T cell receptors with improved pairing

Abstract

To provide approaches to effectively avoid TCR mispairing.SOLUTION: Provided is a modified T cell receptor (TCR) α or β chain, or a fragment or derivative thereof that retains the ability to bind to an antigen-MHC complex, wherein the amino acid at position 44 according to the IMGT numbering of a non-modified chain is substituted with another suitable amino acid that reduces the pairing of the modified α or β chain with an undesired α or β chain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a modified T cell receptor (TCR) α-chain or β-chain, or a heterodimer comprising the same, in which in the variable domain of said modified α-chain or β-chain, the amino acid at position 44 according to the IMGT numbering is replaced with another suitable amino acid to improve desired chain pairing. [Background technology]

[0002] The adaptive immune system consists of antibodies, B cells, and CD4 + and CD8 + T cell receptors consist of T cells that express highly variable alpha (α) and beta (β) chains that are expressed as part of a complex with the invariant CD3 chain molecule on the surface of T cells. A key step in the process of forming TCR heterodimers is called "pairing". T cells that express the paired receptor are called α:β (or αβ) T cells, while a small number of T cells express an alternative receptor formed by variable gamma (γ) and delta (δ) chains and are called γδ T cells.

[0003] The TCR is located on the surface of the T cell and the antigen receptor molecule is involved in the recognition of properly processed antigens presented to the T cell by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC), thus potentially resulting in T cell activation and an immune response to the antigen.

[0004] Each chain of the TCR is composed of two extracellular domains, the variable (V) and constant (C) regions, both of which belong to the immunoglobulin superfamily (IgSF) domain-forming antiparallel β-sheets. The constant regions are adjacent to the cell membrane and are followed by a transmembrane region and a short cytoplasmic tail, whereas the variable regions bind peptide / MHC complexes to antigen-presenting cells.

[0005] The variable domains of both the TCR α and β chains each have three hypervariable or complementarity determining regions (CDRs) that form the antigen binding site. The variable region of the β chain has an additional region of so-called hypervariability (HV4), which does not usually contact the antigen and is therefore not considered a CDR. Importantly, while CDR1 and 2 of both chains are germline coded, CDR3α and β are largely non-template coded and are generated by somatic recombination (Davis & Bjorkman 1988). In humans, the diversity of TCR molecules is achieved by αβ pairing of a set of 47 Vα and 54 Vβ sequences, which combine to achieve the final diversity of CDR3 length and sequence.

[0006] Although CDR3 is the main CDR involved in the recognition of the processed antigen, CDR1 of the α chain has been shown to interact with the N-terminal portion of antigenic peptides (Cole et al. 2009), whereas CDR1 of the β chain occasionally interacts only with the C-terminal portion of peptides.

[0007] CDR2 is believed to primarily recognize MHC. The HV4 region of the β chain is not thought to be involved in antigen recognition, but has been shown to interact with so-called superantigens (Li et al. 1998). Despite the generally accepted paradigm that CDR1 and CDR2 primarily bind MHC and CDR3 binds antigenic peptides, several studies have revealed the true complexity of antigen recognition by TCRs by showing that all CDR regions can occasionally interact with both antigen and MHC (Burrows et al. 2010, Roomp et al. 2011).

[0008] Adoptive transfer approaches have been developed to use this mechanism of cancer therapy (Rosenberg et al. 1988), whereby T cells transduced with genes encoding the α and β chains of tumor-specific T cell receptors (TCRs) mediate antitumor immunity in patients. In recent years, this approach has attracted attention. As one strategy, TCR gene therapy provides patients with autologous T cells genetically engineered with recombinant TCR chains. This technique offers a promising approach for the treatment of cancer and tumors. To do so, TCR α and β chains that detect / bind specific antigen-MHC complexes are cloned into wild-type T cells taken from the patient. The genetically modified T cells are then expanded ex vivo, and the expanded cells are returned to the patient to provide an immune response against, for example, tumors.

[0009] In effect, the transgenic T cells thus generated express both wild-type TCRs with wild-type α and β chains and recombinant TCRs with α and β chains specific for the respective recombinant antigen-MHC complexes. Both wild-type α and β chains and recombinant α and β chains can usually still pair with each other (Shao et al. 2010). This undesirable possibility is called "TCR mispairing" and is a recognized problem in the field of TCR (gene) therapy.

[0010] Mismatches and inaccurate match between the engineered TCRα or β chains and the endogenous TCRβ or α chains, respectively, lead to reduced surface expression of the engineered TCRαβ heterodimer, which in turn reduces the functional avidity of the modified T cells. Moreover, T cells expressing mismatched TCRs and grown under high IL-2 conditions were demonstrated to induce graft-versus-host disease (GvHD) in preclinical models (Bendle et al., 2009).

[0011] Several strategies for optimizing engineered TCR α and β pairing to enhance the functional avidity of therapeutic T cells have been discussed, for example, in Govers et al. (2010).

[0012] These possibilities for avoiding mismatches include the following: 1. Murine TCR: In this approach, the human TCR α and β constant chains are replaced by the corresponding mouse domains. Although human and mouse TCR-C domains show a high degree of homology, subtle differences affect the stability of the TCR / CD3 interaction and therefore the TCR surface expression levels. 2. Cysteine-modified TCR: This approach introduces a cysteine ​​amino acid at a structurally favorable position, thus allowing the formation of an additional disulfide bridge to promote correct pairing between the two TCR chains. Site-specific mutations such as T48C in the TCR α constant chain and S57C in the TCR β constant chain led to a TCR heterodimer linked by two interchain bonds (i.e. the introduced disulfide bridge and the integral transmembrane disulfide bridge (position 95 in the α constant domain and position 131 in the β constant domain). 3. Domain Swapping: Constant domains are swapped between the α and β chains of a tumor-specific T cell receptor to create a domain-swapped (ds) TCR. When correctly paired, these dsTCR chains retain all domains necessary to recruit the CD3 protein to be expressed on the T cell surface and mediate a functional T cell response upon engagement with a target antigen. In contrast, mispaired TCRs containing one dsTCR chain and one wild-type TCR chain lack critical domains required for CD3 recruitment, export, and signaling, and therefore cannot mediate deleterious autoimmunity. 4. Exclusive TCR heterodimers: This approach utilizes steric and electrostatic forces to facilitate correct pairing between TCR α and β transgenes while simultaneously inhibiting pairing between exogenous and endogenous TCR α and β chains. In one example, site-directed mutagenesis is used to introduce S85R into the α constant domain and R88G into the β constant domain to obtain the required electrostatic charge changes, thus generating a "knobs-in-holes" configuration of each other, which is said to minimally distort the secondary and tertiary structures. 5. Use of chimeric TCR-CD3ζ chains, in which each TCR chain is fused to a CD3ζ molecule. 6. Use of single chain TCRs in which the Vα of a defined TCR is fused to the β chain using a flexible peptide linker. 7. Use of shRNA sequences or zinc finger nucleases to knock down expression of endogenous TCR.

