ENGINEERED IMMUNE-MOBILIZING T-CELL RECEPTORS WITH ENHANCED AFFINITY FOR HIV-1 GAG

Abstract

The present invention relates to T cell receptors (TCRs) which bind the HLA-A*02 restricted peptide SLYNTVATL (SEQ ID NO: 1) derived from the HIV Gag gene product, p17. Said TCRs comprise non-natural mutations within the alpha and/or beta variable domains relative to a native HIV TCR. The TCRs of the invention possess unexpectedly high affinity, specificity and sensitivity for a complex of SEQ ID NO: 1 and HLA-A*02, and drive a particularly potent T cell response. Such TCRs are particularly useful in the development of soluble immunotherapeutic reagents for the treatment of HIV infected individuals.

Claims

1-26. (canceled)

27. A T cell receptor (TCR) having the property of binding to SLYNTVATL (SEQ ID No: 1) in complex with HLA-A*02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein (i) the alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 6 or 7 and the beta chain variable domain comprises the amino acid sequence of SEQ ID NO: 8; or (ii) the alpha chain domain comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 6 or 7 and the beta chain variable domain comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 8.

28. The TCR of claim 27, wherein the TCR of (ii) has a KD for the SLYNTVATL (SEQ ID No: 1) HLA-A*02 complex within 50% of the measured K.sub.D of the TCR of (i) when measured using Surface Plasmon Resonance under identical conditions.

29. The TCR of claim 27, wherein the TCR of (ii) has a KD for the SLYNTVATL (SEQ ID No: 1) HLA-A*02 complex within 20% of the measured K.sub.D of the TCR of (i) when measured using Surface Plasmon Resonance under identical conditions.

30. The TCR of claim 27, wherein in the alpha chain variable domain, the sequence of amino acid residues 28-33, 51-56, and 91-101 is selected from the following with reference to the numbering of SEQ ID NO: 2: TABLE-US-00010 CDR1 (28-33) CDR2 (51-56) CDR3 (91-101) SWEGQS (SEQ ID NO: 26) LYADPD (SEQ ID NO: 27) AVRTNSGYALN (SEQ ID NO: 22) SWEGQS (SEQ ID NO: 26) IYSNGD (SEQ ID NO: 21) AVRTNSGYALN (SEQ ID NO: 22)

31. The TCR of claim 27, wherein in the beta chain variable domain, the sequence of amino acid residues 28-32, 50-55, and 93-103 is selected from the following with reference to the numbering of SEQ ID NO: 3: TABLE-US-00011 CDR1 (28-32) CDR2 (50-55) CDR3 (93-103) SGHDT (SEQ ID NO: 23) AVRGVE (SEQ ID NO: 28) ASSDTVSYEQY (SEQ ID NO: 25)

32. The TCR of claim 27, wherein the alpha chain variable domain comprises an amino acid sequence that has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 6 — 7, and the beta chain variable domain comprises an amino acid sequence that has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8.

33. The TCR of claim 27, which is an alpha-beta heterodimer, having an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence, optionally wherein the alpha and beta chain constant domain sequences are modified by truncation or substitution to delete the native disulphide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2, further optionally wherein the alpha and beta chain constant domain sequence(s) are modified by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulphide bond between the alpha and beta constant domains of the TCR.

34. The TCR of claim 27, which is in single chain format of the type Va-L-Vb, Vb-L-Va, Va-Ca-L-Vb, Va-L-Vb-Cb, Va-Ca-L-Vb-Cb or Vb-Cb-L-Va-Ca wherein Va and Vb are TCR alpha and beta variable regions respectively, Ca and Cb are TCR alpha and beta constant regions respectively, and L is a linker sequence.

35. The TCR of claim 27, wherein the TCR is associated with a detectable label, a therapeutic agent, or a pharmacokinetic (PK)-modifying moiety.

