invention

[0001] The present invention relates to (nucleic acid-encoded) inducible T cell receptors, compositions and kits, vectors and host cells comprising such (nucleic acid-encoded) inducible T cell receptors, their use in the preparation of inducible T cell receptors and host cells comprising such T cell receptors, methods for preparing such inducible T cell receptors and for dimerizing T cell receptors, and medical uses of such compounds or pharmaceutical compositions comprising these compounds, in particular for the treatment of cancer. In particular, the present invention relates to a combination comprising one or more nucleic acid molecules, the one or more nucleic acid molecules comprising a nucleic acid sequence A encoding a TCR alpha chain connected to a dimerization domain and a nucleic acid sequence B encoding a TCR beta chain connected to a dimerization domain, as well as proteins encoded by such nucleic acid molecules and corresponding uses and methods as defined herein and in the claims. Background Art

[0002] The development of effective T cell therapy is also accompanied by an increase in the risk of adverse events such as on-target and off-target side effects. As T cell therapy becomes more and more effective, acute toxicity such as cytokine release syndrome becomes more pronounced. In order to obtain safe adoptive T cell therapy, the ability to control genetically modified T cells is crucial. The development of methods for controlling genetically modified T cells has always been a research focus. Usually, the administration of T cell inhibitory drugs is incomplete and non-specific. Other safety switches such as suicide genes are used to engineer T cells, ultimately leading to the depletion of most of these cells. However, the resulting increase in basal cell apoptosis and the expansion of remaining T cells potentially limit its efficacy. Summary of the invention

[0003] The technical problem underlying the present invention is therefore to meet the above-mentioned objects. The above-mentioned technical problem has been solved by means and methods described herein, explained in the embodiments and defined in the claims.

[0004] The inventors of the present application have solved the above problems and developed the modification of recombinant T cell receptor (TCR), thereby producing controllable TCR protein expression. According to the inducible TCR of the present invention, constant α and / or β chain comprises mutation, resulting in the pairing loss of two chains. In addition, the inducible TCR of the present invention comprises a dimerization domain added in constant α and β chain downstream. The dimerization domain is characterized in that the conditional dimerization state of the addition depends on the dimerization agent. After adopting the corresponding dimerization agent to induce dimerization, the membrane expression of a given TCR is achieved.

[0005] Thus, the present invention provides modification of recombinant antigen-specific T cell receptors (TCR), wherein the modification causes control of protein membrane expression of antigen-specific recombinant TCR. On the other hand, the present invention provides nucleic acids encoding such TCR modifications, vectors comprising such nucleic acids, host cells comprising such vectors and nucleic acids, and their various uses and applications.

[0006] Specifically, as surprisingly found in the context of the present invention, point mutations in the C-terminal constant α and / or β chains of TCR lead to an obstruction or inhibition of the natural pairing of the two chains. As a result, the TCR cannot form a complex of correctly paired α and β chains together with the T cell receptor complex CD3. Therefore, no or only a very small number of TCRs are expressed on the surface of recombinant T cells, which results in the inability of T cells to recognize antigens and subsequently trigger function.

[0007] In addition, a dimerization domain is introduced downstream of the constant region of the α and β chains of the TCR to allow dimerization of the TCR after the addition of a dimerization agent, wherein the dimerization domain and the dimerization agent overcome the hindrance / inhibition of TCR pairing caused by point mutations. Thus, the overlaying principle is an induced dimerization, such as chemically induced dimerization (CID) of the TCR α and β chains, in which the chains do not pair in the absence of a dimerization agent.

[0008] Therefore, the present invention relates to an inducible T cell receptor (TCR) having dimerization domains downstream of constant α and β chains, respectively, wherein the constant α and β chains are induced to dimerize by a dimerization agent and are thereby expressed on the cell membrane surface.

[0009] In one aspect, the invention relates to a composition comprising one or more nucleic acid molecules comprising:

[0010] (a) a nucleic acid sequence A comprising (i) a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO: 2, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, wherein the inducible dimerization domain is downstream linked to the nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO: 2; and (b) a nucleic acid sequence B comprising (i) a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO: 2. NO:4 having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5% or 99% identical, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, similarly positioned as the dimerization domain of (a)(ii) linked to a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO:2, wherein the inducible dimerization domain is linked downstream of the nucleic acid sequence encoding the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98.5% or 99% identical to SEQ ID NO:4. NO:4 has a nucleic acid sequence having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical, and the dimerization domain corresponds to the dimerization domain of (a)(ii) linked to a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO:2, wherein the dimerization domain of (a)(i) corresponding to SEQ ID NO:2 is NO:2 has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity, comprising at least one, preferably at least two, amino acid substitutions, and / or compared to SEQ ID NO:4, the amino acid sequence of (b)(i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity with SEQ ID NO:4 comprises at least one, preferably at least two, more preferably at least three amino acid substitutions.

[0011] According to the present invention, the description of nucleic acid sequence A or B, wherein the nucleic acid sequence A or B respectively comprises a nucleic acid sequence encoding an inducible dimerization domain, and the inducible dimerization domain is connected downstream to the nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity with SEQ ID NO: 2 (encoding TCR constant α chain) or with SEQ ID NO: 4 (encoding TCR constant β chain allele 2), also means that the corresponding amino acid sequence (i.e., protein) encoded by the nucleic acid sequence A or B respectively comprises an inducible dimerization domain, and the inducible dimerization domain is connected downstream to the amino acid sequence (i.e., protein) encoded by the nucleic acid sequence, and the nucleic acid sequence encodes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity with SEQ ID NO: 2 or SEQ ID NO:4 has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity. In other words, not only the encoding nucleic acid sequence encoding the TCR constant α chain or β chain and the dimerization domain are connected, but also the encoded and expressed amino acid sequence and the polypeptide are connected (i.e., a protein comprising a TCR constant α chain connected to an inducible dimerization domain and / or a protein comprising a TCR constant β chain connected to an inducible dimerization domain, respectively). As understood in the context of the present invention, the nucleic acid sequence encoding an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identity to SEQ ID NO:2 or 4 encodes a TCR constant α or β chain, respectively. In the context of the present invention, the amino acid sequences (i.e., proteins) encoded by the nucleic acid sequences A and B (and therefore the nucleic acid molecules comprising said sequences) are preferably understood as encoding TCR α and β chains (including corresponding variable and constant regions) respectively comprising an inducible dimerization domain, and are therefore suitable for providing and preparing the inducible TCR described herein.

[0012] According to the present invention, nucleic acid sequence A preferably encodes a TCR alpha chain comprising a variable region and a constant region, and nucleic acid sequence B preferably encodes a TCR beta chain comprising a variable region and a constant region. As known in the art, TCR is mainly composed of an alpha chain and a beta chain, each chain comprising a variable region and a constant region, and the TCR further comprises an adjacent transmembrane region and a cytoplasmic region. The constant region is located downstream of the variable region of TCR and adjacent to the cell membrane, followed by a transmembrane region and a short cytoplasmic region. The constant regions of alpha and beta chains are usually paired by cysteine-based disulfide bonds in the extracellular domain (see, e.g., Glusman et al., Immunity (2001), 15: 337-349; Call et al., Cell (2002), 111: 967-979). The key point of the present invention is that the constant region of the α and / or β chain of the TCR contains one or more mutations that lead to the loss of pairing of the α and β chains (i.e., an amino acid sequence comprised by nucleic acid sequence A that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO: 2 encoding the corresponding TCR constant α chain, and an amino acid sequence comprised by nucleic acid sequence B that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99% identical to SEQ ID NO: 4 encoding the corresponding TCR constant β chain, wherein due to the presence of one or more mutations in the TCR constant α and / or β chain, the chain exhibits impaired pairing ability). Therefore, compared with SEQ ID NO:1 and / or 3, nucleic acid sequence A and / or B include nucleotide differences, and the nucleotide differences result in amino acid substitutions as described herein, preferably resulting in the loss of natural pairing of α and β chains of TCR. According to the present invention, the loss of this natural pairing can be overcome by the inducible (controlled) dimerization of the dimerization domain encoded by the sequence of the introduction of nucleic acid sequences A and B, wherein the dimerization domain dimerizes after adding a suitable dimerization agent as described herein. That is, unless induced by adding a suitable dimerization agent, the dimerization domain will not or at least will not dimerize to a substantial extent, and is therefore also referred to as an inducible dimerization domain. Therefore, the combination of nucleic acid molecules described in the text and provided can be provided or can be used to prepare an inducible TCR (iTCR), which can be induced (i.e., dimerization induction of each α and β chain) after adding a dimerization agent as described herein. As used herein, the term "combination (combination)" may also include or mean a "composition (composition)" in which one or more of the nucleic acid molecules are included or one or more of the nucleic acid molecules are combined together.

[0013] The nucleic acid sequence A of the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99%, preferably at least 97.5%, 98%, 98.5% or 99%, more preferably at least 98.5% or even 99% identical to SEQ ID NO: 2 (SEQ ID NO: 2 encodes the aa sequence of the human constant region of the TCR α chain). The nucleic acid sequence B of the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5% or 99%, preferably at least 97.5%, 98%, 98.5% or 99%, more preferably at least 98% or even 98.5% identity with SEQ ID NO: 4 (SEQ ID NO: 4 encodes the aa sequence of the human constant region of the TCR β chain). In the context of the present invention, the amino acid substitution compared to SEQ ID NO: 2 is preferably a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, the conservative amino acid substitution can be an acidic amino acid substitution for another acidic amino acid, an amino acid with a non-polar side chain substitution for another amino acid with a non-polar side chain, a basic amino acid substitution for another basic amino acid, an amino acid with a polar side chain substitution for another amino acid with a polar side chain, etc. The conservative substitution can be, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues involved. For example, a "conservative" substitution can mean a substitution listed in Table 1 below as "Exemplary Substitutions". As used herein, a "highly conservative" substitution refers to a substitution shown under the heading "Preferred Substitutions" in Table 1 below.

[0014] Table 1 Amino Acid Substitution

[0015] original Exemplary substitutions Preferred replacement Ala(A) val;leu;ile Val Arg(R lys;gln;asn lys Asn(N) gln;his;asp,lys;arg gln Asp(D) glu;asn glu Cys(C) ser;ala ser Gln(Q) asn;glu asn Glu(E) asp;gln asp Gly(G) ala ala His(H) asn;gln;lys;arg arg Ile(I) leu; val; met; ala; phe; leu Leu(L) norleucine;ile;val;met;ala; ile Lys(K) arg;gin;asn arg Met(M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr tyr Pro(P) ala ala Ser(S) thr thr Thr(T) ser ser Trp(W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser Phe Val(V) ile;leu;met;phe;ala; leu

[0016] In one embodiment of the invention, the amino acid sequence of (a)(i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identity to SEQ ID NO:2 comprises an amino acid substitution at position 44 and / or 47 compared to SEQ ID NO:2, and / or the amino acid sequence of (b)(i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identity to SEQ ID NO:4 comprises an amino acid substitution at position 4, 5, 37, 63, 77 and / or 79 compared to SEQ ID NO:4. In one embodiment of the invention, the amino acid substitutions at positions 44 and / or 47 compared to SEQ ID NO: 2 are conservative amino acid substitutions.

