HIV-specific binding molecules and TCR

Abstract

HLA-A derived from the HIV Gag gene product p17 * Specific binding molecules comprising a T cell receptor (TCR) that binds to the 02 restricted peptide SLYNTVATL (SEQ ID NO: 1) are presented. The TCRs of the invention comprise non-naturally occurring mutations within the alpha and / or beta variable domains relative to native TCRs. The specific binding molecules of the invention have improved stability and / or yield, and yet unexpectedly retain the favorable properties of the specific binding molecules from which they are derived. Such specific binding molecules are particularly useful for the development of soluble immunotherapeutic reagents for the treatment of HIV-infected individuals.

Description

Summary of the Invention

[0001] The present invention relates to HLA-A derived from the HIV Gag gene product p17. * The present invention relates to specific binding molecules, such as T cell receptors (TCRs), that bind to the 02 restricted peptide SLYNTVATL (SEQ ID NO: 1). The specific binding molecules may comprise CDR sequences embedded within framework sequences. The CDR and framework sequences may correspond to T cell receptor (TCR) variable domains and may further comprise non-naturally occurring mutations relative to the native TCR variable domain. The specific binding molecules of the present invention have improved stability and / or yield and, moreover, unexpectedly, favorable properties of the specific binding molecules from which they are derived (SEQ ID NO: 1 and HLA-A * 02 and drive particularly potent T cell responses. Such specific binding molecules are particularly useful for the development of soluble immunotherapeutic reagents for the treatment of HIV-infected individuals.

[0002] HLA-A * A specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) which forms a complex with .02, and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain is (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally comprising an N-terminal methionine, The β chain variable domain is Disclosed herein is a specific binding molecule comprising DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO: 5), optionally with an N-terminal methionine, an amino acid sequence (SEQ ID NO: 44).

[0003] In some embodiments, the specific binding molecule is HLA-A * 02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain is (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally having an N-terminal methionine, The β chain variable domain is The amino acid sequence of DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), optionally with an N-terminal methionine: MDAGVTQSPT HLIKTRGQQV TLRCSPKSGH DTVSWYQQAL GQGPQFIFQA VRGVERQRGN FPDRFSGHQF PNYSSELNVN ALLLGDSALY LCASSDTVSY EQYFGPGTRL TVT (SEQ ID NO: 44), has.

[0004] In another embodiment, a nucleic acid molecule encoding a TCR alpha chain and / or a TCR beta chain, wherein the TCR alpha chain is (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and / or comprising a variable domain amino acid sequence selected from the group consisting of: The TCR β chain Disclosed herein is a nucleic acid molecule comprising DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), and optionally a variable domain amino acid sequence having an N-terminal methionine (SEQ ID NO:44).

[0005] In some embodiments, the nucleic acid molecule encodes a TCR alpha chain and / or a TCR beta chain, wherein the TCR alpha chain is (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and / or comprising a variable domain amino acid sequence selected from the group consisting of: The TCR β chain is DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), and optionally the variable domain amino acid sequence (SEQ ID NO:44) with an N-terminal methionine.

[0006] In another embodiment, HLA-A * 1. A pharmaceutical composition comprising a specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) which forms a complex with .02, the specific binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the alpha chain variable domain amino acid sequence being: (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally comprising an N-terminal methionine, The β chain variable domain is Disclosed herein is a pharmaceutical composition comprising DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO: 5), and optionally an amino acid sequence having an N-terminal methionine (SEQ ID NO: 44).

[0007] In some embodiments, the pharmaceutical composition comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain has a TCR alpha variable domain and a TCR beta chain variable domain, and the alpha chain variable domain comprises (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally having an N-terminal methionine, The β chain variable domain is DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), and optionally an amino acid sequence having an N-terminal methionine (SEQ ID NO:44).

[0008] Provided herein is a method for treating HIV infection or AIDS in a human subject, comprising administering to a subject an HLA-A * The method comprises administering a therapeutically effective amount of a specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) in a complex with .02, the specific binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the alpha chain variable domain being (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally comprising an N-terminal methionine, The β chain variable domain is Also disclosed are methods comprising the amino acid sequence DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), optionally with an N-terminal methionine (SEQ ID NO:44).

[0009] In some embodiments, the method comprises: * The method comprises administering a therapeutically effective amount of a specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) in a complex with .02, the specific binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain is (a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), (b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or (c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally having an N-terminal methionine, The β chain variable domain is DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO:5), and optionally an amino acid sequence having an N-terminal methionine (SEQ ID NO:44).

[0010] In yet another aspect, disclosed herein is (a) a TCR expression vector comprising a nucleic acid as disclosed herein in a single open reading frame or in two separate open reading frames encoding the alpha chain and the beta chain, respectively; or (b) a cell comprising a first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as disclosed herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as disclosed herein.

[0011] In yet another aspect, disclosed herein are isolated or non-naturally occurring cells, particularly T cells, that display a TCR as disclosed herein. [Background technology]

[0012] Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS). The virus is an enveloped retrovirus that belongs to the lentivirus group. In 2020, it was estimated that approximately 40 million adults and children worldwide have HIV (Non-Patent Document 1; accessed June 29, 2021, https: / / www.unaids.org). Current treatment relies on the use of combination antiretroviral therapy (ART) to control viral infection. However, although effective, these treatments have not been able to completely eradicate the infection because a reservoir of long-term surviving latently infected CD4+ T cells is rapidly established as a result of stable integration of viral genes into host cell chromosomes (Non-Patent Document 2). Typically, viral rebound occurs upon treatment cessation, meaning that lifelong treatment is necessary. Although the majority of humans living with HIV mount a vigorous T cell response to the HIV Gag protein during the course of infection, these T cells are unable to eliminate long-term surviving CD4+ T cells harboring replication-competent provirus. Immunotherapeutic strategies tested to date have not produced meaningful clinical benefits in terms of post-treatment management and / or reduction of the HIV reservoir (Non-Patent Documents 3 and 4). Thus, novel therapies are needed that have the potential to eradicate the viral reservoir and achieve a functional cure.

[0013] A novel immunotherapeutic approach involves generating a strong immune response against HIV-infected cells by using engineered T cell receptors (TCRs). In reality, T cells and TCRs typically have a weak affinity for antigens, in the low micromolar to nanomolar range. Engineering the TCR by mutating the antigen recognition site can generate an increase in antigen affinity, which can lead to an enhanced immune response in vivo. In the context of HIV, the enhanced response should be sufficient to eradicate replication-competent virus from viral reservoirs where antigen expression is at low levels. Such engineered TCRs can be used in cell therapy applications, together with genetically modified T cells (see Non-Patent Document 5). Alternatively, engineered TCRs can be produced as soluble reagents for the purpose of delivering cytotoxic or immunostimulatory agents to infected cells (Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Patent Document 1). Similar approaches have been successfully developed for the treatment of certain cancers (Non-Patent Document 9).

[0014] For soluble TCRs to be used as therapeutic agents, the affinity for the antigen (K D ) and / or binding half-life that is particularly high, e.g., K in the picomolar range. D and / or a binding half-life of several hours is desirable. Such high affinity is necessary to drive a strong response against target cells presenting low levels of antigen. In all applications involving affinity engineered TCRs, it is essential that the TCR not only has a higher affinity for the antigen than the corresponding wild-type TCR, but also retains a high level of antigen specificity. Loss of specificity in this context can result in off-target effects when such TCRs are administered to patients.

[0015] Affinity maturation typically requires a person skilled in the art to identify specific mutations and / or combinations of mutations, including but not limited to substitutions, insertions and / or deletions, that can be made to the WT TCR sequence to increase antigen recognition. Methods for identifying mutations of a given TCR that confer affinity enhancement are known in the art, for example, using display libraries (Non-Patent Document 10, Non-Patent Document 11). However, to generate a significant increase in the affinity of a given TCR for a given target, a person skilled in the art must select a specific mutation and / or combination of mutations from a large pool of possible alternatives. In many cases, it may not be possible to achieve the desired affinity and specificity. The mutations required for high affinity and specificity also produce TCRs that can be expressed, refolded and purified in reasonable yields and are highly stable in purified form.

[0016] The peptide sequence SLYNTVATL (SEQ ID NO: 1) is derived from the p17 gene product of the Gag gene, one of nine genes that make up the HIV virus, and T cell responses to it have been shown to be particularly effective in controlling viral load, indicating that this epitope is immunodominant (Non-Patent Documents 12, 13, and 14). This peptide (referred to herein as Gag) is expressed by the HLA-A antigen on the surface of HIV-infected cells. * Gag-specific T cells have also been detected, albeit at a lower frequency, in ART-treated (non-aviremic) individuals (Non-Patent Document 15, Non-Patent Document 16, Non-Patent Document 17). Thus, Gag-HLA-A * The 02 complex provides an ideal target for TCR-based recognition of HIV-infected cells.

[0017] Patent Document 2 describes Gag-HLA-A *discloses TCRs that are mutated relative to the WT TCR, recognizing the 02 complex. CD8+ cytotoxic T cells transduced with the affinity-enhanced TCRs were able to control HIV infection in vitro at appropriate effector-target ratios for T cell therapy. These TCRs were able to recognize all the most common viral escape peptides (Non-Patent Document 18). Such TCRs have utility in adoptive T cell therapy, as well as therapeutic agents based on soluble TCRs. Furthermore, Yang et al. describe in vivo studies showing that bispecific molecules incorporating the TCRs disclosed in Patent Document 2 can eliminate HIV-infected CD4+ T cells from ART-treated individuals by redirecting polyclonal (non-HIV-specific) CD8+ T cells and avoiding the potential for HIV-specific immune effectors to become dysfunctional (Non-Patent Document 19).