36. The TCR of claim 35, wherein the TCR is associated with an anti-CD3 antibody covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR, optionally via a linker sequence, optionally wherein the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 12), GGGSG (SEQ ID NO: 13), GGSGG (SEQ ID NO: 14), GSGGG (SEQ ID NO: 15), GSGGGP (SEQ ID NO: 16), GGEPS (SEQ ID NO: 17), GGEGGGP (SEQ ID NO: 18), and GGEGGGSEGGGS (SEQ ID NO: 19).

37. A TCR-anti-CD3 fusion molecule wherein the alpha chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 6 or 7 and the beta chain variable domain comprises the amino acid sequence of SEQ ID NO: 8, and wherein the anti-CD3 antibody is covalently linked to the N-terminus or C-terminus of the TCR beta chain via a linker sequence selected from SEQ ID NOs: 12-19.

38. A TCR-anti-CD3 fusion molecule comprising: a alpha chain amino acid sequence of SEQ ID NO: 9 or 10, and a beta chain amino acid sequence of SEQ ID NO: 11.

39. A nucleic acid encoding the TCR alpha chain and/or the TCR beta chain of claim 27.

40. An expression vector comprising the nucleic acid of claim 39.

41. A cell comprising: (a) a TCR expression vector which comprises nucleic acid encoding the TCR alpha chain and the TCR beta chain of claim 1, wherein the nucleic acid comprises a single open reading frame or two distinct open reading frames encoding the alpha chain and the beta chain; or (b) a first expression vector which comprises a nucleic acid encoding the TCR alpha chain of claim 1 and a second expression vector which comprises nucleic acid encoding the TCR beta chain of claim 1.

42. An isolated or non-naturally occurring cell presenting a TCR of claim 27.

43. A pharmaceutical composition comprising a TCR of claim 27, together with one or more pharmaceutically acceptable carriers or excipients.

44. A method of treatment, comprising administering the TCR of claim 27 to a human subject in need thereof

45. The method of claim 44, wherein the subject has an HIV infection or AIDS.

Description

DESCRIPTION OF FIGURES

[0090] FIG. 1 provides the amino acid sequence of the extracellular part of the alpha and beta chain of a wild type Gag TCR.

[0091] FIG. 2 provides the amino acid sequence of the soluble version of the wild type Gag TCR alpha and beta chain.

[0092] FIG. 3 provides amino acid sequences of mutated TCR alpha chain variable domains

[0093] FIG. 4 provides amino acid sequence of mutated TCR beta chain variable domains

[0094] FIG. 5 provides alpha chain amino acid sequences of TCR anti-CD3 fusion molecules.

[0095] FIG. 6 provides beta chain amino acid sequence of TCR anti-CD3 fusion molecules.

[0096] FIG. 7 provides cellular data demonstrating potency TCR anti-CD3 fusion molecules

[0097] FIG. 8 provides cellular data demonstrating specificity of TCR anti-CD3 fusion molecules

[0098] FIG. 9 provides cellular data demonstrating sensitivity of TCR anti-CD3 fusion molecules

[0099] The invention is further described in the following non-limiting examples.

EXAMPLES

Example 1

Expression, Refolding and Purification of Soluble TCRs

Method

[0100] DNA sequences encoding the alpha and beta extracellular regions of soluble TCRs of the invention were cloned separately into pGMT7-based expression plasmids using standard methods (as described in Sambrook, et al. Molecular cloning. Vol. 2. (1989) New York: Cold spring harbour laboratory press). The expression plasmids were transformed separately into E. coli strain Rosetta (BL21pLysS), and single ampicillin-resistant colonies were grown at 37° C. in TYP (+ampicillin 100 μg/ml) medium to an OD.sub.600 of ˜0.6-0.8 before inducing protein expression with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation. Cell pellets were lysed with BugBuster protein extraction reagent (Merck Millipore) according to the manufacturer's instructions. Inclusion body pellets were recovered by centrifugation. Pellets were washed twice in Triton buffer (50 mM Tris-HCI pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) and finally resuspended in detergent free buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM NaEDTA). Inclusion body protein yield was quantified by solubilising with 6 M guanidine-HCl and measuring OD.sub.280. Protein concentration was then calculated using the extinction coefficient. Inclusion body purity was measured by solubilising with 8M Urea and loading ˜2 μg onto 4-20% SDS-PAGE under reducing conditions. Purity was then estimated or calculated using densitometry software (Chemidoc, Biorad). Inclusion bodies were stored at +4° C. for short term storage and at −20° C. or −70° C. for longer term storage.