[0017] The amino acid sequence shown in SEQ ID NO: 2 may be encoded by the nucleotide sequence shown in SEQ ID NO: 1. Thus, the amino acid sequence as described herein (a)(i) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identity to SEQ ID NO: 2 and encoded by said nucleic acid sequence may be encoded by the nucleotide sequence according to SEQ ID NO: 1, in which the corresponding nucleotides are replaced so as to obtain the corresponding amino acid substitution compared to SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 may also be encoded by the nucleotide sequence according to SEQ ID NO: 1, in which one or more nucleotides are replaced, in which case such replacement is not translated into an amino acid replacement (silent mutation). Thus, a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identical to SEQ ID NO:2 may correspond to the nucleotide sequence of SEQ ID NO:1, in which the corresponding nucleotides are replaced so as to obtain the corresponding amino acid substitutions compared to SEQ ID NO:2, and the nucleotide sequence of SEQ ID NO:1 further comprises additional nucleotide substitutions that do not result in amino acid substitutions.

[0018] Similarly, the amino acid sequence shown in SEQ ID NO: 4 may be encoded by the nucleotide sequence shown in SEQ ID NO: 3. Thus, the amino acid sequence of (b)(i) as described herein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identity to SEQ ID NO: 4 and encoded by said nucleic acid sequence may be encoded by the nucleotide sequence according to SEQ ID NO: 3, in which the corresponding nucleotides are replaced so as to obtain the corresponding amino acid substitution compared to SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 may also be encoded by the nucleotide sequence according to SEQ ID NO: 3, in which one or more nucleotides are replaced, in which case such replacement is not translated into an amino acid replacement (silent mutation). Thus, a nucleic acid sequence encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identical to SEQ ID NO:4 may correspond to the nucleotide sequence of SEQ ID NO:3, in which the corresponding nucleotides are replaced so as to obtain the corresponding amino acid substitutions compared to SEQ ID NO:4, and the nucleotide sequence of SEQ ID NO:3 further comprises additional nucleotide substitutions that do not result in amino acid substitutions.

[0019] Table 1 shows the amino acid changes introduced in both the α and β constant regions of the α and β chains of the human TCR (see SEQ ID NOs: 2 and 4, respectively), wherein, as can be seen from Examples 1-6, the changes in the amino acid sequence compared to SEQ ID NO: 2 (resulting in the TCR constant α chain) and SEQ ID NO: 4 (resulting in the TCR constant β chain) result in impaired pairing of the α chain with the β chain, but still allow correct assembly of the α chain of the TCR with the CD3 ε and δ subunits, and correct assembly of the β chain of the TCR with the CD3 γ and ε subunits. Fig.11 and Fig.12 The sequence listing shown shows the substituted amino acid positions with reference to the IMGT numbering system. According to IMGT numbering, those skilled in the art will appreciate that position 84.1 is between positions 84 and 85 of the amino acid sequence of human TCR (Lefranc, M.-P., Immunology Today 18, 509 (1997)).

[0020] Table 1: Amino acid changes introduced in both the α and β constant regions of the α and β chains of the human TCR.

[0021]

[0022] Table 2 shows the amino acid changes introduced in the beta constant region of a human TCR (SEQ ID NO: 4), wherein, as can be seen from Examples 7 and 8, the changes in the amino acid sequence compared to SEQ ID NO: 4 (resulting in the TCR constant beta chain) result in impaired pairing of the alpha chain with the beta chain, but still allow the correct assembly of the alpha chain of the TCR with the CD3 epsilon and delta subunits, and the correct assembly of the beta chain of the TCR with the CD3 gamma and epsilon subunits. There are two alleles of the TCR beta chain constant region, referred to as C1 and C2. Reference Fig.12 The sequence table shown in Figure 1 shows the amino acid positions of the substitutions with reference to the IMGT numbering system. Table 2 below shows the differences between the two alleles of the β constant chain. Although SEQ ID NO: 4 represents the C2 constant chain, it shows that the amino acids at positions 4, 5 and 37 are K, N and Y (1.3, 1.4 and 29 using IMGT numbering are shown in the table below), Fig.12 The C1 constant chain is shown. Mutations in the constant chain at the positions listed in Table 2 result in reduced pairing of the α and β chains.

[0023] Table 2: Amino acid changes introduced in the beta constant region of human TCR.

[0024]

[0025]

[0026] In one embodiment of the invention, when preferably expressed in the cytoplasmic region after TCR expression and localization, the dimerization domain connected downstream to nucleic acid sequences A and B, respectively, is located at the C-terminus of the TCR. In this context, as known to those skilled in the art, after the formation of the complex by the α and β chains (together with the CD3 complex) (see, e.g., Call et al., supra), i.e. after dimerization of the dimerization domain encoded by the nucleic acid sequences A and B as defined and provided herein, the TCR is typically localized in the cell membrane.

[0027] In one embodiment of the invention, for the combinations (or compositions) described and provided herein, the nucleic acid sequence A and the nucleic acid sequence B may be contained by separate nucleic acid molecules or contained in one nucleic acid molecule. For example, nucleic acid sequences A and B may be located in the same nucleic acid molecule, such as in an expression cassette. In such expression cassettes, preferably nucleic acid sequences A and B may be under the same expression control, such as under the control of the same promoter or the same promoter type, so as to allow for a comparable amount of expression.

[0028] In one embodiment of the invention, the nucleic acid sequence of (a)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identical to SEQ ID NO:2, and compared to the amino acid sequence of SEQ ID NO:2, the amino acid sequence comprises at least one amino acid substitution selected from the group consisting of T44A and T47A; and / or the nucleic acid sequence of (b)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identical to SEQ ID NO:4, and compared to the amino acid sequence of SEQ ID NO:4, the amino acid sequence comprises at least one amino acid substitution selected from the group consisting of L63A, S77A and R79A. In another embodiment, the nucleic acid sequence of (b)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identical to SEQ ID NO:4, and the amino acid sequence comprises at least one amino acid substitution selected from K4V, N5P and Y37K compared to the amino acid sequence of SEQ ID NO:4.

[0029] In a preferred embodiment of the present invention, the nucleic acid sequence of (a)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identical to SEQ ID NO:2, and compared to the amino acid sequence of SEQ ID NO:2, the amino acid sequence comprises two amino acid substitutions, which are T44A and T47A; and the nucleic acid sequence of (b)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identical to SEQ ID NO:4, and compared to the amino acid sequence of SEQ ID NO:4, the amino acid sequence comprises three amino acid substitutions, which are L63A, S77A and R79A.

[0030] In another preferred embodiment, the nucleic acid sequence of (a)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identical to SEQ ID NO:2 and the nucleic acid sequence of (b)(i) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identical to SEQ ID NO:4, which amino acid sequence comprises three amino acid substitutions compared to the amino acid sequence of SEQ ID NO:4, and the three substitutions are K4V, N5P and Y37K.

[0031] In one embodiment of the present invention, the nucleic acid sequence A encodes the amino acid sequence shown in SEQ ID NO:5.

[0032] In one embodiment of the present invention, the nucleic acid sequence B encodes the amino acid sequence shown in SEQ ID NO:6.

[0033] In the context of the present invention, the inducible dimerization domain is a dimerization domain that dimerizes after adding a dimerization agent. In the amino acid sequence (i.e., protein) encoded by nucleic acid sequences A and B, respectively, the dimerization domain is connected to or located at the downstream (i.e., C-terminal) of the constant α or β chain (encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5% or 99% identity with SEQ ID NO:2 or SEQ ID NO:4) that shows impaired pairing ability. Preferably, the inducible dimerization domain is located in the cytoplasmic domain of iTCR. As known to those skilled in the art, TCR is first formed at the endoplasmic reticulum (ER), and after the α and β chains are paired, a complex is formed with CD3, and then positioned on the cell membrane / cell surface by the Golgi apparatus. In the context of the present invention, due to the presence of mutations as described and provided herein in the corresponding constant regions, the α and β chains of the iTCR are not paired, and therefore, they do not leave the ER after dimerization via an inducible dimerization domain (which in turn dimerizes after adding a suitable dimerization agent). In the context of the present invention, the inducible dimerization domain can be a homodimerization domain or a heterodimerization domain, preferably a homodimerization domain.In the context of the present invention, suitable examples of such dimerization domains are known in the art and include, in particular, estrogen receptors (such as ERT2; see Ballister, Publicly Accessible Penn Dissertations (2014), 1202: Inducible Protein Dimerization: New Tools and Applications to Understanding the Mitotic Checkpoint), FKBP (see Ballister, supra), FKBP / calcineurin A (CNA) (see Ho et al., Nature (1996), 382: 822-826); FKBP / CyP-Fas (see Belshaw et al., PNAS (1996), 93: 4304-4607), the FKBP / FRB domain of mTOR (see Ballister, Publicly Accessible Penn Dissertations (2014), 1202: Inducible Protein Dimerization: New Tools and Applications to Understanding the Mitotic Checkpoint), Checkpoint), GyrB (MA Farrar et al., Methods in Enzymology, Volume 327, 2000, pages 421-429), GAI / GID1 (Miyamato et al., Nature Chemical Biology (2012), 8:465-470), Snap-Tag / HaloTag (see Erhart et al., Chem&Biol (2013), 20:549-557) and eDHFR / HaloTag (Ballister et al., Nature Communications 5, Article number: 5475 (2014), doi: 10.1038 / ncomms6475).

[0034] The present invention relates to an expression cassette comprising a nucleic acid sequence A and a nucleic acid sequence B as described and provided herein, or to at least two expression cassettes, wherein at least one expression cassette comprises a nucleic acid sequence A as described and provided herein and at least one expression cassette comprises a nucleic acid sequence B as described and provided herein. In one embodiment of the invention, nucleic acid sequences A and B are under the control of the same element (such as a promoter and / or enhancer, an operator, a repressor and a transcription termination signal, etc.), and if contained by the same expression control, they can be under the common control of the same element. The promoter can be, for example, a constitutive promoter or an inducible promoter. Suitable promoters / enhancers for mammalian host cells are known in the art and include, for example, SV40, AdMLP, metallothionein promoters, 7.5K promoters, MIEP, EF1A, CMV, PGK, etc.

[0035] The present invention further relates to a vector comprising a nucleic acid sequence A and / or B as described herein and provided, or an expression cassette as described herein and provided. Suitable vectors depend on the corresponding host cell into which the vector is to be introduced (transformed, transduced), and are known in the art. The term "vector" as used herein encompasses but is not limited to plasmids, viral vectors (including retroviral vectors, slow viral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV); retroviral and slow viral vectors are preferred in the context of the present invention), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". Engineered vectors typically include an origin for autonomous replication in a host cell (if stable expression of a polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally comprise a promoter / enhancer and other control elements as described herein and known in the art, such as an operator, a repressor and a transcription termination signal, as well as a genetic marker, a reporter gene, a targeting sequence and / or a protein purification tag. As known to those skilled in the art, a large number of suitable vectors are available, and many are commercially available. Examples of suitable vectors are provided in, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012).