[0018] The inventors have found that certain mutations in the TCR disclosed in WO 2007 / 023363 can unexpectedly increase yield and / or stability during production in E. coli without affecting target binding. Such molecules have ideal properties for clinical development. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] WO 03 / 020763 [Patent Document 2] International Publication No. 2017163064 [Non-patent literature]

[0020] [Non-Patent Document 1] AIDS by the Numbers. UNAIDS. 2020 [Non-Patent Document 2] Siliciano et al. 2003 Nat Med, 9, 727 [Non-Patent Document 3] Ward AR et al., Semin Immunol. 2020: 101412 [Non-Patent Document 4] Barr L, et al., Journal of Virus Eradication. 2020; 6: 100010 [Non-Patent Document 5] Vonderheide and June, 2014, Immunol Rev, 257, 7-13 [Non-Patent Document 6] Lissin, et al., (2013). "High-Affinity Monocloncal T-cell receptor (mTCR) Fusions. Fusion Protein Technologies for Biophamaceuticals: Applications and Challenges". S. R. Schmidt, Wiley [Non-Patent Document 7] Boulter, et al., (2003), Protein Eng 16(9): 707-711 [Non-Patent Document 8] Liddy, et al., (2012), Nat Med 8: 980-987 [Non-Patent Document 9] Nathan, P. et al. New Engl J Med 385, 1196-1206 (2021) [Non-Patent Document 10] Li et al., (2005) Nat Biotechnol. 23(3): 349-354 [Non-Patent Document 11] Holler et al., (2000). Proc Natl Acad Sci U S A; 97(10): 5387-5392 [Non-Patent Document 12] Rolland et al. 2008, PLoS One, 3:e1424 [Non-Patent Document 13] Streeck H, et al. J Virol. 2009; 83(15): 7641-7648 [Non-Patent Document 14] Pereyra et al. J Virol. 2014;88(22): 12937-12948 [Non-Patent Document 15] Gray CM, et al. J Immunol. 1999; 162(3): 1780-1788 [Non-Patent Document 16] Ogg GS, et al. J Virol. 1999; 73(1): 797-800 [Non-Patent Document 17] Seth A, et al. J Infect Dis. 2001; 183(5): 722-729 [Non-Patent Document 18] Varela-Rohena et al. 2008, Nat Med, 14(12): 1390-5 [Non-Patent Document 19] Yang H, et al. Mol Ther. 2016; 24(11): 1913-1925 [Brief description of the drawings]

[0021] [Figure 1] FIG. 1a shows the amino acid sequences of the TCR α chain variable region and β chain variable region of the TCR disclosed in Patent Document 2, and FIG. 1b shows the amino acid sequence of the bispecific protein disclosed in Patent Document 2. [Diagram 2] 1 is the amino acid sequence of the TCR α chain variable region of a specific binding molecule of the invention, with the F50K mutation highlighted in grey. [Diagram 3] FIG. 1 is a graph showing the yield of bispecific proteins with different mutations per culture volume. [Figure 4] 1 is a graph showing the binding kinetics of the M49K and F50K mutants. [Diagram 5]Amino acid sequence of the TCR α chain variable region of a specific binding molecule of the invention with the F50K and S96A mutations highlighted in grey. [Figure 6] 1 is an amino acid sequence of a bispecific protein of the invention. [Figure 7] Graphs showing efficacy of T cell redirection as determined by IFNγ (top) and GrB (bottom) release. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In a first aspect, the present invention relates to an HLA-A * A specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) which forms a complex with .02, the specific binding molecule comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, The alpha chain variable domain is a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), or c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), and optionally comprising an N-terminal methionine, The β chain variable domain is A specific binding molecule is provided that comprises the amino acid sequence DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO: 5), optionally with an N-terminal methionine (SEQ ID NO: 44).

[0023] In the above amino acid sequences of the TCR α and β chain variable domains of the specific binding molecules of the invention, the shaded residues indicate the CDRs and the underlined residues indicate mutations relative to the TCR α and β chain variable domains of the TCR disclosed in WO 2007 / 023111 (see Figures 1a and 1b herein, respectively). When an amino acid is referred to herein by its numerical position, the numbering of that position assumes the presence of an optional N-terminal methionine.

[0024] The present invention provides specific binding molecules that can be produced in increased yields and / or have unexpectedly high stability, while also unexpectedly retaining the favorable properties of the specific binding molecules from which they are derived. These include excellent antigen binding properties, including picomolar antigen affinity, long binding half-life, the ability to mediate potent immune activation against HIV-infected cells that present extremely low levels of antigen when fused to an activating moiety, and a high level of specificity. The specific binding molecules of the present invention are particularly suitable for use as soluble targeting reagents in treating HIV-infected individuals.

[0025] Specific binding molecules or binding fragments thereof can be used to generate molecules with ideal therapeutic properties such as supraphysiological affinity for the target, long binding half-life, high specificity for the target, and excellent stability. The present invention also includes bispecific, bifunctional, or fusion molecules incorporating specific binding molecules or binding fragments thereof and T cell redirecting moieties. Such molecules can mediate a strong and specific response against HIV-infected cells by redirecting and activating polyclonal T cell responses. Furthermore, the use of specific binding molecules with supraphysiological affinity promotes the recognition and clearance of HIV-infected cells that present low levels of peptide-HLA. Alternatively, specific binding molecules or binding fragments may be fused to other therapeutic and / or diagnostic agents and / or incorporated into engineered T cells for adoptive therapy.

[0026] TCR domain sequences may be defined according to the IMGT nomenclature, which is widely known and available to those working in the TCR field. See, for example, LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press, Lefranc, (2011), Cold Spring Harb Protoc 2011(6): 595-603, Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10, and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, the αβ TCR consists of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains, a variable domain and a constant domain. A short linking region connects the variable and constant domains, and this linking region is typically considered to be part of the α variable region. In addition, the β chain usually contains a short diversity region next to the joining region, which is also typically considered as part of the β variable region. The variable domain of each chain is located at the N-terminus and contains three complementarity determining regions (CDRs) embedded in framework sequences (FRs). The CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding α chain variable (Vα) regions and several genes encoding β chain variable (Vβ) regions, which are distinguished by the framework, CDR1 and CDR2 sequences, and by the CDR3 sequence, which is partially defined. The Vα and Vβ genes are referred to by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press).Similarly, there are several junction genes, or J genes, designated TRAJ or TRBJ for the α and β chains, respectively, and a diversity gene, or D gene, for the β chain, designated TRBD (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2): 107-114; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). The great diversity of T cell receptor chains arises from combinatorial rearrangements between various V, J and D genes, including allelic variants and junctional diversity (Arstila, et al., (1999), Science 286(5441): 958-961; Robins et al., (2009), Blood 114(19): 4099-4107). The constant regions of the TCR α and β chains, i.e., the C regions, are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

[0027] As used herein, the term "specific binding molecule" refers to a molecule capable of specifically binding to a target antigen. Such molecules may take on a number of different formats as discussed herein and may be bispecific, i.e., they have a first binding region that specifically binds to a first target antigen and a second binding region that specifically binds to a second target antigen. Furthermore, fragments of the specific binding molecules of the present invention are also contemplated. A fragment refers to a portion of a specific binding molecule that retains specific binding to a target antigen.

[0028] The term "mutation" includes substitutions, insertions and deletions. Mutations to a native (also referred to as parent, natural, non-mutated, wild-type, or scaffold) specific binding molecule can confer beneficial therapeutic properties, such as high affinity, high specificity, and high efficacy. For example, mutations include SLYNTVATL (SEQ ID NO: 1)-HLA-A * 02 Binding affinity of a specific binding molecule to a complex (k D ) and / or binding half-life (t 1 / 2 In the present invention, mutations can unexpectedly further increase yield and / or stability while retaining the beneficial properties mentioned above.

[0029] The alpha chain variable domain has the amino acid sequence: a) A KEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIM K L YADPDKEDGR FTAQLNKASQ Y V SLLIRDS Q P SDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), may include.

[0030] We unexpectedly found that mutating the F residue at position 50 to K improved yields during production in E. coli without affecting target binding.

[0031] Alternatively, the alpha chain variable domain has the amino acid sequence: b) A KEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ Y V SLLIRDS Q P SDSATYLCA VRTN A GYALNFGKGTSLVT P (SEQ ID NO: 3), Includes.

[0032] We found that mutating the S residue at position 96 to A improved stability, again unexpectedly, without affecting target binding.

[0033] Preferably, the alpha chain variable domain has the amino acid sequence: c) A KEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIM K L YADPDKEDGR FTAQLNKASQ Y V SLLIRDS Q P SDSATYLCA VRTN A GYALNFGKGTSLVT P (SEQ ID NO: 4), Includes.

[0034] Specific binding members comprising such an alpha chain variable domain unexpectedly have both improved yield and improved stability, while target binding remains unaffected.

[0035] The specific binding molecules of the present invention, especially in soluble form, are suitable for high-yield purification, especially when expressed in E. coli. The yield can be determined based on the amount of correctly folded material obtained at the end of the purification process relative to the original culture volume. In some cases, the yield is determined as in Example 1 herein. High yield typically means a yield of more than 2 mg / L, or more preferably more than 3 mg / L, or more than 4 mg / L, or more than 5 mg / L, or more.