[0101] For soluble TCR refolding, a and f3 chain-containing inclusion bodies were first mixed and diluted into 10 ml solubilisation/denaturation buffer (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 mM NaCl, 10 mM EDTA, 20 mM DTT) followed by incubation for 30 min at 37° C. Refolding was then initiated by further dilution into 1 L of refold buffer (100 mM Tris pH 8.1, 400 mM L-Arginine HCL, 2 mM EDTA, 4 M Urea, 10 mM cysteamine hydrochloride and 2.5 mM cystamine dihydrochloride) and the solution mixed well. The refolded mixture was dialysed against 10 L H.sub.2O for 18-20 hours at 5° C.±3° C. After this time, the dialysis buffer was twice replaced with 10 mM Tris pH 8.1 (10 L) and dialysis continued for another 15 hours. The refold mixture was then filtered through 0.45 μm cellulose filters.

[0102] Purification of soluble TCRs was initiated by applying the dialysed refold onto a POROS® 50HQ anion exchange column and eluting bound protein with a gradient of 0-500 mM NaCl in 20 mM Tris pH 8.1 over 50 column volumes using an Akta® purifier (GE Healthcare). Peak TCR fractions were identified by SDS PAGE before being pooled and concentrated. The concentrated sample was then applied to a Superdex® 75HR gel filtration column (GE Healthcare) pre-equilibrated in Dulbecco's PBS buffer. The peak TCR fractions were pooled and concentrated and the final yield of purified material calculated.

Example 2

Expression, Refolding and Purification of Soluble TCR Anti-CD3 Fusion Molecules

[0103] Preparation of soluble TCR anti-CD3 fusions was carried out as described in Example 1, except that the TCR beta chain was fused via a linker to an anti-CD3 single chain antibody. In addition a cation exchange step was performed during purification following the anion exchange. In this case the peak fractions from anion exchange were diluted 20 fold in 20mM MES (pH6.5), and applied to a POROS® 50HS cation exchange column. Bound protein was eluted with a gradient of 0-500 mM NaCl in 20 mM MES. Peak fractions were pooled and adjusted to 50 mM Tris pH 8.1, before being concentrated and applied directly to the gel filtration matrix as described in Example 1.

Example 3

Antigen Binding Characterisation of Soluble TCR Anti-CD3 Fusion Molecules

[0104] Binding analysis was carried out by surface plasmon resonance, using a BlAcore 3000 or BlAcore T200 instrument. Biotinylated class I HLA-A*02 molecules were refolded with the peptide of interest and purified using methods known to those in the art (O'Callaghan et al. (1999). Anal Biochem 266(1): 9-15; Garboczi, et al. (1992). Proc Natl Acad Sci USA 89(8): 3429-3433). Biotinylated peptide-HLA monomers were immobilized on to streptavidin-coupled CM-5 sensor chips. All measurements were performed at 25° C. in Dulbecco's PBS buffer, supplemented with 0.005% P20.

[0105] Equilibrium binding constants were determined using serial dilutions of soluble TCR/TCR anti-CD3 fusions injected at a constant flow rate of 30 ul min-1 over a flow cell coated with ˜200 response units (RU) of peptide-HLA-A*02 complex. Equilibrium responses were normalised for each concentration by subtracting the bulk buffer response on a control flow cell containing an irrelevant peptide-HLA. The Kd value was obtained by non-linear curve fitting using Prism software and the Langmuir binding isotherm, bound=C*Max/(C+KD), where “bound” is the equilibrium binding in RU at injected TCR/TCR anti-CD3 concentration C and Max is the maximum binding.