[0036] The carrier of the present invention can also be an expression vector. "Expression vector" or "expression construct" can be used for transcription of heterologous polynucleotide sequences, such as those encoding TCR chains (encoded by nucleic acid sequences A and B) described and provided in the context of the present invention in suitable host cells and the translation of their mRNA. In addition to replication origin, selection marker and restriction enzyme cleavage site, expression vectors generally include one or more regulatory sequences operably connected to the heterologous polynucleotide to be expressed. The term "regulatory sequence" refers to the nucleic acid sequence necessary for the operably connected coding sequence of the (heterologous) polynucleotide expressed in a specific host organism or host cell, and therefore includes transcription and translation regulatory sequences. Generally, the regulatory sequence required for expressing heterologous polynucleotide sequences in prokaryotes includes a promoter, an optional operator sequence and a ribosome binding site. In eukaryotes, promoters, polyadenylation signals, enhancers and optional splicing signals are generally required. In addition, specific initiation and secretion signals can also be introduced into the vector to allow the target polypeptide to be secreted into the culture medium. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, particularly on the same polynucleotide molecule, the nucleic acid is "operably connected". For example, when a promoter is capable of achieving expression of a coding sequence of a heterologous gene, the promoter is operably linked to the coding sequence. The promoter is usually placed upstream of the gene encoding the polypeptide of interest and regulates the expression of the gene. Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as CMV promoter / enhancer), simian virus 40 (SV40) (such as SV40 promoter / enhancer), adenovirus (such as, adenovirus major late promoter (AdMLP)) and polyoma virus. As previously described, the expression vector may also include an origin of replication and a selectable marker. The vector of the present invention may further comprise one or more selectable markers. Suitable selectable markers for eukaryotic host cells include, but are not limited to, herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyl transferase (hgprt) and adenine phosphoribosyl transferase (aprt) genes. Other genes include dhfr (methotrexate resistance), gpt (mycophenolic acid resistance), neo (G-418 resistance) and hygro (hygromycin resistance). Vector amplification can be used to increase expression levels. In general, the selection marker gene can be directly linked to the polynucleotide sequence to be expressed, or the selection marker gene can be introduced into the same host cell by co-transformation. In view of the above, the present invention further provides one or more of the nucleotide sequences described herein inserted into (i.e., contained by) a vector. In particular, the present invention provides a (replicable) vector comprising the nucleic acid sequences A and / or B described and provided herein.

[0037] According to the host cell intended for expression of the amino acid sequence (protein) encoded by nucleic acid sequence A and / or B, the technician will be able to easily select a suitable expression vector. Specific examples of suitable expression vectors are viral vectors, such as retroviral vectors, such as MP71 vectors or retroviral SIN vectors; and lentiviral vectors or lentiviral SIN vectors. Viral vectors can, for example, infect lymphocytes, which envision that the lymphocytes subsequently express the amino acid sequence (protein) encoded by nucleic acid sequence A and / or B. Another example of a suitable expression vector is the Sleeping Beauty (SB) transposon transposase DNA plasmid system, SB DNA plasmid. The nucleic acids and / or in particular expression constructs of the present invention can also be transferred to cells by transient RNA transfection. The viral vectors currently used for natural TCR expression generally connect the TCR-α and TCR-β chain genes in one vector to an internal ribosome entry site (IRES) sequence or a 2A peptide sequence derived from tsechovirus, thereby expressing single messenger RNA (mRNA) molecules under the control of a viral promoter in the transduced cells. However, the skilled person will be able to easily select and use other known systems to introduce the nucleotide sequences A and / or B of the present invention into a suitable expression system such as CRISPR / Cas or Sleeping Beauty (SB) transposon.

[0038] The present invention also relates to amino acid sequences and proteins (used interchangeably herein) encoded by nucleic acid sequences described and provided herein, in particular by nucleic acid sequences A and / or B. A combination of amino acid sequences (proteins) encoded by nucleic acids comprising nucleic acid sequences A and B is provided or can be used to prepare iTCRs as described and provided herein. Therefore, the present invention also relates to an iTCR prepared by using an amino acid sequence (protein) encoded by a nucleic acid comprising nucleic acid sequences A and B or by using a nucleic acid comprising nucleic acid sequences A and B. As described herein, the iTCR is a TCR that exhibits impaired pairing of the α and β chains, and that comprises a C-terminal inducible dimerization domain (as described herein) on both the α and β chains, but the inducible dimerization domain dimerizes after adding a suitable dimerization agent. If at least 2-, 3-, 4- or 5-fold less TCR can be detected on the cell membrane of a given TCR-producing cell compared to the amount of native human TCR (having TCR constant α and β chains as shown in SEQ ID NO: 2 and 4, respectively) on the cell membrane, then the pairing ability of the TCR can be considered "impaired" in the context of the present invention. Suitable measurement methods are known in the art and include flow cytometry as exemplified herein.

[0039] The present invention also relates to amino acid sequences and proteins encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identity to SEQ ID NO: 2 and encoding a TCR constant alpha chain that cannot be paired with the natural human TCR constant beta chain according to SEQ ID NO: 4. The present invention also relates to amino acid sequences and proteins encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98% or 98.5%) identity to SEQ ID NO: 4 and encoding a TCR constant beta chain that cannot be paired with the natural human TCR constant alpha chain according to SEQ ID NO: 2.

[0040] As described herein, the inducible dimerization domain dimerizes after adding an appropriate dimerization agent and thus binds the two chains together, and the inducible dimerization domain is connected to an amino acid sequence encoded by nucleic acid A or B (respectively encoding a TCR constant α or β chain with a dimerization domain connected downstream), and the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identical to SEQ ID NO: 2 or 4 (respectively encoding a TCR constant α or β chain). As known to those skilled in the art, such dimerization agents depend on the corresponding dimerization domains of the α and β chains of the iTCR to be prepared using the nucleic acid molecules of the combinations or compositions provided herein. For example, (depending on the corresponding dimerization domain) suitable dimerization agents include tamoxifen (such as 4-hydroxytamoxifen), endoxifen, AP21967, 4-(1-[4-(dimethylaminoethoxy)phenyl]-2-phenyl-1-butenyl)phenol, and 23,27-epoxy-3H-pyrido[2,1-c][1,4]oxazacyclotriacontane, FK1012, FK506, FKCsA, rapamycin, coumermycin, gibberellin, HaXS and TMP-HTag. In one embodiment of the present invention, in particular for estrogen receptors (such as ERT2) encoded by nucleic acid sequences A and B as dimerization domains, the dimerization agent is tamoxifen or endoxifen, preferably endoxifen. Examples of suitable dimerization domain / dimerization agent pairs are shown in Table 3.

[0041] Table 3: Examples of suitable dimerization domain / dimerization agent pairs

[0042] Dimerization system Dimerization Reagents Estrogen receptor Estrogen receptor Tamoxifen FKB FKB FK1012 FKB Calcineurin A (CNA) FK506 FKB CyP-Fas FqV FKB The FRB domain of mTOR Rapamycin G JZ G JZ Coumarin GAI GID1 Gibberellins Snap-tag HaloTag HkDJ eDHFR HaloTag TMP-HTag

[0043] The present invention also relates to a kit, the kit comprising a combination or composition comprising one or more nucleic acid molecules containing nucleic acid sequences A and / or B as described and provided herein, an expression cassette as described and provided herein, a host cell as described and provided herein, and / or a vector as described and provided herein and a dimerization agent, the dimerization agent corresponds to a dimerization domain linked to an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5% or 99% (preferably at least 98.5% or 99%) identity with SEQ ID NO: 2 or 4 (SEQ ID NO: 2 corresponds to nucleic acid sequence A, and SEQ ID NO: 4 corresponds to nucleic acid sequence B), the dimerization domain of the dimerization agent being able to induce dimerization of the dimerization domain. The ability to induce dimerization of the dimerization domain can be measured by methods known in the art and exemplified herein. If, after the addition of an agent, an increase in the expression and localization of an iTCR prepared using the methods, combinations, compositions, compounds, vectors, host cells or kits of the invention can be measured in the cell membrane, then the agent is capable of dimerizing the inducible dimerization domain in the context of the present invention; preferably, there are at least 2-fold, 3-fold, 4-fold or 5-fold (preferably at least 5-fold) more iTCRs in the cell membrane of a given host cell compared to without the addition of the dimerization agent. Suitable methods for measuring TCRs on the surface of cells are known in the art and include flow cytometry (see, e.g., Figure 2 ). Table 3 exemplifies suitable dimerization domains and dimerization agent pairs.

[0044] The present invention also relates to a host cell comprising a nucleic acid molecule comprising nucleic acid sequence A and nucleic acid sequence B, an expression cassette as described and provided herein, and / or a vector as described and provided herein. Suitable host cells are known in the art and include, in particular, prokaryotic and eukaryotic cells, preferably eukaryotic cells, such as mammalian cells. Host cells may include, for example, production host cells or effector host cells.

[0045] According to the present invention, a variety of host cells can be used. As used herein, the term "host cell" encompasses such cells: such cells can be or have been recipients (recipients) of nucleic acid molecules or vectors as described herein and / or express (and optionally secrete) the amino acid sequence (i.e., protein) of the present invention (such as iTCR). Unless otherwise expressly stated, the terms "cell" and "cell culture" are used interchangeably. The term "host cell" also includes "host cell line". In general, the term "host cell" includes prokaryotic or eukaryotic cells, and also includes but is not limited to bacteria, yeast cells, fungal cells, plant cells and animal cells, such as insect cells and mammalian cells, such as mice, rats, macaques or human cells. Based on the above, the present invention thus provides a host cell comprising a polynucleotide or a vector, such as an expression vector comprising a nucleotide sequence encoding an amino acid sequence as described herein (such as iTCR). The polynucleotides and / or vectors of the present invention can be introduced into a host cell using conventional methods known in the art (such as by transfection, transformation, etc.).

[0046] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening transient holes or "holes" in the cell membrane to allow the uptake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0047] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, also into non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic change of bacteria or non-animal eukaryotic cells, which is produced by direct uptake from its surroundings through the cell membrane and subsequent incorporation of exogenous genetic materials (nucleic acid molecules). Transformation can be achieved by artificial means. In order for transformation to occur, cells or bacteria must be in a state of competence, which can be produced as a time-limited response to environmental conditions such as starvation and cell density. For prokaryotic transformation, techniques may include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection and electroporation. For plant transformation techniques include Agrobacterium-mediated transfer (such as by A. tumefaciens), rapidly propelled tungsten or gold microprojectiles, electroporation, microinjection and polyethylene glycol-mediated uptake. Based on the above, the present invention further provides a host cell comprising at least one polynucleotide sequence and / or vector as described herein.

[0048] For the expression of the amino acid sequence (i.e., protein) of the present invention, the host cell of expression and / or modification and processing of gene product (i.e., RNA and / or protein) of the inserted polynucleotide sequence can be selected as needed. Such modification (e.g., glycosylation) and processing (e.g., shearing) of gene product may be important for the function of the TCR. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of gene products. Suitable cell lines or host systems can be selected to ensure that the product is correctly modified and processed. For this reason, eukaryotic host cells with primary transcripts, glycosylation, and phosphorylation cellular mechanisms for correctly processing gene products can be used. It is contemplated herein to provide (a) host cells for expressing and obtaining proteins of the present invention (i.e., iTCR), particularly soluble forms ("production host cells") and (b) host cells expressing TCR of the present invention and having effector functions ("effect host cells"). Such "effect host cells" are particularly suitable for therapeutic applications, and it is contemplated to be applied to subjects in need thereof. Preferred "effector host cells" include lymphocytes, such as cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, γ / δ-T cells.