[0036] The specific binding molecules of the present invention, especially in purified form, may have improved stability. Stability in this context typically refers to the accumulation of degradation products and / or the increase in heterogeneity of the purified material over time. Typically, accelerated stability studies can be performed to provide an indication of long-term molecular stability. In such cases, the purified material may be exposed to stress conditions, such as extremes of temperature or pH. For example, the accumulation of acidic species under high pH conditions may be used as a measure of molecular instability. This process is further described in Example 2. When measured under the conditions described in Example 2, the relative decrease in the main peak and the corresponding increase in acidic species are preferably less than 35%, less than 30%, less than 25%, less than 20%, more preferably less than 15%. Additionally or alternatively, stability may be assessed by measuring the relative abundance of amino acid modifications, such as deamidations, over time. Preferably, there is a change in relative abundance of less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, more preferably less than 1%. Minimizing the risk of molecular instability is essential for successful clinical development.

[0037] In addition to the mutations discussed above, a specific binding molecule of the invention may have one or more further mutations in its α-chain variable domain. These mutations may be selected from A2Q, V73I, Q81K and P82L.

[0038] Combinations of these mutations can be as follows: A2Q, V73I, Q81K, P82L, A2Q and V73I, A2Q and Q81K, A2Q and P82L, V73I and Q81K, V73I and P82L, Q81K and P82L, A2Q, V73I, and Q81K, A2Q, V73I, and P82L, V73I, Q81K, and P82L, or A2Q, V73I, Q81K, and P82L. These mutations may be made to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.

[0039] Within the scope of the present invention are phenotypically silent variants of any of the specific binding molecules of the invention disclosed herein. As used herein, the term "phenotypically silent variants" is understood to refer to specific binding molecules incorporating one or more additional amino acid alterations, including substitutions, insertions and deletions, in addition to those mentioned above, which have a similar phenotype to the corresponding specific binding molecule that does not contain said alteration(s). For purposes of this application, the phenotype of a specific binding molecule is defined as the antigen binding affinity (K D and / or binding half-life), antigen specificity, yield and stability. Phenotypically silent variants have a measured K of the corresponding specific binding molecule that does not contain the above alteration(s) when measured under identical conditions (e.g., 25° C. and on the same SPR chip). D and / or within 50%, or more preferably within 20%, of the binding half-life of SLYNTVATL (SEQ ID NO: 1) HLA-A * K for 02 complex D and / or binding half-life. Suitable conditions are further provided in Example 3 of WO 2007 / 023363. Yield and stability are further defined above. Antigen specificity is further defined below. As known to those skilled in the art, SLYNTVATL (SEQ ID NO: 1) HLA-A * It may be possible to produce specific binding molecules incorporating changes in their variable domains compared to those detailed above without altering the affinity of the interaction with the 02 complex. In particular, such silent mutations may be incorporated in sequence parts known not to be directly involved in antigen binding (e.g. framework regions, or CDR parts that do not contact the peptide antigen). Such trivial variants are included within the scope of the present invention.

[0040] Phenotypically silent variants can be produced by incorporating one or more conservative substitutions and / or one or more tolerated substitutions. By tolerated substitutions, we mean substitutions that do not fall within the definition of conservative as provided below, but are nevertheless phenotypically silent. By conservative substitutions, we mean the replacement of one or more amino acids with alternative amino acids that share similar properties. Those skilled in the art know that various amino acids have similar properties and are therefore "conservative". One or more such amino acids of a protein, polypeptide or peptide can often be replaced by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide or peptide. Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be replaced with each other (amino acids with aliphatic side chains). Of these possible substitutions, it is preferred that glycine and alanine are used to replace each other (because they have relatively short side chains), and valine, leucine and isoleucine are used to replace each other (because they have larger aliphatic side chains that are hydrophobic). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). It is recognized that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on alanine may be replaced with an ethyl group and / or non-significant changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0041] Mutations, including conservative and tolerated substitutions, insertions and deletions, may be introduced into the provided sequences using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. For further details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) CSHL Press. Further information on ligation-independent cloning (LIC) methods can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The specific binding molecule sequences provided by the present invention can be obtained from solid phase synthesis, or any other suitable method known in the art.

[0042] The specific binding molecule of the present invention is SLYNTVATL-HLA-A * The specific binding molecule of the present invention has the property of binding to the SLYNTVATL-HLA-A 02 complex ("SLYNTVATL" disclosed as SEQ ID NO: 1). * The specific binding molecules of the present invention show a high degree of specificity for the 02 complex ("SLYNTVATL" disclosed as SEQ ID NO:1) and are therefore particularly suitable for therapeutic use. Specificity relates to the ability of the specific binding molecules of the present invention to recognize target cells that are antigen positive, while having a minimal ability to recognize target cells that are antigen negative. Antigen positive cells are those that have been determined to be infected with HIV and / or those that are SLYNTVATL-HLA-A. * 02 complex ("SLYNTVATL", disclosed as SEQ ID NO:1), or HLA-A, as discussed herein. * The specific binding molecules of the present invention have been determined to present an escape mutant of SLYNTVATL (SEQ ID NO: 1) presented by HLA-A 02. *When the specific binding molecule of the present invention binds to the HLA-A 02 subtype, it can bind to a complex of target peptides. For example, the specific binding molecule of the present invention can bind to the HLA-A 02 subtype. * When bound to 02:01, the specific binding molecules of the invention are capable of binding to a complex of target peptides and / or HLA-A * 02:05 and / or HLA-A * 02:06 and / or HLA-A * 02:07 and / or HLA-A * When bound to 02:02, it is capable of binding to the target peptide complex.

[0043] Specificity can be measured in vitro, for example in a cellular assay such as that described in Example 5 of WO 2005 / 023901. To test specificity, the specific binding molecule may be in soluble form, associated with immune effectors and / or expressed on the surface of cells, e.g. T cells. Specificity can be determined by measuring the level of T cell activation in the presence of antigen-positive and antigen-negative target cells as defined above. Minimal recognition of antigen-negative target cells is defined as a T cell activation level that is less than 20%, preferably less than 10%, preferably less than 5%, more preferably less than 1% of the level occurring in the presence of antigen-positive target cells, when measured under the same conditions and at a therapeutically relevant specific binding molecule concentration. For soluble TCR associated with immune effectors, a therapeutically relevant concentration is between 10 -9M or less, and / or up to 100-fold, preferably up to 1000-fold greater than the corresponding EC50 or IC50 value. Preferably, for soluble specific binding molecules associated with immune effectors, there is at least a 100-fold, at least a 1000-fold, at least a 10000-fold, at least a 10000-fold difference between the EC50 or IC50 values ​​of T cell activation for antigen-positive cells compared to antigen-negative cells. This difference may be referred to as the therapeutic window. Additionally or alternatively, the therapeutic window may be calculated based on the lowest effective concentration ("LOEL") observed for normal cells and HIV-infected cells. Antigen-positive cells can be obtained by peptide pulsing using an appropriate peptide concentration, resulting in low levels of antigen presentation, comparable to latently infected cells (e.g., 10-fold, as described in Bossi et al., (2013) Oncoimmunol. 1; 2(11): e26840). -9 M peptide), or they can naturally present said peptide. Preferably, both the antigen positive cells and the antigen negative cells are human cells. Preferably, the antigen positive cells are human cells, such as HIV-infected CD4+ T cells. Antigen negative cells preferably include those derived from healthy human tissue, or non-HIV-infected CD4+ T cells.

[0044] The specificity may additionally or alternatively be determined by determining whether the specific binding molecule is SLYNTVATL-HLA-A. * The ability of the peptide to bind to the HLA-02 complex ("SLYNTVATL" disclosed as SEQ ID NO:1) and not to a panel of other peptide-HLA complexes. Preferably, the other peptide-HLA complexes are HLA-A * 02. This can be determined, for example, by the Biacore method of Example 3 of US Pat. No. 5,999,366. The panel may contain at least 5, preferably at least 10, additional peptide-HLA complexes. The additional peptides may share a low level of sequence identity with SLYNTVATL (SEQ ID NO: 1) and may be naturally occurring. The additional peptides are preferably derived from proteins expressed in healthy human tissues (i.e. cells not infected with HIV). SLYNTVATL-HLA-A* Binding of a specific binding molecule to the 02 complex ("SLYNTVATL" disclosed as SEQ ID NO:1) can be at least 2-fold greater than to other naturally occurring peptide-HLA complexes, more preferably at least 10-fold, or at least 100-fold, or at least 1000-fold greater, or at least 3000-fold greater.

[0045] An alternative or additional approach to determining the specificity of a specific binding molecule may be to use serial mutagenesis of a target peptide, e.g., alanine scanning, to identify peptide recognition motifs of the specific binding molecule. Residues that form part of the binding motif are those for which substitutions are not tolerated. Non-tolerant substitutions may be defined as peptide positions at which the binding affinity of the specific binding molecule is reduced by at least 50%, or at least 80%, relative to the binding affinity for the non-mutated peptide. Such approaches are further described in Cameron et al., (2013), Sci Transl Med. 2013 Aug 7; 5 (197): 197ral03 and WO2014096803 in the context of TCR, but it will be appreciated that such methods may also be applied to the specific binding molecules of the present invention. The specificity of the specific binding molecule in this case may be determined by identifying alternative motif-containing peptides, particularly alternative motif-containing peptides in the human proteome, and testing these peptides for binding to the specific binding molecule. Binding of the specific binding molecule to one or more alternative peptides may indicate lack of specificity. In this case, further testing of the specificity of the specific binding molecule through cell assays may be required. A low tolerance for (alanine) substitutions in the central part of the peptide indicates that the specific binding molecule has high specificity and therefore low risk of cross-reactivity with alternative peptides.