[0013] Another approach was proposed by O'Shea et al. (1993), who designed a pair of peptides, called "Velcro", that could pair with each other due to favorable electrostatic interactions in the heterodimeric state. The authors demonstrated that the two peptides were predominantly unfolded in isolated form, but when mixed, they preferentially associated and formed a stable parallel coiled-coil heterodimer. This approach was also applied to generate soluble TCRs by Chang et al. (1994), in which the heterodimeric complex was supported by fusing the peptides to truncated α and β chains, respectively.

[0014] WO 2014 / 153470 A2 discloses methods and compositions for modifying TCR genes using nucleases (zinc finger nucleases or TAL nucleases) to modify the TCR gene by targeted disruption.

[0015] WO 2014 / 083173 relates to a method for producing novel T cell receptors that reduces the risk of adverse events in immunotherapy, in particular in adoptive T cell transfer.

[0016] WO 2016 / 071343 A1 relates to modified T cell receptors (TCRs) and their use in particular in adoptive cell therapy (ACT) for transferring T lymphocytes. The TCRs are mutated in the transmembrane regions of the α and β chains, the mutations favoring correct TCR chain pairing.

[0017] Although the above approaches are promising, several hurdles, including the proper expression of exogenous TCRs, prevent their clinical impact. The poor clinical response indicates that numerous problems remain to be solved. Since T cell functional avidity is primarily determined by both TCR affinity and the number of TCR molecules expressed, significant efforts have been made to improve these biophysical properties in TCR engineered cells using two important approaches: (a) improving TCR affinity, and (b) enhancing TCR expression. To improve TCR affinity, attempts have been made to select high affinity receptors or to increase the affinity of the transferred receptors by point mutations. Alternatively, various approaches have been devised to increase the number of TCRs on the surface of transduced cells. These include engineering expression vectors, using codon-optimized TCR sequences, eliminating glycosylation sites, and improving the pairing of the introduced TCR chains. Summary of the Invention [Problem to be solved by the invention]

[0018] Therefore, there remains a need to provide approaches to effectively avoid TCR mismatching. [Means for solving the problem]

[0019] These additional approaches should be easy to introduce and efficiently reduce the occurrence of mismatched TCR heterodimers while increasing the abundance of correctly matched TCR heterodimers, i.e., recombinant α- and β-chain pairs, in modified T cells. Also, methodologies requiring minimal manipulation of the TCR chains and host T cells would be desirable. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] These and further objects are achieved by the method and means according to the present invention.

[0021] According to a first aspect of the invention, there is provided a modified T cell receptor (TCR) α- or β-chain, or a fragment or derivative thereof, which maintains the ability to bind to an antigen-MHC complex, in which in the variable domain of said modified α- or β-chain, Q or any other amino acid at position 44 according to the IMGT numbering is replaced with another suitable amino acid. As also shown below, the suitable amino acid reduces pairing with undesired α- or β-chains, e.g. unmodified chains, whilst maintaining the pairing specificity of the modified TCR α- and β-chains.

[0022] Since position 44 is located in the FR2 region, it is known not to directly contact the target of the TCR binding site, and therefore it is assumed that substitutions at position 44 are not critical to and / or do not interfere with specific target epitope recognition.

[0023] The amino acids according to the present invention can be selected from the 20 α-amino acids (L-amino acids) used in living organisms. "Suitable" amino acids are intended to include both these amino acids (i.e. the 20 α-amino acids and L-amino acids used in living organisms) and "unusual" amino acids (e.g. S-amino acids, or D-amino acids) or modified amino acids. The 20 α-amino acids used in living organisms are preferred.

[0024] The numbering of amino acid residues in the TCR α and β chains follows the IMGT standard as disclosed in Lefranc et al. (2001). The IMGT standard is a general system of rules for assigning unambiguous numbers to amino acid residues in immunoglobulin molecules, including the TCR α and β variable chains. See Figures 1A-C for a comparison of IMGT numbering (bold) with Kabat numbering. In Figure 1B, Q44 in the α chain (TRAV) and β chain (TRBV) is labeled in grey. The numbering according to the IMGT standard may deviate from the simple numbering of a given amino acid sequence of the TCR variable domain, especially due to blanks in the CDR sequences, as seen in Figures 1A-C.

[0025] Q44 according to IMGT numbering is a highly conserved residue in the framework 2 (FR2) region, shared by almost all TCR α chains except TRAV2, TRAV24, TRAV29 / DV5, and TRAV40, and by all TCR β genes except TRBV14Q44 (Lefranc et al, 2001; Strong et al., 1999). Furthermore, Q44 is very often part of a 4AA motif comprising WYXQ, where X is, for example, R, V, K, or Q (Lefranc et al, 2001).

[0026] Knies et al. (2016) describe an optimized single-chain TCR (scTCR) to inhibit residual TCR mispairing to achieve safe adoptive immunotherapy for bulk endogenous TCRα / β-positive T cells. Prevention of residual mispairing was achieved by a novel artificial disulfide bond designed between the Vα domain and the 3'-tail of the Vβ-proximal linker in the three-domain scTCR.

[0027] Hoffmann et al. (2015) describe a systematic bioinformatics analysis of the structural properties of bound and unbound TCR molecules, focusing on Vα / Vβ relative angles and flexibility. Their results demonstrated the importance of this angle for signal transduction, as several different Vα / Vβ angle-based structural clusters could be observed, and there is greater angular flexibility for the unbound TCR than for the bound TCR. A unique rotation center was identified, and a core region around this point is centrally located between the Vα and Vβ domains, very close to position 44 in both Vα and Vβ.