[0049] The "production host cell" used to express the protein of the present invention is preferably capable of expressing a large amount of recombinant protein. Exemplary mammalian host cells that can be used as "production host cells" include Chinese hamster ovary (CHO cells), including CHO cells lacking DHFR (such as DG44 and DUXBI 1), NSO, COS (derivatives of CVI with SV40 T antigen), HEK293 (human kidney) and SP2 (mouse myeloma) cells. Other exemplary host cell lines include, but are not limited to, HELA (human cervical cancer), CVI (monkey kidney line), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells) and RAJI (human lymphocytes). Host cell lines are generally available from commercial services, American Tissue Culture Collection (ATCC) or self-published literature. Non-mammalian cells (such as bacteria, yeast, insect or plant cells) are also readily available and can also be used as "production host cells" as described above. Exemplary bacterial host cells include Enterobacteriaceae, such as Escherichia coli, Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenza. Other host cells include yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris. Insect cells include, but are not limited to, Spodoptera frugiperda cells. Based on the foregoing, conceivable expression systems (i.e., host cells comprising expression vectors as described above) include microorganisms transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors, such as bacteria (e.g., Escherichia coli, Bacillus subtilis); yeasts (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid).Generally, mammalian expression systems carrying recombinant expression constructs are preferred, and the recombinant expression constructs include promoters derived from mammalian cell genomes (such as metallothionein promoters) or promoters derived from mammalian viruses (such as adenovirus late promoters; vaccinia virus 7.5K promoters, cytomegalovirus (CMV) major immediate early promoter (MIEP) promoters). Suitable mammalian host cells can be selected from known cell lines (such as COS, CHO, BLK, 293, 3T3 cells), but it is also conceivable to use lymphocytes, such as cytotoxic T lymphocytes (CTLs), CD8+T cells, CD4+T cells, natural killer (NK) cells, natural killer T (NKT) cells, γ / δ-T cells.

[0050] According to the foregoing, the present invention also provides a method for producing and obtaining a protein as described herein (such as TCR), the method comprising the following steps: (i) incubating a host cell (i.e., producing a host cell) under conditions that cause expression of the protein and (ii) purifying the protein. The host cell carrying the expression vector is grown under conditions suitable for producing the protein provided herein, in particular the constant α chain and / or β chain encoded by nucleic acid sequences A and B, respectively, as described herein, and analyzing α and / or β chain protein synthesis. For the expression of a double-chain TCR, the vectors encoding the α chain and the β chain can be co-expressed in the host cell to express the entire molecule.

[0051] Once the protein of the invention (i.e., iTCR) is expressed, it can be purified by any purification method known in the art, for example, by chromatography (e.g., ion exchange chromatography (e.g., hydroxyapatite chromatography), affinity chromatography (particularly protein A, protein G, or lectin affinity chromatography), sizing column chromatography), centrifugation, differential solubility, hydrophobic interaction chromatography, or by any other standard technique for protein purification. Based on the individual characteristics of the protein to be recovered, the skilled person will be able to readily select an appropriate purification method.

[0052] As previously mentioned, the present invention also provides "effector host cells" comprising a nucleotide sequence, vector or protein of the present invention. The effector host cell is modified using conventional methods to comprise a nucleic acid sequence encoding a protein of the present invention, and it is envisioned that the effector host cell expresses the protein described herein (particularly on the cell surface). For the purposes of the present invention, "modified host cells expressing proteins of the present invention" generally refer to (effect or production) host cells that are treated or altered to express proteins according to the present invention, for example, by RNA transfection as described in the accompanying examples. Other modification or transfection or transduction methods are also envisioned, such as those described elsewhere herein. Therefore, the term "modified host cell" includes "transfected", "transduced" and "genetically engineered" host cells, preferably the host cells express the protein of the present invention. Preferably, such "(modified) effector host cells" (particularly "(modified) effector lymphocytes") are capable of mediating effector functions by intracellular signal transduction after the protein binds to its specific antigen target. Such effector functions include, for example, the release of perforins (which create holes in the target cell membrane), granzymes (which are proteases that act intracellularly to induce apoptosis), the expression of Fas ligands (which activate apoptosis in target cells carrying Fas), and the release of cytokines, preferably Th1 / Tc1 cytokines such as IFN-γ, IL-2, and TNF-α. Therefore, it is envisioned that effector host cells engineered to express a protein of the invention (which is able to recognize and bind to its antigenic target in the subject to be treated) to achieve the above effector functions, thereby killing target (e.g., cancer) cells. Cell lysis of target cells can be evaluated using, for example, a CTL fluorescent killing assay (CTL, USA), which detects the disappearance of fluorescently labeled target cells during co-culture with transfected recipient T cells.

[0053] In view of the above, the effector host cell preferably expresses a functional iTCR, which generally comprises the iTCR α and β chains described herein, and the signal transduction subunits CD3γ, δ, ε, ζ (CD3 complex). In addition, the expression of co-receptors CD4 or CD8 may also be required. In general, lymphocytes carry the required genes (such as Lck, FYN, CD45 and / or Zap70) involved in antigen binding, receptor activation and downstream signal transduction, and T cells are particularly suitable as effector host cells. However, it is also contemplated herein that such an effector host cell expresses the TCR of the present invention as a "binding domain" without CD3 signal transduction subunits and / or the above-mentioned downstream signal transduction molecules (i.e., it is capable of recognizing the antigen targets described herein, but does not realize the functions mediated by CD3 and / or the above-mentioned downstream signal transduction molecules). It is also contemplated that such effector cells can recognize the antigen targets described herein, and optionally can realize other functions unrelated to the signal transduction of CD3 signal transduction and / or the above-mentioned downstream signal transduction molecules. Examples include NK or NKT cells expressing the protein of the present invention and capable of releasing cytotoxic granules such as when recognizing their antigenic targets. Therefore, according to the present invention, it is believed that cytotoxic T lymphocytes (CTL), CD8+T cells, CD4+T cells, natural killer (NK) cells, natural killer T (NKT) cells, γ / δ-T cells are available lymphocyte effector host cells. This lymphocyte expressing the recombinant iTCR of the present invention is also referred to as "modified effector lymphocytes" herein. However, the technician will easily recognize that any component of the protein signaling pathway that leads to the desired effector function can usually be introduced into a suitable host cell by recombinant genetic engineering methods known in the art. Effector host cells, especially lymphocytes (such as T cells), can be autologous host cells, which are obtained from the subject to be treated and transformed or transduced to express the protein of the present invention. Generally, the recombinant expression of proteins will be completed by using viral vectors as described in the attached examples. The technology for obtaining and separating cells from patients is known in the art. As mentioned above, it is particularly envisioned that the effector host cells provided herein are used for therapeutic applications. In order to improve the therapeutic efficacy, it may be necessary to further genetically modify the host cells. For example, when using autologous CD8+T cells as "effector host cells", additional suitable modifications include downregulation of endogenous TCR, CTLA-4 and / or PD-1 expression; and / or amplification of costimulatory molecules (such as CD28, CD134, CD137). Means and methods for achieving the above genetic modifications have been described in the art.

[0054] Methods for targeted genome engineering of host cells are known in the art and include, in addition to gene knockout using siRNA, the use of so-called "programmable nucleases", such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-cas (CRISPR-associated) systems, in particular as reviewed in Kim & Kim Nature Reviews Genetics 15, 321–334 (2014). For example, programmable nucleases (such as TALENs) can be used to cleave DNA regions encoding "unwanted" proteins (such as PD-1, CTLA-4 or endogenous TCRs) and thereby reduce their expression. When T cells are used as (effector) host cells, downregulation of endogenous TCRs has the advantage of reducing the undesirable "mispairing" of endogenous and exogenous TCR α / β chains.

[0055] The present invention also relates to the use of the dimerization agent described herein for dimerizing the protein encoded by the nucleic acid sequence A described and provided herein and the protein encoded by the nucleic acid sequence B. In one embodiment of the present invention, the dimerization agent to be used for dimerizing the protein encoded by the nucleic acid sequence A described and provided herein and the protein encoded by the nucleic acid sequence B can be Endoxifen.

[0056] The present invention further relates to a method for dimerizing a protein encoded by nucleic acid sequence A described and provided herein and a protein encoded by nucleic acid sequence B, the method comprising the step of adding a dimerization agent described herein (such as tamoxifen or endoxifen, preferably endoxifen) to the protein.

[0057] The present invention further relates to a method for preparing an inducible T cell receptor (iTCR), the method comprising the step of introducing the nucleic acid sequence A and nucleic acid sequence B described and provided herein, the expression cassette described and provided herein, or the vector described and provided herein in vitro into a host cell under conditions that allow expression of the nucleic acid sequence A and nucleic acid sequence B. Suitable transformation / transduction methods and expression methods are known in the art and are also described herein.

[0058] The present invention further relates to the use of a combination or composition described and provided herein, an expression cassette described and provided herein, or a vector described and provided herein for producing modified T cells, such as T lymphocytes.

[0059] The present invention further relates to a combination or composition described and provided herein, an expression cassette described and provided herein, a vector described and provided herein, a host cell described and provided herein, or a protein described and provided herein, for use in T cell therapy.

[0060] The invention further relates to a combination or composition as described and provided herein, an expression cassette as described and provided herein, a vector as described and provided herein, a host cell as described and provided herein, or a protein as described and provided herein, for use in the treatment of cancer. In the present context, treatable cancers include all types of cancer, in particular solid cancers (e.g., non-small cell lung cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, skin cancer, prostate cancer, brain or nervous system cancer, head and neck cancer, testicular cancer, lung cancer, liver cancer, kidney cancer, bladder cancer, gastrointestinal cancer, bone cancer, endocrine system cancer, lymphatic system cancer, fibrosarcoma, neuroectodermal tumors, mesothelioma, epidermoid cancer or Kaposi's sarcoma) and blood cancers (leukemias).

[0061] The present invention further relates to a pharmaceutical composition comprising a combination composition described and provided herein, an expression cassette composition described and provided herein, a vector composition described and provided herein, a host cell composition described and provided herein, or a protein composition described and provided herein.

[0062] The term "pharmaceutical composition" refers in particular to a composition suitable for administration to humans. However, the term also generally covers compositions suitable for administration to non-human animals. The pharmaceutical composition and its components (i.e., active agents and optional excipients) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable composition of the present invention may be, for example, sterile. Specifically, the term "pharmaceutically acceptable" may mean approved by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans. The aforementioned active agent (e.g., host cell or iTCR) is preferably present in the pharmaceutical composition in a therapeutically effective amount. "Therapeutically effective amount" refers to the amount of the active agent that induces the desired therapeutic effect. The pharmaceutical composition may be prepared by standard procedures such as EDTA in cell cultures or experimental animals. 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The dose that is lethal to 50% of the population determines the therapeutic efficacy and toxicity. The dose ratio between therapeutic and toxic effects is the therapeutic index and can be expressed as ED 50 / LD 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

[0063] The exact dosage of nucleic acids, proteins, vectors or host cells can be determined by those skilled in the art using known techniques. A suitable dosage provides a sufficient amount of the active agent of the present invention and is preferably therapeutically effective, i.e., causes the desired therapeutic effect. As is known in the art, for therapeutic purposes (such as maintenance of remission of the disease versus acute onset), the route, time and frequency of administration, the time and frequency of administration of the formulation, age, body weight, general health, sex, diet, severity of the disease state, drug combination, reaction sensitivity and tolerance / response to treatment may be necessary. Suitable dosage ranges can be determined using data obtained from cell culture assays and animal studies, and may include ED 50 . Typically, depending on the route of administration, the dosage can vary between 0.1 and 100,000 micrograms, until the total dose is about 2 g. The range of exemplary dosages of the present active agent is about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 1 mg / kg or about 0.1 mg / kg to about 1 mg / kg. Guidance on specific doses and delivery methods is provided in the literature. It should be recognized that treatment may require a single administration of a therapeutically effective dose or multiple administrations of the present active agent of the present invention. For example, depending on the formulation, half-life and clearance rate of a particular composition, some pharmaceutical compositions may be administered every 3 to 4 days, weekly, or once every two weeks, or once within a month. The pharmaceutical composition may optionally include one or more excipients and / or additional active agents.