[0046] The specific binding molecules of the present invention are *02. Escape mutants of the peptide SLYNTVATL (SEQ ID NO: 1) have been isolated from AIDS patients and include the following (Sewell et al., (1997) Eur J Immunol. 27: 2323-2329): SLFNTVATL (SEQ ID NO:6) SLFNTVAVL (SEQ ID NO: 7) SLSNTVATL (SEQ ID NO:8) SSFNTVATL (SEQ ID NO: 9) SLLNTVATL (SEQ ID NO: 10) SLYNTIATL (SEQ ID NO: 11) SLYNTIAVL (SEQ ID NO: 12) SLFNTIATL (SEQ ID NO: 13) SLFNTIAVL (SEQ ID NO: 14) SLYNFVAVL (sequence number 15).

[0047] The specific binding molecules of the present invention may have an ideal safety profile for use as therapeutic reagents. In this case, the specific binding molecules may be in soluble form and preferably fused to immune effectors. Suitable immune effectors include, but are not limited to, cytokines such as IL-2 and IFN-γ; superantigens and their mutants; chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein; antibodies and antibody-like scaffolds including antibody fragments, derivatives and variants (e.g., anti-CD3, anti-CD28 or anti-CD16) that bind to antigens on immune cells, such as T cells or NK cells; and Fc receptors or complement activators. An ideal safety profile means that in addition to exhibiting excellent specificity, the specific binding molecules of the present invention may pass further preclinical safety tests. Examples of such tests include whole blood assays to ensure that there is minimal cytokine release in the presence of whole blood, thus reducing the risk of potential cytokine release syndrome in vivo, and alloreactivity tests to ensure that there is a low potential for recognition of another HLA type.

[0048] The specific binding molecules of the invention preferably have a binding activity of less than 100 nM, for example, from about 50 nM to about 1 pM, of SLYNTVATL-HLA-A * K for 02 complex ("SLYNTVATL" disclosed as SEQ ID NO:1) D and / or have a binding half-life (T1 / 2) to the complex ranging from about 1 minute to about 50 hours or more. Certain specific binding molecules of the present invention have a K D Certain TCRs of the present invention may have a K for the complex of about 50 pM to about 200 pM. D The specific binding molecules of the invention may have a binding half-life (T1 / 2) to the complex ranging from about 1 minute to about 50 hours or more (e.g., 100 hours), from about 30 minutes to about 50 hours or more (e.g., 100 hours), or from about 6 hours to about 50 hours or more (e.g., 100 hours). All such specific binding molecules, when coupled to a detectable label or a therapeutic agent, are highly suitable for use as therapeutic and / or diagnostic agents. Certain specific binding molecules of the invention may be suitable for adoptive therapy applications, such specific binding molecules having a K for the complex of about 50 nM to about 200 nM. D , and / or may have a binding half-life to the complex of from about 3 seconds to about 12 minutes.

[0049] Binding affinity (equilibrium constant K D Methods for determining the binding affinity (inversely proportional to K) and binding half-life (denoted as T1 / 2) are known to the skilled artisan. In a preferred embodiment, the binding affinity and binding half-life are determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), e.g. using a BIAcore instrument or an Octet instrument, respectively. A preferred method is provided in Example 3 of US Pat. No. 5,999,363. A doubling of the affinity of a specific binding molecule is known as K D It will be recognized that T1 / 2 is the time when ln2 is reduced to half the dissociation rate (k off ) is calculated by dividing T1 / 2 by k off is halved. K D and koff Values ​​are usually measured for soluble forms of TCRs, i.e. truncated to remove cytoplasmic and transmembrane domain residues (single chain TCRs and / or TCRs incorporating non-native disulfide bonds or other dimerization domains). To take into account variability between independent measurements, and in particular interactions with dissociation times of more than 20 hours, the binding affinity and / or binding half-life of a given specific binding molecule may be measured several times, for example three or more times, using the same assay protocol and the results averaged. To compare binding data between two samples (i.e. two different specific binding molecules and / or two preparations of the same specific binding molecule), it is preferred to perform the measurements using the same assay conditions (e.g. temperature), for example those described in Example 3 of WO 2006 / 023991.

[0050] Certain preferred mutant specific binding molecules of the invention are capable of generating very strong T cell responses in vitro against antigen positive cells, particularly against cells presenting low levels of antigen (i.e. around 5-100) typical of HIV-infected CD4 cells. Such specific binding molecules may be in soluble form and linked to immune effectors such as anti-CD3 antibodies. The T cell response measured may be a T cell activation marker, such as release of interferon gamma or granzyme B, or target cell killing, or other measures of T cell activation, such as T cell proliferation. Preferably, the very strong response is of an EC50 or IC50 value in the pM range, such as 100 pM or less, preferably 50 pM or less, for example between 50 pM and 1 pM.

[0051] The specific binding molecules of the invention may comprise a TCR variable domain. Preferably, the TCR variable domain comprises a heterodimer of an α chain and a β chain.

[0052] In the specific binding molecules of the present invention, the variable domain and, if present, the constant domain and / or any other domains may be organized in any suitable format / configuration. Examples of such configurations are well known in the art of antibodies. The skilled artisan is aware of the similarities between antibodies and TCRs and can apply such configurations to TCR variable and constant domains (Brinkman et al., MAbs. 2017 Feb-Mar; 9(2): 182-212). For example, the variable domain may be arranged in a monoclonal TCR format, where the two chains are linked by disulfide bonds, either in the constant domain or in the variable domain, or the variable domain is fused to one or more dimerization domains. Alternatively, the variable domain may be arranged in a single chain format, with or without one or more constant domains, or the variable domain may be arranged in a diabody format.

[0053] The specific binding molecules of the present invention may comprise at least one TCR constant domain or fragment thereof, such as the α-chain TRAC constant domain and / or the β-chain TRBC1 or TRBC2 constant domain. As will be appreciated by those skilled in the art, the terms TRAC and TRBC1 / 2 also include naturally occurring polymorphic variants, such as the N to K variant at position 4 of TRAC (Bragado et al International immunology. 1994 Feb; 6(2): 223-30).

[0054] If present, one or both of the constant domains may contain mutations, substitutions or deletions relative to the native constant domain sequence. The constant domain may be truncated, i.e., may not have a transmembrane or cytoplasmic domain. Alternatively, the constant domain may be full length, meaning that the extracellular, transmembrane and cytoplasmic domains are all present. The TRAC and TRBC domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The α-chain constant domain sequence and / or the β-chain constant domain sequence(s) may have a disulfide bond introduced between the residues of the respective constant domains, as described, for example, in US Pat. No. 5,399,991. Preferably, the α and β constant domains may be modified by substitution of a cysteine ​​residue at Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, which may form a non-native disulfide bond between the TCR α and β constant domains. TRBC1 or TRBC2 may further comprise a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant domains present in the αβ heterodimer of the invention may be further truncated at one or more C-termini, for example by up to 15, or up to 10, or up to 8 or less amino acids. One or both of the extracellular constant domains present in the αβ heterodimer of the invention may be truncated at one or more C-termini, for example by up to 15, or up to 10, or up to 8 amino acids. The C-terminus of the α chain extracellular constant domain may be truncated by 8 amino acids.

[0055] Alternatively, rather than a full-length or truncated constant domain, the TCR constant domain may be absent altogether. Thus, the specific binding molecules of the invention may be composed of the variable domains of the TCR α and β chains, optionally with additional domains as described herein. The additional domains include, but are not limited to, immune effector domains (e.g., antibody domains), Fc domains or albumin binding domains, therapeutic agents or detectable labels.

[0056] Single chain formats include, but are not limited to, αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ types, where Vα and Vβ are the TCR α and β variable regions, respectively, Cα and Cβ are the TCR α and β constant regions, respectively, and L is a linker sequence (Weidanz et al., (1998) J Immunol Methods. Dec 1; 221(1-2): 59-76, Epel et al., (2002), Cancer Immunol Immunother. Nov; 51(10): 565-73, WO 2004 / 033685, WO 9918129). Linker sequences are typically flexible because they are composed primarily of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that are likely to limit flexibility. Alternatively, a linker with greater rigidity may be desirable. The usable or optimal length of the linker sequence can be easily determined. Often, the linker sequence is less than about 12 amino acids in length, such as less than 10, or between 2 and 10 amino acids in length. The linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to, GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010 / 133828) and GGGSGGGG (SEQ ID NO: 24). Further linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28), and TVLSSAS (SEQ ID NO: 29).If present, one or both of the constant domains may be full length or they may be truncated and / or contain mutations as described above. Preferably, the single chain TCR is soluble. In certain embodiments, the single chain TCR of the invention may have disulfide bonds introduced between residues of the respective constant domains as described in WO 2004 / 033685. Single chain TCRs are further described in WO 2004 / 033685, WO 98 / 39482, WO 01 / 62908, Weidanz et al. (1998) J Immunol Methods 221(1-2): 59-76, Hoo et al. (1992) Proc Natl Acad Sci USA 89(10): 4759-4763, Schodin (1996) Mol Immunol 33(9): 819-829.