[0028] Substitutions in the context of the present invention can be generated using standard methods of protein mutagenesis, such as site-directed or random mutagenesis of coding DNA or cDNA, or genome editing techniques such as CRISPR Cas, TALEN, ZFN, Argonaut (NgAgo) or CRISPR Cpf1. Furthermore, such substitutions can be made by simply synthesizing the coding gene, cDNA or mRNA. Nowadays, service providers offer the synthesis of nucleic acids of a given sequence.

[0029] Methods of random or site-directed mutagenesis resulting in site-specific amino acid substitutions are well known to those skilled in the art and are disclosed, for example, in Labrou et al. (2010) and Trehan et al. (2016).

[0030] Methods of genome editing (e.g., CRISPR Cas, TALEN, ZFN, Argonote (NgAgo) or CRISPR Cpf1) for modifying a given amino acid sequence are well known to those of skill in the art and are disclosed, for example, in Maeder & Gersbach (2016). Methods of gene synthesis are well known to those of skill in the art and are disclosed, for example, in Hughes et al. (2011). The disclosures of these references shall be deemed to be incorporated herein by reference in their entirety.

[0031] As discussed above, some prior art methods suggest modification of the TCR α and / or β chains by introducing mouse constant chains. This approach carries the risk of increased immunogenicity due to non-human sequences, which is avoided by the methods and products of the present invention.

[0032] Furthermore, the inventors have shown that substitutions at position 44 can have a modest effect on binding to the target peptide (i.e. affinity for the target antigen / MHC complex), which is likely due to the fact that in the tertiary structure of the α and β variable domains, Q44 is maximally distant from the regions formed by the CDRs.

[0033] According to one embodiment, the modified α- and β-chains do not have any substitutions in their constant domains and / or their transmembrane domains compared to the respective unmodified receptors identified and / or isolated. Since the constant domains contain a large and highly conserved interface between the TCR α- and β-chains, it is difficult to avoid the effects of even minor modifications in terms of pairing behavior, stability and immunogenicity compared to substitutions at positions in the variable domains.

[0034] According to one embodiment, an α chain having a substitution at position 44 will preferably pair with a T cell receptor (TCR) β chain in which position 44 in the variable domain has been substituted with a suitable amino acid. Similarly, in another embodiment, a β chain having a substitution at position 44 will preferably pair with a T cell receptor (TCR) α chain in which position 44 in the variable domain has been substituted with a suitable amino acid.

[0035] Therefore, in T cells modified to express a second heterologous or recombinant TCR having the claimed substitutions, mispairing between the recombinant α chain and the endogenous β chain, or vice versa, is reduced or even avoided.

[0036] In this way, an increased abundance of correctly paired engineered TCR α / β heterodimers can be achieved, which improves the overall avidity of modified T cells and avoids adverse reactions such as induction of graft-versus-host disease (Bendle et al., 2009).

[0037] According to one embodiment of said recombinant T cell receptor (TCR) heterodimer, at least in the α or β chain, the amino acid present at position 44 of the variable domain is substituted with one amino acid selected from the group consisting of Q, R, D, E, K, L, W, and V.

[0038] According to a further embodiment, the α chain has at least a variable domain sequence comprising a framework region of a sequence sharing at least 95% sequence identity with SEQ ID NO: 1 or 3, in which the Q or any other amino acid at position 44 in the variable domain is replaced by another suitable amino acid as disclosed herein, or the β chain has a variable domain sequence comprising a framework region of a sequence sharing at least 95% sequence identity with SEQ ID NO: 2 or 4, in which the Q or any other amino acid at position 44 in the variable domain is replaced by another suitable amino acid as disclosed herein.

[0039] Preferably, the α or β chain has a variable domain sequence comprising at least the framework regions of a sequence that shares (has) at least 96%, more preferably 97%, even more preferably at least 98%, even more preferably at least 99%, or most preferably 100% sequence identity with SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4, respectively.

[0040] SEQ ID NO: 1 shows the amino acid sequence of the TCR alpha chain variable domain of TCR R7P1D5 (also known as TRAV5, Lefranc et al. 2001). SEQ ID NO: 2 shows the amino acid sequence of the TCR beta chain variable domain of R7P1D5 (also known as TRBV12-4, Lefranc et al. 2001). R7P1D5 is disclosed in US Provisional Patent Application No. 62 / 308,944, the contents of which are incorporated herein by reference. Upon binding to MHC, R7P1D5 binds, for example, to a peptide designated "MAG-003" on antigen presenting cells. MAG-003 comprises an amino acid sequence according to the following general formula I: X1X2LEHVVRX3 wherein X1 is selected from amino acids K and Y; X2 is selected from amino acids V, L, and A; and X3 is selected from V, L, A, and I.

[0041] SEQ ID NO: 3 shows the amino acid sequence of the TCR α chain variable domain TRAV8-6 (Lefranc et al. 2001). SEQ ID NO: 4 shows the amino acid sequence of the TCR β chain variable domain TRBV6-5 (Lefranc et al. 2001).

[0042] Note that in Figures 1 and 3, the sequences are shown in IMGT numbering, which deviates from the simple numbering of the respective sequences in the sequence listing. The wavy underlines in Figure 3 indicate spaces that are not occupied by amino acid residues but are taken into account in the IMGT numbering. It is understood that Q44 refers to the IMGT numbering as shown in Figures 1 and 3, and not the numbering as derived from the attached sequence listing.

[0043] R7P1D5 (TRAV5 and TRBV12-4), with its α and β chains, and TRAV8-6 and TRBV6-5 are just four examples of TCR variable domains that can be used in the context of the present invention. Other TRAV and TRBV subgroups are disclosed on the IGMT website. It should be noted that TRAV and TRBV can adopt specificity for different target epitope / MHC complexes, especially by appropriate adaptation of the CDR sequences.

[0044] Please note that the above sequences are shown without the signal sequence, and sometimes sequence databases show slightly deviating sequences, for example in the Uniprot database TRAV8-6 lacks the N-terminal A shown in SEQ ID NO:3 and Figure 1, while TRBV6-5 lacks the N-terminal N and A shown in SEQ ID NO:4 and Figure 1.