[0064] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is within the knowledge of those skilled in the art to select suitable excipients to prepare the pharmaceutical composition desired by the present invention. Exemplary carriers used in the pharmaceutical composition of the present invention include saline, buffered saline, glucose and water. Usually, the selection of suitable excipients depends especially on the desired preparation of the active agent used, the disease to be treated and the pharmaceutical composition.

[0065] The present invention further provides a pharmaceutical composition comprising one or more of the above-mentioned active agents of the present invention (e.g., host cells or iTCR constructs) and one or more other active agents suitable for treating and / or preventing the disease to be treated. Preferred examples of active ingredients suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. The pharmaceutical composition according to the present invention may be suitable for administration by a variety of routes. Typically, administration is completed parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0066] In particular, depending on the active agent used, the pharmaceutical composition of the present invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The process known to the present invention for producing drugs is shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions comprising, for example, host cells as described herein are generally provided in liquid form, and preferably include a pharmaceutically acceptable buffer. After preparing the pharmaceutical compositions of the present invention, they can be placed in suitable containers and labeled for treating a specified condition. Such labels, for example, include dosage, frequency of administration and method of administration.

[0067] In view of the above, the present invention thus provides proteins (such as iTCR), nucleic acids, vectors and / or host cells described herein for use as medicines. The term "treatment" in all grammatical forms includes therapeutic or prophylactic treatments in subjects in need thereof. "Therapeutic or prophylactic treatment" includes prophylactic treatments intended to completely prevent clinical and / or pathological manifestations or therapeutic treatments intended to improve or alleviate clinical and / or pathological manifestations. Therefore, the term "treatment" also includes improving or preventing a disease.

[0068] The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein and refer to any subject in need of treatment, particularly a mammalian subject. Generally speaking, mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc. However, it is readily understood that the proteins (such as iTCRs), nucleic acids, vectors, host cells, and pharmaceutical compositions provided herein are specifically contemplated for use in treating human subjects.

[0069] For treatment, the protein (e.g., iTCR), nucleic acid, vector (e.g., viral vector) or host cell of the invention can be directly administered to a subject in need thereof. The method may include the following steps: (a) providing one or more of the (i) protein (e.g., iTCR), (ii) nucleic acid, (iii) vector, (iv) host cell and / or (v) pharmaceutical composition of the invention; and (b) administering one or more of (i)-(v) to a subject in need thereof. Optionally, the method may also include another step of cancer treatment, such as radiation or administration of one or more anticancer agents.

[0070] The treatment according to the present invention may also include the following steps: (a) providing a sample of a subject, the sample comprising lymphocytes; (b) providing one or more of the proteins (such as iTCR), nucleic acids, vectors, host cells and / or pharmaceutical compositions of the present invention; (c) introducing one or more of (i) to (v) of step (b) into the lymphocytes of step (a), and thereby obtaining modified lymphocytes, (d) administering the modified lymphocytes of step (c) to a subject or patient in need thereof. It is particularly envisaged that the lymphocytes provided in step (a) are "effector host cells" as described above, and are advantageously selected from T cells, NK cells and / or NKT cells, in particular CD8 + T cells; and the lymphocytes can be obtained from a sample (particularly a blood sample) of a subject in the aforementioned step (a') by conventional methods known in the art. However, it is also conceivable to use other lymphocytes that are preferably capable of expressing the protein of the present invention (such as iTCR) and exerting the desired biological effector function as described herein. In addition, the lymphocytes are usually selected to be compatible with the subject's immune system, i.e., preferably the lymphocytes do not induce an immunogenic response. For example, it is conceivable to use "universal recipient cells", i.e., universally compatible lymphocytes that exert the desired biological effector function, which can be grown and amplified in vitro. Therefore, the use of such cells will avoid (obviate) the need to obtain and provide the subject's own lymphocytes in step (a). The ex vivo introduction of step (c) can be implemented by introducing the nucleic acid or vector described herein into lymphocytes via electroporation or by infecting lymphocytes with a viral vector, the viral vector being, for example, a lentiviral or retroviral vector as described above in the context of effector host cells. Other conceivable methods include the use of a transfection agent (such as liposomes) or transient RNA transfection. The transfer of antigen-specific TCR genes into (primary) T cells via, for example, (retro)viral vectors or transient RNA transfection represents a promising tool for generating tumor-associated antigen-specific T cells, which can then be reintroduced into donors where they specifically target and destroy tumor cells expressing the antigen.

[0071] In view of the above, therefore, another aspect of the invention is the use of proteins (such as iTCR), nucleic acid sequences, vectors and / or host cells described elsewhere herein for producing modified lymphocytes. Means and methods for introducing nucleic acids and vectors into lymphocytes are known in the art and described above.

[0072] It should be specifically noted that as used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or substitute the methods described herein.

[0073] Unless otherwise indicated, the term "at least" in front of a series of elements or a minimum number of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine using no more than routine experiments, many equivalents to the specific embodiments of the invention described herein. It is expected that such equivalents are encompassed by the present invention. In addition, the term "at least" includes the exact amount and upper limit, for example, "at least 5" also includes the meaning of "just 5", or "at least 3" includes the meaning of "just 3".

[0074] The term "and / or" wherever used herein includes the meanings of "and," "or," and "all or any other combination of the elements connected by the term."

[0075] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, it also includes specific numbers, such as "about 20" includes 20.

[0076] The terms "less than," "at least," or "greater than" include specific numbers. For example, less than 20 means less than or equal to 20. Likewise, more than or greater than means more than or equal to, or greater than or equal to, respectively.

[0077] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising" will be understood to mean including the stated integers or steps, or sets of integers or steps, but not excluding any other integers or steps, or sets of integers or steps. When used herein, the term "comprising" can be replaced by the terms "containing" or "including", or sometimes when used herein, the term "having". In addition, "comprising" and variations thereof also include the meaning of "consisting of".

[0078] As used herein, "consisting of" excludes any element, step or ingredient not specifically specified in the claimed element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0079] In each instance herein, any one of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by any one of the other two terms.

[0080] It should be understood that the present invention is not limited to the specific methodology, protocols, materials, reagents and substances, etc. described herein, as these can vary. The terminology used herein is only used for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0081] All publications and patents cited throughout this specification (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether preceding or following, are incorporated herein by reference in their entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. In the event that material incorporated by reference is inconsistent or inconsistent with this specification, this specification shall supersede any such material.

[0082] As used herein, the terms "polynucleotide" or "nucleic acid" include sequences of polyribonucleotides and polydeoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form, linear or circular, or mixtures thereof (including hybrid molecules). Therefore, nucleic acids according to the present invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations thereof or derivatives thereof (such as PNA). The terms "nucleic acid sequence" or "(poly)nucleotide sequence" are used interchangeably herein. Similarly, as will be apparent to those skilled in the art, the terms "amino acid sequence", "polypeptide" or protein are used interchangeably herein.

[0083] The polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The polynucleotides of the invention may also contain one or more modified bases, such as, for example, tritylated bases and unusual bases (e.g., inosine). Other modifications are also contemplated, including chemical, enzymatic, or metabolic modifications, as long as the binding molecules of the invention can be expressed from the polynucleotides. The polynucleotides may be provided in an isolated form as defined elsewhere herein. The polynucleotides may include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

[0084] The present invention further relates to a method for screening or testing iTCR function, the method comprising the following steps: using a vector (such as a lentiviral vector, such as pCDH) to introduce a reporter gene (such as a fluorescent protein, such as GFP, YFP, CFP, eGFP, ieGFP; preferably ieGFP) into a host cell (such as TCR - Host cells; preferably T lymphocytes, such as Jurkat-76), further using a vector (such as a lentiviral vector, such as pCDH; preferably the same vector as the reporter gene) to introduce one or more nucleic acid molecules comprising nucleic acid sequences A and B described herein and provided (preferably on an expression cassette; "iTCR construct") into the same host cell, using an antigen peptide recognized by (i) TCR and a dimerization agent described herein (such as tamoxifen or endoxifen for estrogen receptors (such as ETR2) as dimerization domains) to incubate the host cells, and compare the signal transduction of the reporter gene with the signal transduction of the reporter gene of a control cell, in which the same vector construct has been introduced except for the human natural TCR gene expression cassette (i.e., TCR constant α and β chains according to SEQ ID NOs: 2 and 4, respectively, without a dimerization domain connected downstream of the constant α and β chains). The expression of the same vector construct can be performed by methods known in the art, such as by flow cytometry as exemplified herein (see e.g. Figure 4 and Example 3) measuring (i) the introduction of TCR into the cell membrane. Comparable signaling of host cells treated with iTCR constructs and those treated with native TCRs indicates successful iTCR function according to the present invention. "Comparable" in this context can mean, for example, that the signaling intensity of host cells with iTCR constructs is at least 60%, 70%, 80% or 90% of the signaling intensity of host cells with native TCR constructs, preferably at least 93%, 95%, 96%, 98%, 98.5% or 99% of the signaling intensity of host cells with native TCR constructs. Sequence Listing <110> Gene Medical Immunotherapy, LLC <120> Inducible T cell receptor and its uses <130> LC20310006P <150> EP 18161766.3 <151> 2018-03-14 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 420 <212> DNA <213> Homo sapiens <400> 1 atccagaatc cggaccccgc cgtgtaccag ctgagagaca gcaagagcag cgacaagagt 60 gtgtgcctgt tcaccgactt cgactcccag accaacgtgt cccagagcaa ggacagcgac 120 gtgtacatca ccgacaagac cgtgctggac atgcggagca tggacttcaa gagcaacagc 180 gccgtggcct ggtccaacaa gagcgatttc gcctgcgcca acgccttcaa caacagcatt 240 atccccgagg acacattctt cccaagcccc gagagcagct gcgacgtgaa gctggtggaa 300 aagagcttcg agacagacac caacctgaat ttccagaacc tgagcgtgat cggcttcaga 360 atcctgctgc tgaaggtggc cggcttcaac ctgctgatga ccctgcggct gtggtccagc 420 <210> 2 <211> 140 <212> PRT <213> Homo sapiens <400> 2 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 3 <211> 537 <212> DNA <213> Homo sapiens <400> 3 gaagatctga agaacgtgtt ccccccagag gtggccgtgt tcgagcctag cgaggccgag 60 atcagccaca cccagaaagc caccctcgtg tgcctggcca ccggcttcta tcccgaccac 120 gtggaactgt cttggtgggt caacggcaaa gaggtgcaca gcggcgtgtc caccgatccc 180 cagcctctga aagaacagcc cgccctgaac gacagccggt actgcctgag cagccggctg 240 agagtgtccg ccaccttctg gcagaacccc cggaaccact tcagatgcca ggtgcagttc 300 tacggcctga gcgagaacga cgagtggacc caggacagag ccaagcccgt gacccagatc 360 gtgtctgccg aagcctgggg cagagccgat tgcggcttta ccagcgagag ctaccagcag 420 ggcgtgctga gcgccaccat cctgtacgag atcctgctgg gcaaggccac cctgtacgcc 480 gtgctggtgt ctgccctggt gctgatggct atggtcaagc ggaaggacag ccggggc 537 <210> 4 <211> 179 <212> PRT <213> Homo sapiens <400> 4 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro 1 5 10 15 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu 65 70 75 80 Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105 110 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 115 120 125 Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser 130 135 140 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 145 150 155 160 Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 165 170 175 Ser Arg Gly <210> 5 <211> 140 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of nucleic acid sequence A <400> 5 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Ala Asp Lys Ala Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 6 <211> 179 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of nucleic acid sequence B <400> 6 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro 1 5 10 15 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Ala Lys 50 55 60 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ala Ser Ala Leu 65 70 75 80 Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105 110 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 115 120 125 Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser 130 135 140 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 145 150 155 160 Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 165 170 175 Ser Arg Gly <210> 7 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> ERT2 <400> 7 atgagagccg ccaatctgtg gcccagcccc ctgatgatca agcggagcaa gaagaactcc 60 ctggccctga gcctgaccgc cgaccagatg gtgtcagctc tgctggatgc cgagccccct 120 atcctgtaca gcgagtacga ccccacccgg ccttttagcg aggccagcat gatgggcctg 180 ctgaccaatc tggccgaccg cgagctggtg cacatgatca actgggccaa gcgggtgccc 240 ggcttcgtgg atctgacact gcacgaccag gtgcacctgc tggaatgcgc ctggctggaa 300 atcctgatga ttggcctcgt gtggcggagc atggaacacc ccgtgaagct gctgttcgcc 360 cccaacctgc tgctggaccg gaaccagggc aaatgcgtgg aaggcatggt ggaaatcttc 420 gacatgctgc tggccacctc cagccggttc agaatgatga acctgcaggg cgaagagttc 480 gtgtgtctga agtccatcat cctgctgaac agcggcgtgt acaccttcct gagcagcacc 540 ctgaagtccc tggaagagaa ggaccacatc caccgggtgc tggacaagat caccgacacc 600 ctgatccacc tgatggctaa ggccggactg accctgcagc agcagcatca gagactggcc 660 cagctgctgc tgatcctgag ccacatccgg cacatgagca acaagggaat ggaacatctg 720 tacagcatga agtgcaagaa cgtggtgccc ctgtacgatc tgctgctgga agccgccgat 780 gcccacagac tgcacgcccc tacatctaga ggcggagcca gcgtggaaga gacagaccag 840 tctcacctgg ccacagccgg ctccacaagc tctcacagcc tgcagaagta ctacatcacc 900 ggcgaggccg agggctttcc tgcaacagcc 930 <210> 8 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> ERT2 <400> 8 Met Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser 1 5 10 15 Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser 20 25 30 Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro 35 40 45 Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu 50 55 60 Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro 65 70 75 80 Gly Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys 85 90 95 Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu 100 105 110 His Pro Val Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn 115 120 125 Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu 130 135 140 Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe 145 150 155 160 Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe 165 170 175 Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg 180 185 190 Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala 195 200 205 Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu 210 215 220 Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu 225 230 235 240 Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu 245 250 255 Glu Ala Ala Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly 260 265 270 Ala Ser Val Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser 275 280 285 Thr Ser Ser His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu 290 295 300 Gly Phe Pro Ala Thr Ala 305 310 <210> 9 <211> 15 <212> PRT <213> artificial sequence <220> <223> Related "TDA" peptide <400> 9 Thr Asp Ala Trp Arg Phe Ala Met Asn Tyr Pro Arg Asn Pro Thr 1 5 10 15 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Unrelated to "PEV" peptides <400> 10 Pro Glu Val Trp Ile Leu Ser Pro Leu Leu Arg His Gly 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Unrelated T2 peptide <400> 11 Val Leu Asp Gly Leu Asp Val Leu Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Related T2 peptides <400> 12 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1A schematic diagram of an inducible TCR is shown. Point mutations ("asterisks") are introduced into the constant ("c") alpha ("α") and beta ("β") chains of a given TCR. Due to the mutations, the TCRα-CD3 and TCRβ-CD3 complexes cannot pair in the endoplasmic reticulum. A dimerization domain ("DD") or alternatively a heterodimerization domain ("HD") is inserted into the c-terminus of both the constant α and β chains of the same TCR. After the introduction of a dimerization or heterodimerization agent ("DA or HA"), α and β chain pairing can occur, and the subsequent formation of a functional cluster comprising CD3 ("CD3") and TCR, including CD3 ζ-chains ("ζ-chains, zeta-chains"), and thus the TCR can be expressed on the cell surface.