[0057] The TCR variable domains can be arranged in a diabody format, in which two single-chain fragments dimerize in a head-to-tail configuration, resulting in a compact molecule with a molecular mass similar to that of a tandem scFv (approximately 50 kDa).

[0058] The present invention also includes particles that exhibit the specific binding molecules of the present invention, and the inclusion of said particles in particle libraries. Such particles include, but are not limited to, phages, yeast cells, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (see, for example, WO 2004 / 044004, WO 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

[0059] The specific binding molecules of the invention are useful for delivering detectable labels or therapeutic agents to antigen-presenting cells and tissues that contain antigen-presenting cells. They may thus be associated (covalently or otherwise) with detectable labels (for diagnostic purposes, where the specific binding molecules are used, e.g., to detect the presence of cells presenting the cognate antigen); and / or therapeutic agents, including immune effectors; and / or pharmacokinetic (PK)-modifying moieties.

[0060] Examples of PK modifying moieties include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28; 16(10): 25831-64 and Jevsevar et al., (2010) Biotechnol J.Jan; 5(1): 113-28), PASylation (Schlapschy et al., (2013) Protein Eng Des Sel. Aug; 26(8): 489-501), albumin and albumin binding domains (Dennis et al., (2002) J Biol Chem. Sep 20; 277(38): 35035-43), and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12): 1186-90). Further PK modifying moieties include antibody Fc fragments, which may be utilized to extend the in vivo half-life of specific binding molecules of the invention.

[0061] When an immunoglobulin Fc domain is used, it may be any antibody Fc region. The Fc region is the tail region of an antibody that interacts with cell surface Fc receptors and some proteins of the complement system. The Fc region typically comprises two polypeptide chains, each with two or three heavy chain constant domains (termed CH2, CH3, and CH4), and a hinge region. The two chains are linked by a disulfide bond in the hinge region. The Fc domains from immunoglobulin subclasses IgG1, IgG2, and IgG4 bind to FcRn and undergo FcRn-mediated recycling, resulting in a long circulating half-life (3 to 4 weeks). The interaction of IgG with FcRn is localized to the portion of the CH2 and CH3 domains that comprise the Fc region. Preferred immunoglobulin Fc's for use in the present invention include, but are not limited to, Fc domains from IgG1 or IgG4. Preferably, the Fc domain is derived from a human sequence. The Fc region may also preferably include KiH mutations that promote dimerization, as well as mutations that prevent interaction with activating receptors, i.e., functionally silent molecules. The immunoglobulin Fc domain may be fused to the C-terminus or N-terminus of other domains (i.e., TCR variable domains and / or TCR constant domains and / or immune effector domains) in any suitable order or configuration. The immunoglobulin Fc may be fused to one or more of the other domains (i.e., TCR variable domains and / or TCR constant domains and / or immune effector domains) through a linker. The linker sequence is typically flexible, since it is composed primarily of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that are likely to limit flexibility. Alternatively, a linker with greater rigidity may be desirable. The usable or optimal length of the linker sequence may be readily determined. Often, the linker sequence is less than about 12, e.g., less than 10, or between 2 and 10 amino acids in length. The linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.Examples of suitable linkers that may be used in the multidomain binding molecules of the present invention include, but are not limited to, GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010 / 133828) and GGGSGGGG (SEQ ID NO: 24). Further linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29). When an immunoglobulin Fc is fused to a TCR, it may be fused to either the α or β chain, with or without a linker. Additionally, individual chains of the Fc may be fused to individual chains of the TCR.

[0062] Preferably, the Fc region may be derived from an IgG1 or IgG4 subclass. The two chains may comprise the CH2 and CH3 constant domains and all or part of the hinge region. The hinge region may substantially or partially correspond to a hinge region from an IgG1, IgG2, IgG3 or IgG4. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region. Preferably, the hinge region contains at least one disulfide bond linking the two chains.

[0063] The Fc region may contain mutations relative to the WT sequence. Mutations include substitutions, insertions and deletions. Such mutations may be made for the purpose of introducing desired therapeutic properties. For example, knob-into-hole (KiH) mutations may be engineered into the CH3 domain to promote heterodimerization. In this case, one chain is engineered to contain a bulky protruding residue (i.e., knob), e.g., Y, and the other chain is engineered to contain a complementary pocket (i.e., hole). Suitable locations for KiH mutations are known in the art. Additionally or alternatively, mutations may be introduced that abolish or reduce binding to Fcγ receptors and / or increase binding to FcRn and / or prevent Fab arm exchange or remove protease sites. Additionally or alternatively, mutations may be made to improve manufacturability, e.g., by removing or modifying glycosylation sites.

[0064] The PK modifying moiety may also be an albumin binding domain, which may also act to extend half-life. As known in the art, albumin has a long circulating half-life of 19 days, due in part to its size exceeding the renal threshold, and due to specific interactions and recycling through FcRn. Attachment to albumin is a known strategy to improve the circulating half-life of therapeutic molecules in vivo. Albumin can be attached non-covalently through the use of specific albumin binding domains, or covalently by conjugation or direct gene fusion. Examples of therapeutic molecules utilizing attachment to albumin for half-life improvement are provided in Sleep et al., Biochim Biophys Acta. 2013 Dec; 1830(12): 5526-34.

[0065] The albumin binding domain may be any moiety capable of binding to albumin, including any known albumin binding moiety. The albumin binding domain may be selected from endogenous or exogenous ligands, small organic molecules, fatty acids, peptides and proteins that specifically bind to albumin. Examples of preferred albumin binding domains include short peptides such as those described in Dennis et al., J Biol Chem. 2002 Sep 20; 277(38): 35035-43 (e.g., the peptide QRLMEDICLPRWGCLWEDDF (SEQ ID NO: 37)); proteins that have been engineered to bind albumin, such as antibodies, antibody fragments and antibody-like scaffolds, such as Albudab™ marketed by GSK (O'Connor-Semmes et al., Clin Pharmacol Ther. 2014 Dec; 96(6): 704-12), and Nanobody™ marketed by Ablynx (Van Roy et al., Arthritis Res Ther. 2015 May 20; 17: 135), and proteins based on albumin binding domains found in nature, such as the streptococcal protein G protein (Stork et al., Eng Des Sel. 2007 Nov; 20(11): 569-76), such as Albumod™ marketed by Affibody.

[0066] Preferably, the albumin is human serum albumin (HSA). The affinity of the albumin binding domain for human albumin can be in the picomolar to micromolar range. Considering the very high concentration of albumin in human serum (35 mg / ml to 50 mg / ml, approximately 0.6 mM), it is calculated that substantially all of the albumin binding domain will bind to albumin in vivo.

[0067] The albumin binding moiety may be fused to the C-terminus or N-terminus of the other domains (i.e., TCR variable domain and / or TCR constant domain and / or immune effector domain) in any suitable order or arrangement. The albumin binding moiety may be fused to one or more of the other domains (i.e., TCR variable domain and / or TCR constant domain and / or immune effector domain) through a linker. The linker sequence is typically flexible, as it is composed primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains that are likely to limit flexibility. Alternatively, a linker with greater rigidity may be desirable. The usable or optimal length of the linker sequence may be readily determined. Often, the linker sequence is less than about 12, e.g., less than 10, or between 2 and 10 amino acids in length. The linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to, GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010 / 133828) and GGGSGGGG (SEQ ID NO: 24). Further linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29). When the albumin binding moiety is linked to a specific binding molecule, it may be fused to either the α or β chain, with or without a linker.

[0068] Detectable labels for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes and imaging agents.

[0069] For some purposes, the specific binding molecules of the present invention may aggregate into complexes containing several specific binding molecules to form multivalent specific binding molecule complexes. There are several human proteins that contain multimerization domains that can be used in producing multivalent specific binding molecule complexes. For example, the tetramerization domain of p53 has been utilized to produce tetramers of scFv antibody fragments that show increased serum persistence and significantly reduced off-rates compared to monomeric scFv fragments (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392). Hemoglobin also has a tetramerization domain that can be used for this type of application. The multivalent specific binding molecule complexes of the present invention may have enhanced binding ability to complexes compared to the non-multimerized native (also referred to as parent, natural, non-mutated, wild-type, or scaffold) T cell receptor heterodimers of the present invention. Thus, multivalent complexes of the specific binding molecules of the present invention are also included in the present invention. Such multivalent specific binding molecule complexes according to the invention are particularly useful for tracking or targeting cells presenting specific antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent specific binding molecule complexes having such uses.

[0070] Therapeutic agents that may be associated with the specific binding molecules of the present invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforin) or chemotherapeutic agents (e.g., cisplatin). To ensure that the therapeutic effect is exerted at the desired location, the therapeutic agent may be placed into a liposome or other nanoparticulate structure linked to the specific binding molecule so that the compound is released slowly. This will prevent damaging effects during transport within the body and ensure that the therapeutic agent has its maximum effect after the specific binding molecule binds to the appropriate antigen-presenting cells.