[0106] For high affinity interactions, binding parameters were determined by single cycle kinetics analysis. Five different concentrations of TCR anti-CD3 fusion were injected over a flow cell coated with ˜100-200 RU of peptide-HLA complex using a flow rate of 50-60 μl min-1. Typically, 60-200μl of TCR anti-CD3 fusion was injected at a top concentration of 100-300 nM, with successive 2 fold dilutions used for the other four injections. The lowest concentration was injected first. To measure the dissociation phase buffer was then injected until 10% dissociation occurred, typically after 1-3 hours. Kinetic parameters were calculated using BlAevaluation® software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant Kd was calculated from koff/kon.

Results

[0107] The binding parameters (dissociation constant (Kd) and half-life (T½)) of two mutant TCRs of the invention fused to anti-CD3 are shown in the table below. The Kd value of a soluble form of the none mutated TCR, without anti-CD3, was previously determined as 85 nM (WO2006103429 and Varela-Rohena et al. 2008, Nat Med, 14(12)1390-5).

TABLE-US-00008 Alpha chain Beta chain TCR-anti- variable variable CD3 fusion sequence sequences Kd T½ m121 SEQ ID NO: 9 SEQ ID No: 11  64 pM .sup. 94 h (with Q at position 1 and the optional 8 residues present at the C terminus of TRAC) m134 SEQ ID NO: 10 SEQ ID No: 11 193 pM 16.6 h (with Q at position 1 and the optional 8 residues present at the C terminus of TRAC)

COMPARATIVE EXAMPLE

[0108] The previously known highest affinity mutant of the WT TCR (termed a11b6, (Varela-Rohena et al. 2008, Nat Med, 14(12):1390-5)) was prepared as an anti-CD3 fusion and assessed for binding under the same conditions as m121 and m134 above. In this case the binding affinity (Kd) was calculated as 360 pM and the half-life was 3.5 h.

[0109] These results demonstrate that mutant TCRs of the invention have unexpectedly high affinity and long binding half-life for the SLYNTVATL-HLA-A*02 complex, compared to the WT TCR, and other previously disclosed high affinity variants, making them particularly suitable for use as soluble therapeutic agents for redirecting a T cell response against HIV infected cells presenting low levels of SLYNTVATL-HLA-A*02 complex.

Example 4

Potent T Cell Redirection against Cells Presenting SLYNTVATL-HLA-A*02 Complex by TCR Anti-CD3 Fusion Molecules

[0110] The ability of the TCR anti-CD3 fusions of the invention to drive a potent T cell response in the presence of cells presenting the SLYNTVATL-HLA-A*02 complex was investigated using an ELISPOT assay, with IFNγ secretion as a read out for T cell activation.

[0111] Assays were carried out using the IFNγ ELISPOT assay kit (BD Biosciences, cat no 551849), as directed by the manufacturer. T2 cells (American Type Culture Collection) pulsed with 1 nM SLYNTVATL peptide were used as target cells, and plated at 5×10.sup.4 cells/well in 50 μl. Titrated concentrations of TCR-anti CD3 fusions were then added at final concentrations of 10, 1, 0.1, 0.01, 0.001, 0.0001 nM in 50 pl. Effector cells (CD8+ T cells) from donor PBMCs were isolated by Ficoll-Hypaque density gradient separation (Lymphoprep, Nycomed Pharma AS), utilising Lymphoprep (Axis-Shields, cat no NYC-1114547) and Leucosep tubes (Greiner, cat no 227290). CD8+ T cells were enriched from PBMC by negative selections using magnetic bead immunodepletion, in accordance with the manufacturer's instructions (MACS, Miltenyi Biotec). Effector cells were plated at 8×10.sup.4 cells/well in 50 μl. The final volume of each well was made up to 200 μl with assay buffer (10% FCS, 88% RPMI, 1% glutamine, 1% penicillin/streptomycin).