[0086] Figure 2 TCR transduced with inducible TCR G11-3 (iTCR-G11-3, MHC-II restricted TCR) is shown - Jurkat-76 (Jurkat 76 cells are TCR α- and β-derivatives of the CD8-negative human T-cell lymphoma Jurkat cell line, kindly provided by M. Heemskerk) cells and tested for membrane expression of CD3-TCR complexes by flow cytometry 6 h after induction with 10 μM 4-hydroxytamoxifen (4-hydroxytamoxifen, 4-(1-[4-(dimethylaminoethoxy)phenyl]-2-phenyl-1-butenyl)phenol, Sigma-Aldrich, 5 mM stock in DMSO). 4-Hydroxytamoxifen was diluted to 10 μM in cell culture medium containing cells (middle panel) or no 4-hydroxytamoxifen was added ("no tamoxifen", leftmost and rightmost panels). Untransduced Jurkat-76 cells were used as negative controls for TCR expression and Jurkat-76 transduced with wt G11-3 (Milosevic S. et al., J Virol 2006 Nov 80(21): 10357-64) were used as positive controls for G11-3 TCR expression. Cells were stained with anti-CD3-PECy7 and anti-panTCR-PE. FlowJo V10 was used to generate each figure.

[0087] Figure 3a TCR transduced with iTCR-G11-3 is shown - Jurkat-76, and membrane expression of the CD3-TCR complex was assessed by flow cytometry after induction with 10 μM 4-hydroxytamoxifen or without tamoxifen. -Jurkat-76 cells were used as a negative control for TCR expression. Cells were stained with anti-CD3-PECy7 and anti-panTCR-PE and subjected to flow cytometry at 0.5 h, 1 h, 4 h, 6 h and 24 h after treatment with 4-hydroxytamoxifen. Figure 3b The kinetics of iTCR-G11-3 induction over time (0.5h, 1h, 4h, 6h and 24h) using 5μM or 10μM 4-hydroxytamoxifen are shown. Cells were stained with anti-CD3-PECy7 and anti-panTCR-PE and flow cytometry was performed at 0.5h, 1h, 4h, 6h and 24h after treatment with 4-hydroxytamoxifen. GraphPad Prism 7 was used for drawing. Each figure was generated using the analysis tool FlowJo V10.

[0088] Figure 4 Jurkat-76 transduced with iTCR-G11-3 and inducible NFAT-responsive GFP reporter (ieGFP) is shown and induced with 5 μM 4-hydroxytamoxifen for 6 h. LCL loaded with relevant TDAWRFAMNYPRNPT (" TDA ") peptide or irrelevant PEVWILSPLLRHG (" PEV ") peptide is added to the culture. The ratio of LCL:Jurkat-76 is 1: 1 in co-culture. Jurkat-76 transduced with wt (wild type) G11-3 is used as a positive control. Flow cytometry is performed 24 h after co-incubation and CD3, TCR and GFP expression of cells are evaluated. Each figure is generated using analysis tool FlowJo V10.

[0089] Figure 5a Jurkat-76 transduced with iTCR-G11-3 and ieGFP are shown and induced overnight with 1 μM 4-hydroxytamoxifen or left untreated (none). LCLs loaded with an irrelevant PEV peptide were added to the culture and GFP induction was followed over time (shown as 1 h, 1.5 h, and 3.5 h) using an Incucyte Zoom instrument (IncuCyte Zoom HD / 2CLR system, Essen Bioscience). Figure 5b Jurkat-76 transduced with iTCR-G11-3 and ieGFP are shown and induced overnight with 1 μM 4-hydroxytamoxifen or left untreated (None). LCL loaded with the relevant TDA peptide were added to the culture and GFP induction was followed over time (1 h, 1.5 h and 3.5 h shown) using the Incucyte Zoom instrument. Figure 5c: Jurkat-76 transduced with iTCR-G11-3 and ieGFP were induced overnight with 0.1 μM, 1 μM or 5 μM 4-hydroxytamoxifen. LCL loaded with the relevant TDA peptide was added to the culture and GFP induction was tracked for 13 h using the Incucyte Zoom device. Jurkat-76 transduced with wt-G11-3 was used as a positive control. The kinetics of the first 14 hours are shown. Plotted using Excel. GFP fluorescence intensity is plotted on the y-axis in green calibration units (GCU) x mm 2 express.

[0090] Figure 6 : Jurkat-76 transduced with iTCR-G11-3 and ieGFP were induced overnight with 0.05 μM or 0.1 μM 4-hydroxytamoxifen. Samples were washed and LCL loaded with the relevant TDA peptide was added after 1 h, 2 h and 4 h of washing, and GFP induction was tracked using the IncucyteZoom device. For the positive control (picture in the leftmost column), LCL loaded with the relevant TDA peptide was added to the culture and no washing was performed.

[0091] Figure 7a TCR transduced with the inducible TCR iTCR NY-ESO (iTCR-NY-ESO, MHC-I restricted TCR, upper panel) or with wt-NY-ESO TCR (lower panel) are shown. - Jurkat-76 cells and the membrane expression of CD3-TCR complexes are tested by flow cytometry. The cells transduced also express BFP (blue fluorescent protein, the vector encoding iTCR also includes blue fluorescent protein (mTag-BFP) as a reporter gene, which is independently expressed, so only the cells transduced are BFP positive) (left side small figure). The right side small figure shows CD3 and TCR staining of BFP+ cells. Anti-CD3-PECy7 and anti-panTCR-PE are used to dye cells. Each figure is generated using analysis tool FlowJo V10. Figure 7b TCR transduced with inducible TCR NY-ESO (iTCR-NY-ESO, MHC-I restricted TCR, upper panel) or with wt-NY-ESO TCR (lower panel) are shown. -Jurkat-76 cells, and induced with 1 μM of 4-hydroxytamoxifen (5mM stock solution in DMSO).4-Hydroxytamoxifen is diluted to 1 μM in a cell culture medium containing cells.The transduced cells also express BFP (left side small figure).The right side small figure shows CD3 and TCR staining of BFP+ cells.Anti-CD3-PECy7 and anti-panTCR-PE are used to stain cells.Each figure is generated using analysis tool FlowJo V10.

[0092] Figure 8a : Jurkat-76 transduced with iTCR-NY-ESO-mTag-BFP (MHC-I restricted TCR) and ieGFP were incubated with T2 cells loaded with irrelevant VLDGLDVLL peptide or loaded with the relevant peptide SLLMWITQC. The ratio of T2:Jurkat-76 in the co-culture was 1:1. Flow cytometry was performed 24h after co-incubation to stain for CD19 and measure fluorescence in blue, APC and FITC channels. The gating strategy was CD19 - (negative) (therefore excluding CD19 + (positive) T2-target cells) and BFP + Cells were gated and the fluorescence of ieGFP was measured in the FITC channel of FACS. The graphs were generated using the analysis tool FlowJo V10. Figure 8b :The Jurkat-76 transduced with iTCR-NY-ESO (TCR restricted by MHC-I) and ieGFP is induced overnight with 1 μM of endoxifen ((E / Z)-endoxifen hydrochloride hydrate, Sigma-Aldrich)), then incubated with the T2 cells of loading irrelevant VLDGLDVLL (" T2-irr.Pept. ") peptide or loading related peptide SLLMWITQC (" T2-rel.pept. "). The ratio of T2:Jurkat-76 is 1:1 in co-culture. After incubation, flow cytometry is carried out for 24h and BFP (transducer), CD3, TCR and GFP expression of assessment cell are evaluated. Each figure is generated using analysis tool FlowJo V10.