[0071] Examples of suitable therapeutic agents include, but are not limited to, the following: Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16); alternative protein scaffolds with antibody-like binding properties (e.g., DARPins); Immunostimulants, i.e. immune effector molecules that stimulate the immune response, such as cytokines, e.g., IL-2 and IFN-γ; Chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; Complement pathway activators or Fc receptors; Checkpoint inhibitors, such as those targeting PD1 or PD-L1; Small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells having a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals capable of having cytotoxic effects. In addition, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Examples of such cytotoxic agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer sodium photofrin II, temozolomide, topotecan, trimetreate arbourate, auristatin E, vincristine, and doxorubicin; Peptide cytotoxins, i.e. proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase; Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles, or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213; chelating agents may be used to facilitate the association of these radionuclides to the TCR or its multimers; Superantigens and their mutants; peptide-HLA complexes derived from common human pathogens, e.g., Epstein-Barr virus (EBV); Heterologous protein domains, allologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0072] Soluble specific binding molecules of the invention that are associated with an immune effector (usually by fusion to the N- or C-terminus of the α- or β-chain, or both, in any suitable configuration) are preferred. The N-terminus of the TCR may be linked to the C-terminus of the immune effector polypeptide.

[0073] Particularly preferred immune effectors are anti-CD3 antibodies, or functional fragments or variants of said anti-CD3 antibodies. Specific binding molecules of the invention comprising such antibodies are bispecific and may be referred to herein as "fusion molecules". As used herein, the term "antibody" includes such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031 and 12F6. Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, diabodies, Fab fragments, F(ab') 2 Further examples included within the term antibody include Nanobodies™ (these constructs marketed by Ablynx (Belgium) which contain synthetic single immunoglobulin variable heavy domains derived from Camelidae (e.g. camel or llama) antibodies), Domain Antibodies (Domantis, Belgium) which contain affinity matured single immunoglobulin variable heavy or light domains, and alternative protein scaffolds that exhibit antibody-like binding properties, such as Affibodies (Affibody, Sweden) which contain engineered Protein A scaffolds, or Anticalins (Pieris, Germany) which contain engineered anticalins, or DARPins (Molecular Partners, Switzerland) which contain designed ankyrin repeat proteins.

[0074] The anti-CD3 antibody may be covalently linked to the C-terminus or N-terminus of the TCR α or β chain. The anti-CD3 antibody may be covalently linked to the C-terminus or N-terminus of the TCR β chain of the TCR via a linker sequence.

[0075] Preferably, the anti-CD3 is an scFv fragment. Alternatively, the heavy chain variable domain fragment and the light chain variable domain fragment can be arranged in a diabody orientation. Particularly preferred anti-CD3 sequences are provided in WO2020157210 and WO2020157210.

[0076] Examples of preferred configurations of fusion molecules include those disclosed in WO2010133828, WO2020157211, WO2019012138 and WO2019012141. The formats described in WO2010133828 are particularly preferred.

[0077] The specific binding molecules of the present invention include a first polypeptide chain comprising an alpha chain variable domain and a first binding region of an antibody variable domain; a second polypeptide chain comprising a β chain variable domain and a second binding region of the variable domain of said antibody; and The respective polypeptide chains associate such that the specific binding molecule is capable of simultaneously binding to the SLYNTVATL-HLA-A2 complex ("SLYNTVATL," disclosed as SEQ ID NO:1) and the antigen of the antibody.

[0078] A bispecific polypeptide molecule selected from the group of molecules comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first binding region (VD1) of a variable domain of an antibody that specifically binds to a cell surface antigen of a human immune effector cell; a first binding region (VR1) of the variable domain of the TCR that specifically binds to an MHC-associated peptide epitope; A first linker (LINK1) that links the above domains; Including, the second polypeptide chain comprising a second binding region (VR2) of a variable domain of a TCR that specifically binds to an MHC-associated peptide epitope; a second binding region (VD2) of the variable domain of an antibody that specifically binds to a cell surface antigen of a human immune effector cell; A second linker (LINK2) that links the above domains; Including, the first binding domain (VD1) and the second binding domain (VD2) associate to form a first binding site (VD1)(VD2) that binds to a cell surface antigen of a human immune effector cell; the first binding region (VR1) and the second binding region (VR2) associate to form a second binding site (VR1)(VR2) that binds to the MHC-associated peptide epitope; the two polypeptide chains are fused to a human IgG hinge domain and / or a human IgG Fc domain or a dimerization portion thereof, the two polypeptide chains are linked by covalent and / or non-covalent bonds between the hinge domains and / or Fc domains, The bispecific polypeptide molecule is capable of simultaneously binding to a cell surface molecule and an MHC-associated peptide epitope, wherein the order of the binding regions in the two polypeptide chains is selected from VD1-VR1 and VR2-VD2, or VD1-VR2 and VR1-VD2, or VD2-VR1 and VR2-VD1, or VD2-VR2 and VR1-VD1, the domains are linked by either LINK1 or LINK2, the MHC-associated peptide epitope is the SLYNTVATL (SEQ ID NO: 1) complex, and the MHC is HLA-A. * 02, Bispecific polypeptide molecules are also provided herein.

[0079] The linkage between the specific binding molecule and the anti-CD3 antibody can be through a covalent bond or a non-covalent bond. The covalent bond can be direct or indirect through a linker sequence. The linker sequence is typically flexible, as it is composed primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains that are likely to limit flexibility. Alternatively, a linker with greater rigidity may be desired. The usable or optimal length of the linker sequence can be easily determined. Often, the linker sequence is less than about 12, e.g., less than 10, or between 2 and 10 amino acids in length. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to, GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010 / 133828) and GGGSGGGG (SEQ ID NO: 24). Further linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28), and TVLSSAS (SEQ ID NO: 29).

[0080] A preferred specific binding molecule of the invention has the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS FTGYAMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF TFSVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL NDSRYALSSR LRVSATFWQD PRNHFRCQVQ FYGLSENDEW TQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 30), The beta strand comprises:

[0081] An alternative specific binding molecule of the invention has the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS FTGYTMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF TISVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL NDSRYALSSR LRVSATFWQD PRNHFRCQVQ FYGLSENDEW TQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 31), The beta strand comprises:

[0082] The preferred alpha chain has the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT PHIQKPDPAV YQLRDSKSSD KSVCLFTDFD SQTNVSQSKD SDVYITDKCV LDMRSMDFKS NSAVAWSNKS DFACANAFNN SIIPEDT (SEQ ID NO: 32), Includes.

[0083] Preferred specific binding molecules of the invention comprise the amino acid sequences of SEQ ID NO:30 and SEQ ID NO:32.

[0084] Also included within the scope of the present invention are functional variants (also known as phenotypically silent variants) of the above-mentioned specific binding molecules, including anti-CD3.

[0085] In a further aspect, the invention provides nucleic acids encoding the TCR alpha and / or beta chains of the invention. Nucleic acids encoding the specific binding molecules of the invention, including molecules fused to an anti-CD3 antibody or fragment thereof, are also provided. In some embodiments, the nucleic acid is a cDNA. In some embodiments, the nucleic acid may be an mRNA, such as an mRNA encoding a bispecific molecule (Stadler et al., Nat Med. 2017 Jul; 23(7): 815-817). In some embodiments, the invention provides nucleic acids comprising a sequence encoding a TCR alpha chain variable domain of a specific binding molecule of the invention. In some embodiments, the invention provides nucleic acids comprising a sequence encoding a TCR beta chain variable domain of a specific binding molecule of the invention. The nucleic acid may be non-naturally occurring and / or purified and / or engineered. The nucleic acid sequence may be codon-optimized according to the expression system utilized. As known to the skilled artisan, the expression system may include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or may be cell-free expression systems. In some embodiments, the molecule may be an mRNA encoding a bispecific antibody.

[0086] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.Preferably, the vector is a TCR expression vector.Suitable TCR expression vectors include, for example, gamma-retroviral vectors, or more preferably, lentiviral vectors.Further details can be found in Zhang 2012 and references therein (Zhang et al,. Adv Drug Deliv Rev. 2012 Jun 1; 64(8): 756-762).

[0087] The present invention also provides a cell harboring a vector of the present invention, preferably a TCR expression vector. Suitable cells include mammalian cells, preferably immune cells, even more preferably T cells. The vector may comprise the nucleic acid of the present invention, encoding the alpha and beta chains, respectively, in a single open reading frame, or in two separate open reading frames. Another aspect provides a cell harboring a first expression vector comprising a nucleic acid encoding the alpha chain of a specific binding molecule of the present invention, and a second expression vector comprising a nucleic acid encoding the beta chain of a specific binding molecule of the present invention. Such cells are particularly useful for adoptive therapy. The cells of the present invention may be isolated and / or recombinant and / or non-naturally occurring and / or engineered.

[0088] Since the specific binding molecules of the invention have utility in adoptive therapy, the invention includes non-naturally occurring and / or purified and / or engineered cells, particularly T cells, that present the specific binding molecules of the invention. The invention also provides an expanded population of T cells that present the specific binding molecules of the invention. There are several suitable methods for transfecting T cells with nucleic acids (e.g., DNA, cDNA or RNA) that encode the specific binding molecules of the invention (see, e.g., Robbins et al., (2008) J Immunol. 180: 6116-6131). T cells expressing the specific binding molecules of the invention would be suitable for use in adoptive therapy-based treatment of cancer. As known to those skilled in the art, there are several suitable methods by which adoptive therapy may be carried out (see, e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4)).