[0045] According to another embodiment of the recombinant T cell receptor (TCR) heterodimer of the present invention, the TCR comprises one of the preferred substitution pairs selected from the following list: αQ44D / βQ44R;αQ44R / βQ44D;αQ44E / βQ44K;αQ44K / βQ44E;αQ44D / βQ44K;αQ44K / βQ44D;αQ44E / βQ44R;αQ44R / βQ44E;αQ44L / βQ44W;αQ44W / βQ44L;αQ44V / βQ44W;and αQ44W / βQ44V; αW44D / βQ44R;αW44R / βQ44D;αW44E / βQ44K;αW44K / βQ44E;αW44D / βQ44K;αW44K / βQ44D;αW44E / βQ44R;αW44R / βQ44E;αW44L / βQ44W;αW44 / βQ44L;αW44V / βQ44W;and αW44 / βQ44V; αH44D / βQ44R;αH44R / βQ44D;αH44E / βQ44K;αH44K / βQ44E;αH44D / βQ44K;αH44K / βQ44D;αH44E / βQ44R;αH44R / βQ44E;αH44L / βQ44W;αH44W / βQ44L;αH44V / βQ44W;andαH44W / βQ44V; αK44D / βQ44R;αK44R / βQ44D;αK44E / βQ44K;αK44 / βQ44E;αK44D / βQ44K;αK44 / βQ44D;αK44E / βQ44R;αK44R / βQ44E;αK44L / βQ44W;αK44W / βQ44L;αK44V / βQ44W;andαK44W / βQ44V; αE44D / βQ44R;αE44R / βQ44D;αE44 / βQ44K;αE44K / βQ44E;αE44D / βQ44K;αE44K / βQ44D;αE44 / βQ44R;αE44R / βQ44E;αE44L / βQ44W;αE44W / βQ44L;αE44V / βQ44W;andαE44W / βQ44V; αQ44D / βR44;αQ44R / βR44D;αQ44E / βR44K;αQ44K / βR44E;αQ44D / βR44K;αQ44K / βR44D;αQ44E / βR44;αQ44R / βR44E;αQ44L / βR44W;αQ44W / βR44L;αQ44V / βR44W;and αQ44W / βR44V; αW44D / βR44;αW44R / βR44D;αW44E / βR44K;αW44K / βR44E;αW44D / βR44K;αW44K / βR44D;αW44E / βR44;αW44R / βR44E;αW44L / βR44W;αW44 / βR44L;αW44V / βR44W; and αW44 / βR44V; αH44D / βR44;αH44R / βR44D;αH44E / βR44K;αH44K / βR44E;αH44D / βR44K;αH44K / βR44D;αH44E / βR44;αH44R / βR44E;αH44L / βR44W;αH44W / βR44L;αH44V / βR44W; andαH44W / βR44V; αK44D / βR44;αK44R / βR44D;αK44E / βR44K;αK44 / βR44E;αK44D / βR44K;αK44 / βR44D;αK44E / βR44;αK44R / βR44E;αK44L / βR44W;αK44W / βR44L;αK44V / βR44W; andαK44W / βR44V; αE44D / βR44; αE44R / βR44D; αE44 / βR44K; αE44K / βR44E; αE44D / βR44K; αE44K / βR44D; αE44R / βR44E; αE44L / βR44W; αE44W / βR44L; αE44V / βR44W; and αE44W / βR44V.

[0046] In the above, for example, "αQ44R / βQ44D" is intended to mean, for example, that in the variable domain of the α chain Q44 is replaced by R, while in the variable domain of the β chain Q44 is replaced by D.

[0047] A preferred embodiment of said recombinant T cell receptor (TCR) heterodimer comprises a) the modified α-chain preferably pairs with a modified β-chain compared to an unmodified β-chain having a Q or any other suitable amino acid at position 44 of the variable domain; and / or b) the modified β-chain pairs preferentially with a modified α-chain compared to an unmodified α-chain having a Q or any other suitable amino acid at position 44 in the variable domain.

[0048] According to another aspect of the present invention, there is provided a nucleic acid molecule encoding a modified T cell receptor (TCR) α or β chain according to the above description, and / or a recombinant T cell receptor (TCR) heterodimer according to the above description. In one embodiment, the nucleic acid molecule comprises a promoter operably linked to said one or more coding nucleic acid molecules, and / or A signal sequence operably linked to said one or more coding nucleic acid molecules. The composition further comprises at least one of the following:

[0049] The signal sequence encodes a signal peptide that targets the α or β chain to the cell surface of T cells, where it is anchored by its transmembrane domain and the α and β chains are displayed on the extracellular surface. Signal sequences for different α and β chain variable domain subtypes have been disclosed in the art.

[0050] According to another aspect of the present invention there is provided a plasmid or vector comprising at least one of the nucleic acid molecules described above.

[0051] In one embodiment, the vector is preferably a viral vector, preferably a retroviral or lentiviral vector. Methods for transducing alpha or beta T cell receptor (TCR) genes into T cells using viral vectors are disclosed, for example, in Pogulis and Pease (1998), or Zong et al. (2010). The use of lentiviral vectors for gene transfer into human T cells is disclosed in Verhoeyen et al. (2009).

[0052] In another embodiment, the vector comprises a transposon, such as piggyback or sleeping beauty, which can then deliver the respective nucleic acid to the T cell. Methods of using transposons to genetically engineer T cells are disclosed, for example, in Huang et al. (2008).

[0053] In another embodiment, T cells can be transiently transfected, e.g., by introducing one or more RNAs encoding the α and β chains, e.g., by electroporation. Such methods are disclosed, e.g., in Kim and Eberwine (2010).

[0054] According to another aspect of the invention, there is provided a method for preparing modified T cells, said method comprising the steps of: Obtaining T cells from a subject; transducing or transfecting said T cells with one or more nucleic acid molecules according to the invention, or with a plasmid or vector according to the invention; The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0055] In a preferred embodiment, the T cells are obtained from an HLA allele negative donor. For example, the modified T cells are HLA-A * If it is intended to detect antigens presented by serotype 02 APCs, the source intended to be modified is preferably HLA-A * 02 sero-negative donors. In this way, cross-reactivity between endogenous TCRs and target antigen / MHC complexes is reduced or even avoided.