[0093] Fig. 9 : CD8 + PBL or CD8 expressing wt-NY-ESO +PBL is induced overnight with 1,5,10 or 20 μM of Endoxifen or is placed without induction (without Endoxifen).24h after induction, the T2 cells loaded with related peptide SLLMWITQC or irrelevant peptide VLDGLDVLL are added to culture.24h after stimulation with T2 target, by carrying out standard IFN-γELISA with culture supernatant, IFN-γ secretion is detected.48h after induction, flow cytometry is carried out and BFP (transducer), CD3 and BV6-5 of assessment cell are assessed.The exemplary little figure shown is CD8 transduced by iTCR-NY-ESO without induction or with 20 μM Endoxifen induction + PBL. The figures were generated using the analysis tool FlowJo V10. The figures were drawn using graphPad Prism7.

[0094] Fig.10 : CD8 + PBL or CD8 expressing wt-NY-ESO + PBL is induced with 10 μM of Endoxifen for 20h (+ induction) or placed untreated (no induction). The cells are then incubated with T2 cells (+ pept) expressing NuclightRed loaded with relevant peptides or with T2 NuclightRed cells (+ ctrl) loaded with irrelevant control peptides. The killing of T2 cells is tracked over time using Incucyte Zoom equipment. A) shows a photo of 42h after T cells are incubated with target cells. In the small figure above, T cells are not induced with Endoxifen. The small figure below shows T cells induced with Endoxifen. T2 NuclightRed cells appear in the middle, dark gray. Effector cells are light gray. B) Figure shows the density of T2 NuclightRed cells over time (up to 42h). Simulation (Mock) is CD8+PBL without transduction.

[0095] Fig.11 : TCR α constant region using the amino acid numbering nomenclature of IMGT for the human constant α region:

[0096] http: / / www.imgt.org / IMGTrepertoire / Proteins / alleles / index.php?species=Homo%20sapiens&group=TRAC&gene=TRAC1.

[0097] Fig.12 : TCR beta constant region using the amino acid numbering nomenclature of IMGT for human constant beta region allele C1:

[0098] http: / / www.imgt.org / IMGTrepertoire / Proteins / alleles / index.php?species=Homo%20sapiens&group=TRBC&gene=TRBC1.

[0099] Fig.13 : TCR transduced with wt TCR recognizing PRAME antigen (wt TCR) and TCR transduced with TCR recognizing PRAME antigen (amino acid residues 4, 5 and 37 in the constant β chain (SEQ ID 4) were mutated to V, P and K, respectively) - Jurkat-76 cells. Mock controls were untransduced Jurkat-76 TCR- / - cells. Cells were evaluated for CD3 and TCR expression by flow cytometry. FlowJo V10 was used to generate the graphs.

[0100] Fig.14 : TCR containing ieGFP reporter was transduced using G11-3 TCR carrying estrogen receptor at the C-terminus of both constant α and β chains (amino acid residues 4, 5 and 37 in the constant β chain (SEQ ID 4) were mutated to V, P and K, respectively) - Jurkat-76 cells. The cells were induced with 1 μM of Endoxifen for 24 h. The cells were then incubated with LCL cells loaded with the relevant TDAWRFAMNYPRNPT peptide (relevant) or the irrelevant PEVWILSPLLRHG peptide (irrelevant). The ratio of LCL:Jurkat-76 in co-culture was 1:1. The flow cytometry evaluation of the eGFP induced after 24 h of co-incubation was implemented. Each figure was generated using the analysis tool FlowJo V10.

[0101] The following examples illustrate the invention but do not limit the scope of the invention and the claims. Example

[0102] Abbreviations and synonyms

[0103] APC Antigen presenting cell

[0104] BFP blue fluorescent protein

[0105] ER Endoplasmic reticulum

[0106] ERT2 Estrogen receptor (mutated variant)

[0107] GFP Green Fluorescent Protein

[0108] iTCR Inducible TCR

[0109] ieGFP inducible enhanced GFP

[0110] LCL Lymphoblastoid cell line

[0111] PBL peripheral blood lymphocytes

[0112] TCR T cell receptor

[0113] 4-OH-Tamoxifen 4-Hydroxytamoxifen

[0114] Example 1: Making T cell receptor inducible

[0115] In order to damage the pairing of TCRα and β chains, the inventors searched for amino acid mutations in the TCRα constant and TCRβ constant regions, which will damage the pairing of TCRα and β chains, but will not interfere with the pairing of TCRα chains with CD3ε and CD3δ and the pairing of TCRβ chains with CD3γ and CD3ε. In order to identify such amino acids, the inventors conducted a large number of literature studies and considered the crystal structure of known TCRs in order to retain the interaction of TCRα and β chains with CD3 subunits unchanged (Table 1 and Table 2). In order to make the TCR discussed inducible, dimerization (e.g., ERT2) or heterodimerization (e.g., FKBP, FRB) domains are inserted into the C-terminal of the unpaired α and β constant regions of the corresponding mutations. By exposing cells carrying this iTCR to a dimerizing agent (e.g., 4-hydroxytamoxifen, 4-(1-[4-(dimethylaminoethoxy)phenyl]-2-phenyl-1-butenyl)phenol, Sigma-Aldrich) or a heterodimerizing agent (e.g., AP21967, 23,27-epoxy-3H-pyrido[2,1-c][1,4]oxazacyclotriacontane; Sigma-Aldrich), the TCRα-CD3εδ complex should pair with the TCRβ-CD3εγ complex in the endoplasmic reticulum, and then TCRα-CD3εδ-TCRβ-CD3εγ should assemble with CD3ζ in the Golgi apparatus and be transported to the cell surface (Feige MJ., et al., J Biol Chem. 2015 Oct 30;290(44):26821-31). Similar to the original non-inducible TCR, the iTCR should recognize its pMHC complex presented by the antigen presenting cell (APC) upon reaching the cell surface. Figure 1 ).

[0116] Example 2: Testing for Impairment of MHC Class II-Restricted iTCR α and β Chain Pairing in Jurkat-76 Cells and Induction of α and β Pairing with 4-Hydroxytamoxifen

[0117] The synthetic cassette containing iTCR-G11-3 was cloned into the lentiviral vector pCDH and transduced into 0.5x10 6 TCR - / -In Jurkat-76 cells ( Figure 2-6 ). The iTCR-G11-3 TCR cassette contains the V-α and V-β sequences of the MHC class II restricted TCR G11-3 (Milosevic S. et al., J Virol 2006 Nov 80 (21): 10357-64), the mutations in the α and β constant regions shown in Table 1, and ERT2 in the C-terminus of each α and β constant region. ERT2 is a mutant form of the human ligand binding domain of the estrogen receptor that can bind to tamoxifen active metabolites but not to estradiol (Feil R., et al., Biochem Biophys Res Commun. 1997 Aug 28; 237 (3): 752-7). The ERT2 nucleotide and amino acid sequences are depicted in SEQ ID NOs: 7 and 8, respectively. The lack of iTCR-G11-3 expression was tested by flow cytometry by staining uninduced Jurkat cells carrying iTCR-G11-3 with anti-CD3 (CD3-PECy7, SK7, 557851, BD) and anti-TCR (panTCR-PE, IP26, B49177, Beckman Coulter). Without 4-hydroxytamoxifen induction, no CD3 or TCR could be detected on the cell surface. In contrast, by inducing Jurkat cells carrying iTCR-G11-3 with 10 μM 4-hydroxytamoxifen, TCR and CD3 ( Figure 2 To determine the earliest and latest time points at which surface expression of the iTCR could be detected, Jurkat cells carrying iTCR-G11-3 were induced with 5 or 10 μM 4-hydroxytamoxifen, and CD3 and TCR expression were analyzed at 0.5 h, 1 h, 4 h, 6 h, and 24 h after treatment ( Figure 3a and 3b ). Uninduced Jurkat and untransduced TCR - / - Jurkat was used as a negative control for TCR / CD3 surface expression. Surface iTCR expression was detectable as early as 4 hours after induction and remained expressed even after 24 hours.

[0118] Example 3: Testing the MHC class II restricted iTCR function after inducing iTCR α and β chain pairing using 4-hydroxytamoxifen in Jurkat-76 cells

[0119] Since TCR-transduced Jurkat cells do not usually secrete cytokines after pMHC recognition, the inventors utilized an inducible NFAT-responsive GFP reporter cassette (ieGFP) as a readout of functional TCR signaling. The ieGFP cassette was cloned into the lentiviral vector pCDH (System Biosciences) and transduced into 0.5×10 6 TCR - / - The same Jurkat cells carrying ieGFP were then transduced with a lentiviral vector carrying the iTCR-G11-3 cassette ( Figure 4-6 ). The wt G11-3 TCR was also transduced into Jurkat cells carrying ieGFP and served as a positive control for G11-3 TCR signaling ( Figure 4 and 5c LCL cells (used as antigen presenting cells) loaded with an irrelevant peptide “PEV” (PEVWILSPLLRHG) or loaded with a peptide “TDA” (TDAWRFAMNYPRNPT) recognized by the G11-3 TCR were incubated with iTCR-G11-3-ieGFP Jurkat cells or with wt-G11-3-ieGFP Jurkat cells. -5 As expected, GFP signals and the resulting functional TCR signaling were observed only after induction with 5 μM 4-hydroxytamoxifen and incubation with LCL+TDA in Jurkat cells expressing wt-G11-3 or iTCR-G11-3. Figure 4 ). Anti-CD3 (CD3-PECy7, SK7, 557851, BD) and anti-TCR (panTCR-PE, IP26, B49177, Beckman Coulter) staining were performed and flow cytometry analysis was performed.

[0120] Example 4: Determination of the speed of iTCR signaling after pMHC recognition and the speed of shutdown of the TCR signaling cascade after removal of 4-hydroxytamoxifen

[0121] To determine the signaling rate of iTCR-G11-3, 1x10 4 Jurkat cells carrying iTCR-G11-3 and ieGFP were induced overnight (ON) with 1 μM 4-hydroxytamoxifen. To monitor the lack of TCR expression in the uninduced condition, Jurkat cells carrying iTCR-G11-3 and ieGFP were left untreated as a control. LCLs loaded with PEV-independent peptides ( Figure 5a ) or LCL loaded with TDA-related peptides ( Figure 5b ) were added to the culture and the GFP signal was tracked over time using the Incucyte instrument. GFP signal appeared 1 hour after incubation with APC (antigen presenting cells) loaded with the relevant peptide ( Figure 5b and 5c ), and the signal continued to increase until about 7 h after incubation ( Figure 5c ). -5 Interestingly, over time, the GFP signal elicited by the iTCR-G11-3 TCR was always higher than that elicited by the wt-G11-3 TCR ( Figure 5c ). When irrelevant peptides were presented to T cells expressing iTCR on LCLs as APCs, no induction of GFP was observed.

[0122] To analyze how quickly iTCR can be downregulated after removal of 4-hydroxytamoxifen, Jurkat cells expressing iTCR-G11-3 and ieGFP were first induced overnight with 0.05 μM or 0.1 μM 4-hydroxytamoxifen. The next day, 4-hydroxytamoxifen was washed out or not (positive control) from the cells and incubated with LCL loaded with the relevant TDA peptide 1 h, 2 h, or 4 h after removal of 4-hydroxytamoxifen, followed by detection of GFP signal using Incucyte ( Figure 6 ). Compared to the unwashed control, a reduction in the GFP signal was already visible 1 h after washing away the dimerization agent in the cells induced by 0.05 μM 4-hydroxytamoxifen and 4 h after washing away 0.1 μM 4-hydroxytamoxifen. Thus, iTCR can be rapidly induced and can also be rapidly downregulated after removal of the dimerization agent.