[0089] As is known in the art, in vivo production of proteins, including those containing the specific binding molecules of the present invention, can result in post-translational modifications. Glycosylation is one such modification, and involves the covalent attachment of oligosaccharide moieties to defined amino acids in a polypeptide chain. For example, asparagine residues, or serine / threonine residues are well-known positions for oligosaccharide attachment. The glycosylation state of a particular protein depends on several factors, including protein sequence, protein conformation, and availability of specific enzymes. Furthermore, the glycosylation state (i.e., oligosaccharide type, covalent bonds, and total number of attachments) can affect protein function. Thus, glycosylation control is often desirable when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapeutics (Jefferis et al., (2009) Nat Rev Drug Discov Mar; 8(3): 226-34). For the specific binding molecules of the invention, glycosylation can be controlled, for example, by using specific cell lines (including but not limited to mammalian cell lines, such as Chinese Hamster Ovary (CHO) cells or human embryonic kidney (HEK) cells) or by chemical modification. Such modifications may be desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott, (2005) Pharm Sci.Aug; 94(8): 1626-35). In some cases, mutations may be introduced to control and / or modify post-translational modifications.

[0090] For administration to a patient, the specific binding molecule of the invention (preferably associated with a detectable label or therapeutic agent, e.g., anti-CD3, or expressed on transfected T cells), the nucleic acid, expression vector, or cell of the invention may be provided as part of a sterile pharmaceutical composition together with one or more pharma- ceutically acceptable carriers or excipients. This pharmaceutical composition may take any suitable form (depending on the desired method of administration to a patient). It may be provided in unit dosage form, typically provided in a hermetically sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. The kit may include a plurality of the unit dosage forms described above.

[0091] Pharmaceutical compositions may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the pharmaceutical art, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0092] The dosage of the substance of the invention varies widely depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and a suitable dosage range for the specific binding molecule-anti-CD3 fusion molecule may be in the range of 25 ng / kg to 50 μg / kg, or 1 μg to 1 g. The physician will ultimately determine the appropriate dosage to be used. Examples of suitable dosing regimens are provided in WO2017208018.

[0093] A single dose can be administered. Alternatively, multiple doses can be administered, for example, two or more doses, or three or more doses. When multiple doses are administered, the same dose can be administered each time, or reduced doses can be administered for the first and / or subsequent doses.

[0094] The specific binding molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

[0095] The present invention also provides: a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell for use in medicine, preferably in a human subject, preferably for use in a method of treating HIV infection or AIDS in a human subject; Use of a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention in the manufacture of a medicament for treating HIV infection or AIDS in a human subject; A method of treating HIV infection or AIDS, comprising administering to a subject in need of treatment a therapeutically effective amount of a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention; An injectable formulation for administration to a human subject comprising a specific binding molecule, a nucleic acid, a vector, a pharmaceutical composition or a cell of the invention.

[0096] The specific binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the invention may be administered by injection or infusion. Particularly preferred is administration by intravenous or subcutaneous injection. * It may be of the 02 subtype.

[0097] The treatment may further comprise administering, separately, in combination or sequentially, one or more additional antiviral agents and / or one or more additional immunotherapeutic agents and / or one or more soluble bispecific binding proteins. For example, the treatment may comprise administration of two or more bispecific binding proteins, including a specific binding molecule of the invention and an additional specific binding molecule that recognizes another HIV protein.

[0098] The treated patient may be undergoing antiretroviral therapy (ART). Alternatively, the treated patient may be off ART, for example, such patient may be undergoing analytical therapy interruption (ATI). Alternatively, the treated patient may be ART naive.

[0099] The terms "treatment," "treat," "treating," and the like are meant to include slowing, halting, or reversing the progression of HIV and / or AIDS. These terms also include the alleviation, amelioration, attenuation, elimination, or reduction of one or more symptoms of a disorder or condition, even if the disorder or condition is not actually eliminated, and even if the progression of the disorder or condition itself is not slowed, halted, or reversed.

[0100] "Therapeutically effective amount" means the amount of a compound, or a pharma- ceutically acceptable salt thereof, administered to a subject that will elicit a biological or medical response in the subject or a desired therapeutic effect in the subject.

[0101] Therapeutically effective amounts can be easily determined by the attending physician, who is skilled in the art, by using known techniques and observing the results obtained under similar circumstances. In determining the effective amount for a subject, the attending physician will take into account several factors, including, but not limited to, size, age, and general health; the specific disease or disorder involved; the degree or involvement or severity of the disease or disorder; the response of the individual subject; the specific compound administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; the use of concomitant drugs; and other relevant circumstances.

[0102] Preferred features of each aspect of the invention apply as with each of the other aspects mutatis mutandis. Prior art documents referred to herein are incorporated by reference to the fullest extent permitted by law.

[0103] The invention is further described in the following non-limiting examples, with reference to the accompanying drawings. EXAMPLES

[0104] Example 1 - Identification of single TCR α chain mutations that do not negatively affect binding affinity to the target and result in higher yields Bispecific molecules containing high affinity HIV TCR sequences fused to anti-CD3 scFv were produced as previously described (Non-Patent Document 19, Patent Document 2). The TCR end of the molecule was engineered to bind the HIV Gag protein (Gag 78-85 )-derived HLA-A * It recognizes the 02 restricted 9-mer peptide SLYNTVATL (SEQ ID NO: 1). The sequence of the TCR domain is shown in Figure 1a (SEQ ID NO: 33 and SEQ ID NO: 34) and the full-length bispecific molecule is shown in Figure 1b (SEQ ID NO: 35 and SEQ ID NO: 36). During preclinical studies, this molecule was found to have low yields when produced in E. coli (approximately 1 mg / L). Production yields in this range were not considered adequate for the manufacture of reagents to support clinical development of the molecule. Attempts were made to improve the yield through optimizing refolding and purification conditions using standard methods, but without success. Unexpectedly, a single mutation in the TCR α chain variable domain was found to increase the yield without decreasing the binding affinity to the cognate pMHC target.

[0105] method Single-site mutagenesis was performed to introduce an F to K mutation at position 50 of the TCR α chain variable domain as shown in the sequence shown in Figure 2 (Note - in this example, residue numbering is based on the inclusion of the N-terminal methionine). A bispecific protein containing this mutation was produced in E. coli. Yields and binding parameters of the mutants were compared to the corresponding non-mutated versions.

[0106] Bispecific proteins were produced as described previously. Briefly, α- and β-chains were expressed separately as inclusion bodies in E. coli strain BL21-DE3(pLysS) by induction with 0.5 mM IPTG at mid-log phase. Inclusion bodies were isolated by sonication followed by successive washing and centrifugation steps using 0.5% Triton X-100. Soluble proteins were refolded by rapid dilution of the mixture of dissolved α- and β-chain inclusion bodies into 5 M urea, 0.4 M L-arginine, 100 mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM β-mercaptoethylamine. The refold mixture was dialyzed against 10 volumes of demineralized water for 24 hours and then against 10 volumes of 10 mM Tris pH 8.1. The refolded protein was filtered and purified through three successive chromatographic steps: anion exchange (AIEX), cation exchange (CIEX) and size exclusion (SEC) pre-equilibrated in phosphate buffered saline (PBS). Fractions containing the main peak were pooled and further analyzed. The final purified molecule was analyzed by SDS-PAGE under reducing and non-reducing conditions. Yields and recovery were determined across all purification steps (AIEX, CIEX and SEC).

[0107] The purified bispecific molecule was subjected to surface plasmon resonance (SPR) analysis using the BIAcore™ system as previously described (see, e.g., Non-Patent Document 19 and Patent Document 2) to identify its target peptide-HLA complex (SLYNTVATL (SEQ ID NO: 1) HLA-A * 02) was determined. Briefly, biotinylated pHLA was immobilized on a streptavidin-coupled CM5 sensor chip. Flow cell 1 was loaded with free biotin only, which served as the control surface. K D Values ​​were calculated and data were analyzed using a 1:1 binding model (Biacore Insight Evaluation v2.0.15.12933 for single cycle kinetic analysis).

[0108] result Table A shows the total yield and % recovery over all purification steps. Figure 3 shows the yield per culture volume. For F50K, the total yield and % recovery over all purification steps were substantially improved over the non-mutated (WT) version, and furthermore, the yield per culture volume showed a 4.5-fold improvement for F50K over the wild type. Table B shows the target binding parameters for each bispecific protein. These data show that F50K has a significantly improved K D Including T1 / 2 (K D = low pM, and T1 / 2≧24 hr), indicating that they retain similar binding properties to the non-mutated WT protein.

[0109] As a comparison, three additional single-site mutations (L47P, M49K, A53E) were made in the same region as F50 and tested using the same method. As shown in Table A and Figure 3, mutations L47P and A53E did not result in a substantial improvement in yield compared to F50K, while M49K showed a slightly higher improvement than F50K (4.9-fold). Interestingly, all three alternative mutations, including M49K, showed a substantial decrease in binding properties to the target compared to WT. Figure 4 shows a side-by-side comparison of the binding kinetics of M49K and F50K.

[0110] TIFF2025508738000001.tif44170

[0111] TIFF2025508738000002.tif48170

[0112] These data demonstrate the challenge of identifying bispecific proteins with improved developability. A single mutation (F50K) in the TCR α chain variable domain was shown to confer improved yields and retain high affinity binding to the target.

[0113] Example 2 - Identification of additional TCR α chain mutations that increase protein stability without negatively affecting binding affinity to the target Further evaluation of the F50K bispecific protein indicated that the developability of the molecule may be affected by reduced stability. Unexpectedly, a single mutation in the TCR α chain variable region was found to improve protein stability.

[0114] method Single-site mutagenesis was performed to introduce an S to A mutation at position 96 of the TCR α chain variable domain, as shown in Figure 5. (Note - in this example, residue numbering is based on the inclusion of the N-terminal methionine.) Bispecific proteins containing this mutation were produced in E. coli and binding parameters analyzed as previously described.