[0056] Preferably, the modified T cells thus obtained are suitable for autologous T cell therapy of said subject.

[0057] According to another aspect of the present invention, there is provided a modified T cell carrying a set of nucleic acids encoding the α and β chains of a recombinant T cell receptor (TCR) heterodimer according to the above description.

[0058] In one embodiment, the cells are prepared by the method described above.

[0059] According to another aspect of the invention there is provided a use of the modified T cells as described above for treating a patient suffering from, at risk of developing and / or diagnosed with a neoplastic, inflammatory, infectious or autoimmune disease, which use comprises providing or administering to a patient in need thereof a preparation comprising said modified T cells.

[0060] Alternatively, there is provided a method of treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic, inflammatory, infectious or autoimmune disease using modified T cells as described above, which method comprises providing or administering to a patient in need thereof a preparation comprising said modified T cells.

[0061] In this context, it is important to understand that the specificity of each modified TCR, or each modified T cell, to detect antigens presented by the MHC of an antigen-presenting cell depends on the CDR sequences in the variable domains of the α and β chains.

[0062] As described in Loset et al. (2014), T cell receptors for specific antigen-MHC complexes can be obtained by T cell-associated phage display. In such an approach, the Q44 substitution in the variable domains of the α and β chains can be achieved in all members of the library forming the basis of the phage display, or can be introduced subsequently, i.e. once a unique TCR has been found that detects a specific antigen-MHC complex.

[0063] According to a further aspect of the present invention, there is provided a use, a method, a T cell or a T cell receptor (TCR) heterodimer according to the above description, wherein the antigen presented by the MHC complex detected by the T cell receptor is selected from cancer-specific tumor associated antigen (TAA) peptide epitopes. These peptides are known in the art and comprise, as an example, the peptide MAG-003 used in the examples. These peptides can be found, for example, in the Epitope Database (http: / / www.iedb.org / ) or, preferably, in the Epitope Database (http: / / www.iedb.org / ) or, more preferably, in the Epitope Database (http: / / www.iedb.org / ), ... ret;WO 2016 / 107740;WO 2015 / 193359;WO 2015 / 169945;WO 2015 / 063302;WO 2015 / 018805;WO 2012 / 069462;WO 2012 / 079878;WO 2012 / 038463;WO WO 2011 / 151403;WO 2011 / 128448;WO 2011 / 113882;WO 2011 / 113872;WO 2011 / 113819;WO 2011 / 073215;WO 2010 / 037514;WO 2009 / 138236;WO 2007 / 02 WO 8574;WO 2007 / 028573;WO 2006 / 114307;WO 2005 / 116051;WO 2005 / 076009;WO 2004 / 085461;WO 03 / 100432;WO 03 / 102023;WO 2009 / 015843;WO 2009 / 015842;WO 2009 / 015841;WO 2016 / 202963;WO 2016 / 207164;WO 2017 / 001491;WO 2017 / 005733;WO 2017 / 021527;WO 2017 / 036936;WO Such peptides may be found in publications such as those disclosed in any of Publication Nos. WO 2017 / 060169; WO 2017 / 060201; WO 2017 / 097602; WO 2017 / 097699; WO 2017 / 108345; or WO 2017 / 009400 (all of which are incorporated herein by reference with respect to the peptides disclosed therein).

[0064] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered as exemplary or illustrative and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention and / or from a study of the drawings, disclosure, and appended claims.

[0065] It is further to be understood that the present invention is not limited to the specific components or structural features of the described devices or compositions, or process steps of the described methods, since they may vary. It is also to be understood that the terminology used in this specification is intended to describe specific embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope. It should be noted that in the usage of this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents, unless the context clearly dictates otherwise. Furthermore, in the claims, the word "comprises" does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. It is further to be understood that where a parameter range delimited by numerical values ​​is given, the range is deemed to include these limits.

[0066] It is further understood that the embodiments disclosed herein are not intended to be understood as separate embodiments unrelated to each other. Features discussed in one embodiment are also intended to be disclosed in relation to other embodiments shown herein. In some cases, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature is not intended to be disclosed with the other embodiments. A person skilled in the art will understand that it is the gist of the present application to disclose the feature also for the other embodiments, but that this has not been done merely for the purpose of clarity and to keep the specification manageable.

[0067] All amino acid sequences disclosed herein are shown in N-terminal to C-terminal direction; all nucleic acid sequences disclosed herein are shown in 5'→3' direction.For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.This refers in particular to prior art documents that disclose standard or routine methods.In that case, incorporation by reference is primarily intended to provide sufficient to enable the disclosure and to avoid lengthy repetition. [Brief description of the drawings]