[0123] Example 5: Testing MHC class I restricted iTCR expression and function after induction of iTCR α and β chain pairing using 4-hydroxytamoxifen in Jurkat-76 cells

[0124] The synthetic cassette containing the iTCR-NY-ESO TCR and mTag-BFP separated by P2A was cloned into the lentiviral vector pCDH and transduced into 0.5x10 6 TCR - / -In Jurkat-76 cells (Figures 7 and 8). Since the vector encoding the iTCR also contains an independently expressed blue fluorescent protein (mTag-BFP) as a reporter gene, all transduced cells are BFP positive. The iTCR-NY-ESO TCR box contains the V-α and V-β sequences of the MHC class I restricted TCR that recognizes the NY-ESO antigen (Benchmark NY-ESO TCR; see WO 2005 / 113595), mutations in the α- and β-constant regions shown in Table 1, and ERT2 in the C-terminus of each α and β constant region. The lack of iTCR-NY-ESO expression was determined by staining Jurkat cells carrying iTCR-NY-ESO without induction with anti-CD3 and anti-TCR for flow cytometry, while CD3 or TCR ( Figure 7a In contrast, by inducing Jurkat cells carrying iTCR-NY-ESO with 1 μM 4-hydroxytamoxifen, both TCR and CD3 could be detected on the cell surface ( Figure 7b ). Anti-CD3 (CD3-PECy7, SK7, 557851, BD) and anti-TCR (panTCR-PE, IP26, B49177, Beckman Coulter) staining were performed and flow cytometry analysis was performed.

[0125] To determine whether iTCR-NY-ESO is functional after induction, Jurkat cells carrying ieGFP were transduced with a lentiviral vector carrying the iTCR-NY-ESO cassette. The wt NY-ESO TCR was also transduced into Jurkat cells carrying ieGFP and served as a positive control for NY-ESO TCR signaling. Figure 8a ) or loaded with the related peptide SLLMWITQC( Figure 8b ) were incubated with iTCR-NY-ESO-ieGFP Jurkat or wt-NY-ESO-ieGFP Jurkat. -5 As expected, GFP signal, and therefore functional TCR signaling, was observed only in Jurkats expressing wt-NY-ESO or in Jurkats expressing iTCR-NY-ESO after induction with 1 μM 4-hydroxytamoxifen and incubation with T2+-related peptides. The gating strategy was to gate CD19 阴性, BFP-positive cells were gated, and then the fluorescence of ieGFP (CD19-APCeF780, HIB19, 47-0199-42, eBioscience) was measured.

[0126] Example 6: Testing CD8 + MHC class I-restricted iTCR expression and function after endoxifen-induced iTCR α and β chain pairing in PBL

[0127] A commercial cell isolation kit (untouched CD8 + ) Enrichment of CD8 PBL from healthy donors + Cells were transduced with a lentiviral vector carrying the iTCR-NY-ESO cassette or a lentiviral vector carrying wt-NY-ESO (as a positive control). Since induction of iTCR-NY-ESO with 4-hydroxytamoxifen did not induce iTCR-NY-ESO in PBLs at any concentration attempted (data not shown), the inventors used another active tamoxifen metabolite called endoxifen. CD8 + Cells were induced with 20 μM endoxifen overnight or left untreated. The cells were then stained for CD3 (CD3-PECy7, SK7, 557851, BD) and specific V-β family BV6-5 (antibodies: TRBV6-5-PE, IMMU 222, IM2292, Beckman Coulter). Treatment with endoxifen resulted in the induction of iTCR-NY-ESO ( Fig. 9 ). If no treatment is performed, only endogenously expressed BV6-5 positive cells can be observed.

[0128] To determine whether iTCR-NY-ESO is functional when induced in PBL, T2 cells loaded with the relevant peptide SLLMWITQC or the irrelevant peptide VLDGLDVLL were incubated with 1, 5, 10, or 20 μM endoxifen-induced iTCR-NY-ESO CD8+ PBL or with uninduced iTCR-NY-ESO CD8+ PBL or with CD8+ PBL carrying wt-NY-ESO as a positive control. -5 The peptide was loaded at a concentration of 100 M. One day later, the culture supernatant was collected and subjected to a standard IFN-γ ELISA. IFN-γ was detected at high levels in wt-NY-ESO regardless of endoxifen treatment, whereas IFN-γ was detected at high levels in iTCR-NY-ESO only after endoxifen induction ( Fig. 9). These results indicate that iTCR-NY-ESO can be induced on the surface of PBL and has the same functional properties as wt-NY-ESO TCR.

[0129] In further experiments, it was found that T cells carrying i-TCR were cytotoxic and could eliminate targets. Again, 10 μM of endoxifen was used to induce CD8 + PBL or CD8 expressing wt-NY-ESO + PBL were either left untreated for 20 h (+induced) or left untreated (no induction). Cells were then incubated with T2 cells expressing NuclightRed loaded with the relevant peptide (+pept) or T2 NuclightRed cells loaded with an irrelevant control peptide (+ctrl). T2 cell killing was tracked over time using the Incucyte Zoom instrument. Fig.10 The picture in A shows 42 hours after incubation of T cells and target cells. In the upper panel, T cells were not induced with endoxifen. The lower panel shows T cells induced with endoxifen. T2 NuclightRed cells appear in the middle, dark gray. Effector cells are light gray. Fig.10 In B, the graph shows the density of T2 NuclightRed cells over time (up to 42 h). Fig.10 The cells marked as Mock are non-transduced CD8+ PBL.

[0130] Example 7: Testing of CD3 and TCR expression in TCR- / - cells transduced with wt TCR and with TCR carrying mutations K4V, N5P and Y37K in the β chain (SEQ ID NO 4, see Table 2)

[0131] Jurkat-76TCR- / - were transduced with wt TCR recognizing PRAME antigen (wt TCR) and with TCR recognizing PRAME carrying amino acid mutations K4V, N5P and Y37K in the constant β chain (SEQ ID NO: 4). The mock control was Jurkat-76 TCR- / - cells that were not transduced. Flow cytometry was performed and the expression of CD3 and TCR of the cells was evaluated. The analysis tool FlowJo V10 was used to generate the figures. From Fig.13 As can be seen, the mutant TCR showed no CD3 and TCR expression, indicating that the mutant TCR cannot be expressed on the membrane.

[0132] Example 8: Mutations K4V, N5P and Y37K in the β chain (SEQ ID NO: 4, Table 2) disrupt TCR expression, which can be rescued by dimerization of the estrogen receptor contained in the C-terminus of the α and β constant chains

[0133] Jurkat-76 containing ieGFP reporter is transduced using G11-3 TCR carrying estrogen receptor at the C-terminal of constant α and β chains and having amino acid residues K4V, N5P and Y37K in constant β chains (SEQ ID NO:4, Table 2). Cells are induced with 1 μM of Endoxifen for 24 h. Then cells are incubated with LCL cells loaded with irrelevant peptides or related peptides. The ratio of LCL:Jurkat-76 is 1:1 in co-culture. Flow cytometry is implemented 24 h after co-incubation and the eGFP expression of cells is evaluated. Each figure is generated using analysis tool FlowJo V10. Fig.14 It is shown that ieGFP is expressed in response to the related peptide. This shows that after the dimerization induced by Endoxifen, the TCR carrying mutations at 4, 5 and 37 of the β constant chain is still functional. Therefore, the amino acid mutations at 4, 5 and 37 of the constant β chain (SEQ ID 4) can be made without destroying the formation of the TCR-CD3 complex after the dimerization induced by Endoxifen.

Claims

1. A nucleic acid molecule encoding an inducible T cell receptor, comprising: (a) A nucleic acid sequence A consisting of: (i) a nucleic acid sequence encoding an amino acid sequence having two amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2, wherein the two amino acid substitutions are T44A and T47A, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, the inducible dimerization domain being linked downstream to the nucleic acid sequence of (a)(i), wherein the inducible dimerization domain is ERT2; and (b) a nucleic acid sequence B consisting of: (i) a nucleic acid sequence encoding an amino acid sequence having three amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 4, wherein the three amino acid substitutions are L63A, S77A and R79A, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, the inducible dimerization domain being linked downstream to the nucleic acid sequence of (b)(i), wherein the dimerization domain corresponds to the dimerization domain linked to the nucleic acid sequence of (a)(i), wherein the inducible dimerization domain is ERT2, Wherein, in the presence of a dimerizing agent, the amino acid sequences encoded by nucleic acid sequences A and B pair with each other, wherein the dimerizing agent is 4-hydroxytamoxifen or endoxifen.

2. A nucleic acid molecule encoding an inducible T cell receptor, comprising (c) a nucleic acid sequence A consisting of: (i) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, the inducible dimerization domain being linked downstream to the nucleic acid sequence of (c)(i), wherein the inducible dimerization domain is ERT2; and (d) a nucleic acid sequence B consisting of: (i) a nucleic acid sequence encoding an amino acid sequence having three amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 4, wherein the three amino acid substitutions are K4V, N5P and Y37K, and (ii) a nucleic acid sequence encoding an inducible dimerization domain, the inducible dimerization domain being linked downstream to the nucleic acid sequence of (d)(i), wherein the dimerization domain corresponds to the dimerization domain linked to the nucleic acid sequence of (c)(i), wherein the inducible dimerization domain is ERT2, Wherein, in the presence of a dimerizing agent, the amino acid sequences encoded by nucleic acid sequences A and B pair with each other, wherein the dimerizing agent is 4-hydroxytamoxifen or endoxifen.

3. The nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid sequence A and the nucleic acid sequence B are contained by separate nucleic acid molecules or are contained in one nucleic acid molecule.

4. An expression cassette comprising the nucleic acid molecule according to any one of the preceding claims 1 to 3.

5. A vector comprising an expression cassette as defined in claim 4. The vector of claim 5 , wherein the vector is a retroviral vector or a lentiviral vector.

7. A kit comprising i) a nucleic acid molecule according to any one of claims 1 to 3, an expression cassette according to claim 4 or a vector according to claim 5 or 6, and ii) a dimerizing agent, wherein the dimerizing agent is 4-hydroxytamoxifen or endoxifen.

8. A host cell comprising a nucleic acid molecule as defined in any one of claims 1 to 3, an expression cassette as defined in claim 4 or a vector as defined in claim 5 or 6.

9. The host cell of claim 8, wherein the host cell is a T lymphocyte, and the host cell comprises 4-hydroxytamoxifen or endoxifen.

10. A protein encoded by the nucleic acid molecule of any one of claims 1 to 3.

11. Use of a dimerizing agent for dimerizing a protein encoded by a nucleic acid sequence A as defined in any one of claims 1 to 3 and a protein encoded by a nucleic acid sequence B, wherein the dimerizing agent is 4-hydroxytamoxifen or endoxifen.

12. A method for dimerizing a protein encoded by a nucleic acid sequence A as defined in any one of claims 1 to 3 and a protein encoded by a nucleic acid sequence B, said method comprising the step of adding 4-hydroxytamoxifen or endoxifen to said proteins.

13. A method for preparing an inducible T cell receptor, the method comprising the step of introducing a nucleic acid molecule as defined in any one of claims 1 to 3, an expression cassette as defined in claim 4 or a vector as defined in claim 5 or 6 in vitro into a host cell under conditions that allow the expression of the nucleic acid sequence A and the nucleic acid sequence B.

14. A pharmaceutical composition comprising a nucleic acid molecule as defined in any one of claims 1 to 3, an expression cassette as defined in claim 4, a vector as defined in claim 5 or 6, a host cell as defined in claim 8 or 9 or a protein as defined in claim 10.

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