[0115] Analytical anion exchange chromatography (AIEX-UPLC) was used to assess protein stability after 3 days of exposure to high pH (Tris pH 9). Proteins were separated on an anion exchange UPLC column, eluted in order of increasing net surface negative charge and detected by FLD detection (excitation 295 nm, emission 348 nm). The charge profile of the test samples was monitored by integrating the area under the curve of all peaks. The relative decrease in the main peak and the corresponding increase in acidic species were used as a measure of stability. Non-reduced peptide mapping was used to assess deamidation. Briefly, test samples and reference proteins were denatured and digested at +37°C with a combination of trypsin and LysC. The resulting peptides were separated on a C18 HPLC column and detected by UV absorbance at 214 nm. Fragmentation spectra generated for each peptide were searched against a library of potential peptides based on protein sequence, mass accuracy and potential modifications using PMI Byonic software.

[0116] result Table C shows the target binding parameters. These data show that S96A produces similar target binding parameters compared to F50K alone. For comparison, three alternative single-site mutations were introduced into the same protein region as S96A. Of these, only T94L, but not N95Q or S96V, produced similar target binding parameters compared to F50K alone.

[0117] TIFF2025508738000003.tif83170

[0118] Table D summarizes the results of the AIEX-UPLC analysis. These data indicate that S96A is more stable than F50K alone or T94L. Further analysis of both S96A and T94L by peptide mapping indicated that the increased stability is likely the result of reduced deamidation at position N95.

[0119] TIFF2025508738000004.tif79170

[0120] TIFF2025508738000005.tif40170

[0121] Example 3 - Improved bispecific molecules recognize common viral escape mutants and exhibit potent and specific killing of HIV-infected cells The F50K+S96A integrated bispecific protein was further analyzed to evaluate additional properties, including recognition of common viral escape mutants and potency against antigen-positive cell lines. In this example, the TCR was fused to an alternative anti-CD3 domain as further described in WO2020157210. The full length of the bispecific molecule is shown in FIG.

[0122] method SPR analysis was performed on a Biacore instrument as previously described. Briefly, mutants of the HIV peptides listed here were *The biotinylated complexes were then immobilized on a Biacore CM5 chip preloaded with streptavidin. Five increasing concentrations of the soluble bispecific proteins were injected over the immobilized HLA. After the fifth injection, dissociation was measured for 2 hours. The Biacore instrument was used to measure the association and dissociation of molecules from each HLA complex. HLAA * The relative half-life of each interaction was calibrated to the index peptide in 02:01.

[0123] Cytokine enzyme-linked immunosorbent spot (ELISPOT) assays were used to determine potency and detect interferon gamma (IFN-γ) or granzyme B (GrB) secreted by T cells upon activation. 77-85 HLAA pulsed with peptide * 02:01 Lymphoma-derived cancer cell line (T2) and peptide-pulsed HLAA * A 02:01 / β2m transduced T cell line (C8166 A2B2M) was used as target cells for IFN-γ and GrB assays, respectively. PBMCs obtained from HIV-uninfected donors were used as effectors. Cytokine release (IFN-γ) was used to measure T cell activation and GrB release was used as a surrogate for T cell-mediated target cell killing. Target cells were incubated overnight (IFN-γ specific, top panel) or for 40-48 hours (GrB specific, bottom panel) with PBMCs from different HIV-uninfected donors (CTL013, CTL014, CTL018, SC009, SC001B) and increasing concentrations of IMCM113V.

[0124] result Table F shows that bispecific proteins have affinities in the low nanomolar to picomolar range (K D) and a half-life (t1 / 2) of several hours to each of the mutant peptides tested. While affinity was reduced for the mutants, especially those containing the Y3F and T8V substitutions, the weakest interaction between IMCM113V and the peptide-HLA complex (Y3F T8V) was still considered very strong compared to the wild-type TCR. These data indicate that the bispecific protein strongly recognizes common escape mutants.

[0125] TIFF2025508738000006.tif67170

[0126] Figure 7 shows the HLA * 02:01 Positive peptide (HIV Gag 77-85 ) When cultured with pulsed T2 and C8166 A2B2M target cells, the bispecific protein redirects effector T cells to release IFN-γ and GrB in a dose-dependent manner. ECs from responsive donors 50 Values ​​are shown on the graph and range from 1.0 pM to 12.3 pM for IFN-γ release and 0.5 pM to 81.1 pM for GrB release. These data indicate that the bispecific protein * 02:01Positive Gag 77-85 We show that it can specifically redirect T cell activity in the presence of positive cells to activate T cells and release cytokines in a dose-dependent manner with potency in the low picomolar range.

Claims

1. HLA-A ＊ A specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) which forms a complex with .O2, and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the alpha chain variable domain a) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), b) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2), or c) AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), and optionally having an N-terminal methionine, the beta chain variable domain DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO: 5), and optionally an amino acid sequence having an N-terminal methionine (SEQ ID NO: 44), Specific binding molecules.

2. The α chain variable domain comprising the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), The specific binding molecule of claim 1 , comprising:

3. The α chain variable domain comprising the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4), Including, The beta chain variable domain has the amino acid sequence: DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT (SEQ ID NO: 5), The specific binding molecule of claim 1 , comprising:

4. The alpha chain variable domain has the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P (SEQ ID NO: 2); The specific binding molecule of claim 1 , comprising:

5. The alpha chain variable domain has the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3), The specific binding molecule of claim 1 , comprising:

6. 6. The specific binding molecule of any one of claims 1 to 5, which is an αβ heterodimer having an α chain TRAC constant domain and a β chain TRBC1 constant domain or a β chain TRBC2 constant domain.

7. 7. The specific binding molecule of claim 6, wherein the amino acid sequences of the alpha and beta chain constant domains have been modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

8. 7. The specific binding molecule of claim 6, wherein the amino acid sequence(s) of the α and β chain constant domains are modified by substitution of a cysteine ​​residue for Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, said cysteine ​​forming a disulfide bond between the TCR α and β constant domains.

9. 6. The specific binding molecule of any one of claims 1 to 5, which is in a single-chain format of Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ, or Vβ-Cβ-L-Vα-Cα, wherein Vα and Vβ are TCR alpha and beta variable regions, respectively, Cα and Cβ are TCR alpha and beta constant regions, respectively, and L is a linker sequence.

10. The specific binding molecule of any one of claims 1 to 5, which is associated with a detectable label, a therapeutic agent, or a PK-modifying moiety.

11. The specific binding molecule of claim 10, which is associated with an anti-CD3 antibody covalently bound to the C-terminus or N-terminus of the TCR α or β chain.

12. The specific binding molecule of claim 11 , wherein the anti-CD3 antibody is covalently linked to the C-terminus or N-terminus of the TCR β chain via a linker sequence.

13. 13. The specific binding molecule of claim 12, wherein the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23).

14. the beta chain having the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLISSLQ PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS FTGYAMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF TFSVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL NDSRYALSSR LRVSATFWQD 13. The specific binding molecule of claim 12, comprising: PRNHFRCQVQ FYGLSENDEW TQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 30).

15. The beta chain of claim 1, wherein the beta chain has the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLISSLQ PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS FTGYAMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF TFSVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL NDSRYALSSR LRVSATFWQD PRNHFRCQVQ FYGLSENDEW TQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 30), Including, the alpha chain having the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT PHIQKPDPAV YQLRDSKSSD KSVCLFTDFD SQTNVSQSKD SDVYITDKCV LDMRSMDFKS NSAVAWSNKS DFACANAFNN SIPEDT (SEQ ID NO: 32), Including, The specific binding molecule of claim 12.

16. the beta chain having the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLISSLQ PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS FTGYTMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF TISVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL NDSRYALSSR LRVSATFWQD 13. The specific binding molecule of claim 12, comprising: PRNHFRCQVQ FYGLSENDEW TQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 31).

17. the alpha chain having the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT PHIQKPDPAV YQLRDSKSSD KSVCLFTDFD SQTNVSQSKD SDVYITDKCV LDMRSMDFKS NSAVAWSNKS DFACANAFNN SIPEDT (SEQ ID NO: 32), The specific binding molecule of claim 14, comprising:

18. A nucleic acid molecule encoding a TCR alpha chain and / or a TCR beta chain as defined in claim 1.

19. 20. An expression vector comprising the nucleic acid of claim 18.

20. (a) a TCR expression vector comprising the nucleic acid of claim 18 in a single open reading frame or in two separate open reading frames encoding the α chain and the β chain, respectively; or (b) a first expression vector comprising a nucleic acid encoding the α chain of the TCR of claim 1; and a second expression vector comprising a nucleic acid encoding the β chain of the TCR of claim 1; Cells that house

21. An isolated or non-naturally occurring cell, particularly a T cell, presenting the TCR of claim 1.

22. A pharmaceutical composition comprising the specific binding molecule of claim 1, the nucleic acid of claim 18, the vector of claim 19, and / or the cell of claim 20 or 21, together with one or more pharmaceutically acceptable carriers or excipients.

23. A specific binding molecule according to claim 1, a nucleic acid according to claim 18, a vector according to claim 19, and / or a cell according to claim 20 or 21 for use in medicine in a human subject.

24. A specific binding molecule as described in claim 1, a nucleic acid as described in claim 18, a vector as described in claim 19, and / or a cell as described in claim 20 or 21 for use in a method for treating HIV infection or AIDS in a human subject.

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