[0112] Plates were incubated for 18-20 h at 37° C. and 5% CO2 and quantified after development using an automated ELISpot reader (Immunospot Series 5 Analyzer, Cellular Technology Ltd).

[0113] Target cells presenting an irrelevant peptide were added as a negative control in the presence of effectors and 1 nM TCR-anti CD3 fusion. Additional control samples were prepared with effectors cell plus target cells and effectors alone. Data were analysed using Prism 5.0 software (Graph Pad, Software) to calculate EC50 values

Results FIG. 7 shows the response curves produced by m121 and m134. The EC.sub.50 values derived from the curves are in the table below

TABLE-US-00009 Alpha chain Beta chain variable variable TCR ID sequence sequence EC.sub.50 (pM) m121 SEQ ID NO: 9 SEQ ID NO: 11 6 (with Q at position 1 and the optional 8 residues present at the C terminus of TRAC) m134 SEQ ID NO: 10 SEQ ID NO: 11 8 (with Q at position 1 and the optional 8 residues present at the C terminus of TRAC)

COMPARATIVE EXAMPLE

[0114] The previously known highest affinity mutant of the WT TCR (termed a11b6, (Varela-Rohena et al. 2008, Nat Med, 14(12):1390-5)) was prepared as an anti-CD3 fusion and the EC.sub.50 value determined at the same time and under identical conditions as for m121 and m134 above. The EC.sub.50 value for al l b6 was calculated to be 135 pM.

[0115] These data demonstrate that the TCR-anti CD3 fusions of the invention are, unexpectedly, highly potent at redirecting T cells against cells presenting SLYNTVATL-HLA-A*02 complex, and are therefore ideal as therapeutic reagents for targeting HIV infected cells presenting low levels of SLYNTVATL-HLA-A*02 complex.

Example 5

Specific T Cell Redirection by TCR Anti-CD3 Fusions

[0116] The specificity of TCR-anti CD3 fusions was assessed by IFNγ ELISPOT assay using antigen-negative, HLA-A*02-positive, human cancer cell lines as target cells (melanoma cells MeI526 (Thymed) and bladder cancer cells J82 (American Type Culture Collection)). The assays were carried out using the same procedure as described in Example 4. Various concentrations of TCR anti-CD3 fusions were used as indicated in FIG. 8.

Results

[0117] The data presented in FIG. 8 indicate that low-levels of non-specific T cell activation could only be detected at high concentrations of TCR-anti CD3 fusions, i.e. at least 1000-fold greater than EC50 values, and outside of the expected therapeutic range (greater than 10.sup.−9M). Therefore the TCRs of the invention have an unexpectedly high level of target specificity.

Example 6

T Cell Redirection by TCR anti-CD3 Fusions Occurs Even at Low Peptide Concentrations

[0118] To determine the sensitivity of TCR anti-CD3 fusions of the invention to peptide HLA-complex, peptide titration experiments were carried out using T2 cells pulsed with various concentrations of peptide. T cell activation was determined using the same IFNγ ELISPOT procedure as described in Example 4. TCR anti-CD3 fusions were used at a concentration of 1 nM. Peptide concentrations are indicated in FIG. 9.

Results

[0119] The data presented in FIG. 9 demonstrate that TCR anti-CD3 fusions are sensitive to low nanomolar concentrations of exogenously loaded peptide. It has been shown in the art that pulsing T2 cells at peptide concentrations of 10-9 M results in low number of epitopes per cell (>50) and corresponds to the number of epitopes presented on the surface of cancer cells (Bossi et al. Oncoimmunology, 2013, 2(11): e26840); therefore, the TCRs of the invention are unexpectedly highly sensitive to cells presenting very low number of epitopes. Such a high level of sensitivity may facilitate the clearance of reservoirs of virally infected cells in vivo.