[0068] In the drawings and the accompanying sequence listing: [Figure 1A] Figure 1 shows the numbering of amino acid residues in the variable domains of the TCR α and β chains. The figure has been adapted and modified from Lefranc et al. (2003). Sequences are shown for example for TRAV8-6 (Gene ID: 28680) (α chain) and TRBV6-5 (Gene ID: 28602) (β chain). The numbering of amino acid residues in the α and β chains follows the IMGT standard (bold) compared to Kabat numbering. [Figure 1B] Q44 in the α chain (TRAV) and β chain (TRBV) is labeled with a grey undercross. Q44 according to IMGT numbering is a highly conserved residue in the framework 2 (FR2) region and is shared by about 80% of all TCR genes (Strong et al., 1999), including 1G4 (pdbID: 2BNR (Chen et al., 2005)); TRAV8-6 (Gene ID: 28680) (α chain) and TRBV6-5 (Gene ID: 28602), (β chain) and R7P1D5 (comprising TRAV5 and TRBV12-4, Lefranc et al. 2001). Q44 is very often part of a four AA motif comprising WYXQ, where X is any amino acid, e.g., R, V or K. W41 is even more highly conserved (>95%) among the TCR α and β chains (Strong et al., 1999). [Figure 1C] Same as above [Diagram 2]A: Structural motif of αQ44 / βQ44 from the variable domain of TCR 1G4 (pdbID: 2BNR (Chen et al., 2005)). B: Computer-simulated double mutant based on the crystal structure of 1G4 TCR. We manually selected and engineered possible pairs from the wild-type αQ44 / βQ44 pair that maintain a high level of molecular contacts (polar or non-polar) while breaking the steric and / or charge symmetry. Mutants were generated using UCSF Chimera (Pettersen et al., 2004). Throughout the figures, the α and β chains of the TCR are represented by dark blue and cyan ribbons, respectively. Side chains of interest are highlighted in magenta and all heavy atoms are shown. [Diagram 3] Shown are the sequences of the TCR variants R7P1D5 α and β chains (TRAV5 and TRBV12-4); TRAV8-6 (α chain variable domain); and TRBV6-5 (β chain variable domain), which detect a peptide designated MAG-003 when bound to MHC. Grey undercrosses mark the approximate locations of CDR1, CDR2, and CDR3, along with the framework regions FR1, FR2, and FR3. As can be seen, Q44 is located in FR2. Note that, like TR4V6-6 and TRBV6-5, Q44 is also conserved in the other TCR variants. Like the latter, Q44 is in the R7P1D5 portion of the four AA motif comprising WYXQ. Depending on the respective antigen being targeted, the CDR sequences may of course vary. [Figure 4A] Calculated mutation energies are shown for selected computationally engineered mutants. Double mutations are selected for resulting shape and / or charge complementarity at the TRAV / TRBV interface. Selection of charged amino acids (D, E, K, R) are shown. [Figure 4B]A selection of neutral amino acids (W, V, L) is shown. For each proposed pair of complementary mutations, we tested double mutants (green) as well as individual single mutants (orange) to mimic the pairing of the engineered strand with the wild-type strand. Mutation energies higher than 0.5 kcal / mol are considered destabilizing, mutation energies lower than -0.5 kcal / mol are considered stabilizing, and mutation energies between -0.5 and 0.5 kcal / mol are considered neutral (between the red lines - software default parameters). Mutations were performed on the variable domain of the 1G4 TCR (Chen et al., 2005) using Discovery Studio (Dassault Systemes, BIOVIA, 2017) and the mutation energy algorithm described by Spassov and Yan (2013). [Diagram 5] MAG-003 (exemplary peptide):HLA-A*02 tetramer or NYESO1-001 (control peptide):HLA-A*02 tetramer staining of CD8+ T cells electroporated with wild-type and mutant α- and β-chain RNA of TCR R7P1D5, respectively. R7P1D5 detects MAG-003 peptide but not NYESO1 peptide upon binding to MHC. Mock-electroporated CD8+ T cells (without TCR) served as control. Donors (left panel: Donor-A, right panel: Donor-B). [Figure 6] Figure 1 shows IFN release by MAG-003 as an exemplary peptide of HLA-A*02 in the context of CD8+ T cells electroporated with wild-type and mutant α- and β-chain RNA of TCR R7P1D5. All mutants (αQ44R / βQ44D, αQ44E / βQ44K, αQ44K / βQ44E, αQ44D / βQ44K, αQ44K / βQ44D, αQ44E / βQ44R) show improved MAG-003 recognition when compared to unmodified R7P1D5 wild-type. [Figure 7]PRAME-004-specific TCRs R11A and R17A and their corresponding α44K / β44E mutants were transduced into human T cells, respectively, via lentiviral transduction. The activity of the TCR-transduced T cells was assessed by co-incubation with T2 cells loaded with decreasing concentrations of PRAME-004 peptides. The mutant TCR R11KEA shows significantly improved PRAME-004 recognition when compared to the unmodified R11 TCR. [Figure 8] PRAME-004-specific TCRs R17A and R11A and their corresponding 44K / 44E mutants (R11KEA) were transduced into human T cells from two donors through lentiviral transfer. The cytotoxicity of the transduced T cells against tumor cell lines A375 and U2OS expressing PRAME-004 was evaluated through an IncuCyte imaging system. MAG-003-specific TCRs were used as controls (A375 and U2OS also express MAG-003). When compared to the unmodified R11A TCR, the mutant TCR R11KEA shows an increased killing rate of PRAME-004-positive cell lines. EXAMPLES

[0069] It should be noted that the numbering according to the IMGT standard may deviate from the simple numbering of a given amino acid sequence of a TCR variable domain, as seen in Figures 1A-C, especially due to gaps in the CDR sequences. This applies, for example, to TRAV8-6 and TRBV6-5, where the wavy underlines indicate gaps that are not occupied by amino acid residues but are taken into account in the IMGT numbering. These sequences are provided only as examples and, although preferred, are not intended to limit the claims to specific embodiments. This means that the teachings of the present invention are applicable to other TCR α and β chains or TCR αβ heterodimers with other sequences, especially in the variable domains, especially in the CDRs.

[0070] Furthermore, the teachings of the present invention are also applicable to other TCR α and β chains or TCR αβ heterodimers that bind to other target antigen / MHC complexes, preferably, but not exclusively, when they comprise the naturally occurring Q44 amino acid, preferably when they comprise the WYXQ motif. Depending on the respective antigen to be targeted, the CDR sequences may vary.

[0071] Generally, methods for cloning and expressing T cell receptors are disclosed in Walchli et al. (2011). Methods for random or site-directed mutagenesis resulting in site-specific amino acid substitutions are well known to those skilled in the art and are disclosed, for example, in Labrou (2010) and Trehan et al. (2016).

[0072] Methods of genome editing (e.g., CRISPR Cas, TALEN, ZFN, Argonote (NgAgo) or CRISPR Cpf1) for modifying a given amino acid sequence are well known to those of skill in the art and are disclosed, for example, in Maeder and Gersbach (2016). Methods of gene synthesis are well known to those of skill in the art and are disclosed, for example, in Hughes et al. (2011). The disclosures of these references are incorporated by reference in their entirety.

[0073] Computer simulation method By visually inspecting the variable domain of 1G4 TCR (pdb ID: 2BNR (Chen et al., 2005)), we manually selected pairs of mutations for the α44 / β44 motif that would potentially maintain a high level of molecular contacts (polar or non-polar) while breaking the steric and / or charge symmetry. The mutation pairs were selected such that (i) the total charge of the pair was zero, (ii) the two amino acids potentially showed good shape complementarity and / or formed hydrogen bonds and / or salt bridges, and (iii) the two amino acids had different molecular weights (i.e., one large and one small). Discovery Studio software (Dassault Systemes, BIOVIA, 2017) was used to further investigate the effect of the engineered positions on the αβ pairing of TCR. Mutation energies were calculated using the algorithm described by Spassov et al. (Spassov and Yan, 2013) and the antibody was designed by computer simulation. The mutation energies were predicted to reflect the effect of the mutations relative to the wild-type motif αQ44 / βQ44. We tested the double mutant as well as each single mutant paired with the wild-type strand, In the case of double mutants, the mutation energy is predicted to be neutral or stabilizing; For a single mutant paired with a wild-type strand, the mutational energy was predicted to be destabilizing in at least one of two ways.

[0074] Human primary CD8+ T cells were electroporated without RNA (no TCR) or with equal amounts of RNA encoding peptide-specific (e.g. MAG-00, e.g. peptide "p286" disclosed in Wu et al. Scandinavian journal of immunology 74:6 2011 Dec pages 561-7) T cell receptor chains α and β in their wild type (R7P1D5 TCR wt) or mutant form incorporating the Q44D mutation in the TCR α variable domain and the Q44R mutation in the TCR β variable domain (R7P1D5 TCR αQ44D / βQ44R). After overnight culture, the T cells were transfected with MAG-003:HLA-A* Binding of the 02 tetramer (Figure 5, top row) and the irrelevant peptide NYESO1-001:HLA-A * Binding of the 02 control tetramer (Figure 5, bottom row) (peptide NYESO-001; epitope ID 59283 in the epitope database as above) was analyzed. The percentage of tetramer-positive T cells is shown for two individual donors (Figure 5, left panel: donor A, right panel: donor B).

[0075] TCR R7P1D5 encoding tumor-specific TCR-α and TCR-β chains was isolated and amplified from T cells of healthy donors. Cells from healthy donors were stimulated in vitro as previously described (Walter et al., 2003) and expressed HLA-A. * Target-specific cells were single-cell sorted using 02 multimers and then used for subsequent TCR isolation. TCR sequences were isolated through 5'RACE by standard methods, as described, for example, in Molecular Cloning a laboratory manual fourth edition by Green and Sambrook. The α and β variable regions of TCR R7P1D5 were sequenced and cloned for further functional characterization. TCR R7P1D5 binds to HLA-A * Derived from 02 positive donor.

[0076] References cited: DavisMM, Bjorkman PJ T-cellantigen receptor genes and T-cell recognition. (1988). 334(6181), 395~402. ColeDK et al. Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor. (2009). 284(40),27281~27289. LiH et al. Structure-functionstudies of T-cellreceptor-superantigeninteractions. (1998). 163, 177~186. BurrowsSR Hard wiring of T cell receptor specificity for themajor histocompatibility complex is underpinned by TCR adaptability. (2010). 107(23),10608~10613. Roomp K Domingues FS (2011). Predicting interactions between T-cell receptors and MHC-peptidecomplexes. Mol Immunol 48:553~562. Rosenberg SA et al. (1988). Use of tumor-infiltrating lymphocytes and interleukin- 2in the immunotherapy of patients with metastatic melanoma. A preliminaryreport. N Engl J Med 319:1676~1680. Shao H et al. TCR mispairing in genetically modified T cells wasdetected by fluorescence resonance energy transfer. (2010). 37(8),3951~3956. LefrancMP et al. IMGT unique numbering for immunoglobulin and T cell receptor variabledomains and Ig superfamily V-likedomains. Dev. Comp. Immunol. (2003) 27, 55-77 LefrancMP et al. The T cell receptor facts book. (2001) Bendle GM et al. Preclinical development of T cellreceptor gene therapy. Curr. Opin. Immunol. (2009). 21, 209~214 PogulisRJ, Pease LR. A retroviral vector that directs simultaneous expression of α andβ T cell receptor genes. Hum Gene Ther. (1998) Oct 10;9(15): 2299-304. Zhong S etal. Retroviral Transduction of T-cell Receptors in Mouse T-cells J Vis Exp. (2010); (44): 2307 WälchliS et al. A Practical Approach to T-Cell Receptor Cloning and Expression. PLOS ONE (2011)6(11): e27930 O’SheaEK et al. Peptide “Velcro”: design of a heterodimeric coiled coil. (1993). 3(10),658~667. ChangHC et al. A general method for facilitating heterodimeric pairing between twoproteins: application to expression of alpha and beta T-cellreceptor extracellular segments. (1994). 91(24), 11408~11412. VarrialeS et al. An evolutionary conserved motif is responsible for immunoglobulinheavy chain packing in the B cell membrane. (2010). GoversC et al. T cell receptor gene therapy: strategies for optimizing transgenic TCRpairing. Trends Mol Med. (2010) Feb;16(2):77-87 LøsetGA et al., Phage Display Engineered T Cell Receptors as Tools for the Study ofTumor Peptide-MHC Interactions. Front Oncol (2014); 4: 378. HughesRA et al. Gene synthesis: methods and applications. Methods Enzymol. (2011);498: 277-309 Labrou NE.Random mutagenesis methods for in vitro directed enzyme evolution. Curr ProteinPept Sci. (2010) Feb;11(1):91-100. Trehan A etal. REPLACR-mutagenesis, a one-step method for site-direc Scientific Reports (2016) 6, Article number:19121 Maeder ML& Gersbach CA. Genome-editingTechnologies for Gene and Cell Therapy. Molecular Therapy (2016); 24 3,430~446. Verhoeyen E et al. Lentiviral vector gene transferinto human T cells. Methods Mol Biol. 2009;506:97-114 Huang X et al. Sleeping Beauty Transposon-mediated Engineering of Human Primary T Cells forTherapy of CD19+ Lymphoid Malignancies. Molecular Therapy (2008); 16 3,580~589. Kim TK & Eberwine JH. Mammalian cell transfection:the present and the future. Anal Bioanal Chem. 2010 Aug; 397(8): 3173~3178. Chen, JL et al. (2005). Structural and kinetic basisfor heightened immunogenicity of T cell vaccines. The Journal of ExperimentalMedicine, 201(8), 1243~1255. Pettersen EF et al. (2004). UCSF Chimera--a visualization system for exploratory research andanalysis. Journal of Computational Chemistry, 25(13), 1605~1612. Spassov VZ & Yan L (2013). pH-selective mutagenesis of protein-protein interfaces: in silico design of therapeutic antibodieswith prolonged half-life.Proteins: Structure, Function, and Bioinformatics, 81(4), 704~714. Cohen CJ et al. Enhanced antitumor activity of murine-human hybrid T-cellreceptor (TCR) in human lymphocytes is associated with improved pairing and TCR / CD3stability. Cancer Res. 2006 Sep 1;66(17):8878-86. Cohen CJ et al. Enhanced antitumor activity of T cellsengineered to express T-cellreceptors with a second disulfide bond. Cancer Res. 2007 Apr 15;67(8):3898-903. Voss RH et al. Molecular design of the Calphabetainterface favors specific pairing of introduced TCRalphabeta in human T cells.J Immunol. 2008 Jan 1;180(1):391-401. Walter et al. (2003) J Immunol., Nov 15;171(10):4974-8. Knies et al. An optimized single chain TCR scaffoldrelying on the assembly with the native CD3-complex prevents residual mispairing with endogenousTCRs in human T-cells. Oncotarget. 2016 Apr 19; 7(16):21199~21221. ThomasHoffmann et al. Quantitative Analysis of the Association Angle between T-cell ReceptorVα / Vβ Domains Reveals Important Features for Epitope Recognition. PLOS, July17, 2015, http: / / dx.doi.org / 10.1371 / journal.pcbi.1004244

Claims

1. A recombinant T cell receptor (TCR) heterodimer comprising a modified alpha chain or an antigen-MHC complex binding fragment thereof comprising a variable domain and a modified beta chain or an antigen-MHC complex binding fragment thereof comprising a variable domain, wherein the TCR maintains the ability to bind to an antigen-MHC complex, and the modifications of the alpha and beta chain are within the variable domain and are selected from the group of substitution pairs consisting of α44D / β44R; α44R / β44D; α44E / β44K; α44K / β44E; α44D / β44K; α44K / β44D; and α44E / β44R, and wherein position 44 is defined by IMGT numbering.

2. The modification of the α and β chains is αQ44D / βQ44R; αQ44R / βQ44D; αQ44E / βQ44K; αQ44K / βQ44E; αQ44D / βQ44K; αQ44K / βQ44D; αQ44E / βQ44R; R; αW44R / βQ44D; αW44E / βQ44K; αW44K / βQ44E; αW44D / βQ44K; αW44K / βQ44D; αW44E / βQ44R; αH44D / βQ44R; αH44R / βQ44D; αH44E / βQ44K; α H44K / βQ44E; αH44D / βQ44K; αH44K / βQ44D; αH44E / βQ44R; αK44D / βQ44R; αK44R / βQ44D; αK44E / βQ44K; αK44 / βQ44E; αK44D / βQ44K; αK44 / βQ44D; αK44E / βQ44R; αE44D / βQ44R; αE44R / βQ44D; αE44 / βQ44K; αE44K / βQ44E; αE44D / βQ44K; αE44K / βQ44D; αE44 / βQ44R; αQ44R / βR44D; αQ44E / βR44K; αQ44K / βR44E; αQ44D / βR44K; αQ44K / βR44D; αQ44E / βR44; αW44D / βR44; αW44R / βR44D; αW44E / βR44K; αW4 4K / βR44E; αW44D / βR44K; αW44K / βR44D; αW44E / βR44; αW44 / βR44V; αH44D / βR44; αH44R / βR44D; αH44E / βR44K; αH44K / βR44E; αH44D / βR4 4K; αH44K / βR44D; αH44E / βR44; αK44D / βR44; αK44R / βR44D; αK44E / βR44K; αK44 / βR44E; αK44D / βR44K; αK44 / βR44D; αK44E / βR44; αE44D / βR44; αE44R / βR44D; αE44 / βR44K; αE44K / βR44E; αE44D / βR44K; αE44K / βR44E; αE44D / βR44K; αE44K / βR44D.

3. A recombinant T cell receptor (TCR) heterodimer as described in claim 1, wherein the modification of the alpha chain and beta chain is the substitution pair α44K / β44E.

4. A recombinant T cell receptor (TCR) heterodimer as described in claim 1, wherein the modifications of the α chain and β chain are substitution pairs αQ44K / βQ44E, αW44K / βQ44E, αH44K / βQ44E, αK44 / βQ44E, αE44K / βQ44E, αQ44K / βR44E, αW44K / βR44E, αH44K / βR44E, αK44 / βR44E or αE44K / βR44E.

5. A nucleic acid molecule or a set of nucleic acid molecules encoding a modified T cell receptor (TCR) alpha chain or beta chain or a recombinant T cell receptor (TCR) heterodimer described in any one of claims 1 to 4.

6. A plasmid or expression vector comprising at least one of the nucleic acid molecules described in claim 5.

7. A method for preparing modified T cells, comprising the step of transducing or transfecting T cells obtained from a subject with one or more nucleic acid molecules described in claim 5, or a plasmid or expression vector described in claim 6.

8. A modified T cell produced according to claim 7.

9. The modified T cells of claim 8 for use in autologous T cell therapy for a subject in need of therapy.

10. The modified T cells of claim 8 for use in treating a subject in need of treatment who is suffering from, or at risk of developing, and / or has been diagnosed with a neoplastic disease, inflammatory disease, infectious disease or autoimmune disease, comprising the step of administering to the subject in need of treatment a formulation comprising the modified T cells.

11. A recombinant T cell receptor (TCR) heterodimer described in any one of claims 1 to 4, wherein the TCR specifically binds to an antigen presented by an MHC complex, and the antigen is selected from epitopes of tumor-associated antigens (TAA).

12. A heterodimer comprising a modified TCR alpha chain and a modified TCR beta chain, In the variable domains of the modified TCR alpha chain and the modified TCR beta chain, the amino acid at position 44 according to the IMGT numbering is replaced with another amino acid; the α and β chain modifications are selected from substitution pairs in the group consisting of α44D / β44R; α44R / β44D; α44E / β44K; α44K / β44E; α44D / β44K; α44K / β44D; and α44E / β44R; A heterodimer in which the modified α chain and the modified β chain maintain the ability to bind to the antigen-MHC complex.

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