BIFUNCTIONAL BINDING POLYPEPTIDES

Abstract

The present invention provides bifunctional binding polypeptide comprising a pMHC binding moiety and a PD-1 agonist.

Claims

1. A bifunctional binding polypeptide comprising a pMHC binding moiety and a PD-1 agonist.

2. A bifunctional binding polypeptide according to claim 1, wherein the pMHC binding moiety comprises TCR variable domains and/or antibody variable domains.

3. A bifunctional binding polypeptide according to claim 1, wherein the pMHC binding moiety is a T cell receptor (TCR) or a TCR-like antibody.

4. A bifunctional binding polypeptide according to any preceding claim, wherein the pMHC binding moiety is a heterodimeric alpha/beta TCR polypeptide pair.

5. A bifunctional binding polypeptide according to any preceding claim, wherein the pMHC binding moiety is a single chain alpha/beta TCR polypeptide.

6. A bifunctional binding polypeptide according to any one of claims 3-5, wherein the TCR comprises a non-native di-sulphide bond between the constant region of the alpha chain and the constant region of the beta chain.

7. A bifunctional binding polypeptide according to any one of claims 3-6, wherein the TCR binds specifically to a peptide antigen.

8. A bifunctional binding polypeptide according to any preceding claim, wherein the PD-1 agonist is PD-L1 or a functional fragment thereof.

9. A bifunctional binding polypeptide according to claim 8, wherein the PD-L1 comprises or consists of the sequence: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHS SYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY

10. A bifunctional binding polypeptide according to any one of claims 1-7, wherein the PD-1 agonist is a full-length antibody or fragment thereof.

11. A bifunctional binding polypeptide according to claim 10, wherein the PD-1 agonist is a scFv antibody.

12. A bifunctional binding polypeptide according to any preceding claim, wherein the PD-1 agonist is fused to the C or N terminus of the pMHC binding moiety.

13. A bifunctional binding polypeptide according to any preceding claim, wherein the PD-1 agonist is fused to the pMHC binding moiety via a linker.

14. A bifunctional binding polypeptide according to claim 13, wherein the linker is 2, 3, 4, 5, 6, 7 or 8 amino acids in length.

15. A pharmaceutical composition comprising the bifunctional binding polypeptide according to any one of claims 1-14.

16. A nucleic acid encoding the bifunctional binding polypeptide according to any one of claims 1-14.

17. An expression vector comprising the nucleic acid of claim 16.

18. A host cell comprising the nucleic acid of claim 16 or the vector of claim 17, optionally wherein the nucleic acid encoding the bifunctional binding polypeptide is present as a single open reading frame or two distinct open reading frames encoding the alpha chain and beta chain respectively.

19. A method of making the bifunctional binding polypeptide according to any one of claims 1-14 comprising maintaining the host cell of claim 18 under optional conditions for expression of the nucleic acid and isolating the bifunctional binding peptide.

20. A bifunctional binding polypeptide according to any one of claims 1-14, a pharmaceutical composition of claim 15, a nucleic acid of claim 16 and/or a vector of claim 17, for use in medicine, particularly for treating autoimmune disease or use in the treatment or prophylaxis of pain, particularly pain associated with inflammation

21. A bifunctional binding polypeptide, pharmaceutical composition, nucleic acid and/or vector for use according to claim 20, wherein the autoimmune disease is one of Alopecia Areata, Ankylosing spondylitis, Atopic dermatitis, Grave's disease, Multiple sclerosis, Psoriasis, Rheumatoid arthritis, Systemic lupus erythematosus, Type 1 diabetes and Vitiligo, Inflammatory Bowel Disease, Crohn's disease, ulcerative colitis, coeliac disease, eye diseases (e.g. uveitis), cutaneous lupus and lupus nephritis, and autoimmune disease in cancer patients caused by PD-1/PD-L1 antagonists.

22. A method of treating an autoimmune disorder comprising administering the bifunctional binding polypeptide according to any one of claims 1-14, the pharmaceutical composition of claim 15, the nucleic acid of claim 16 and/or the vector of claim 17 to a patient in need thereof.

Description

[0070] The invention is now described with reference to the following non-limiting examples and figures in which:

[0071] FIG. 1 shows dose-dependent inhibition of NFAT reporter activity with a bifunctional polypeptide of the invention comprising a soluble TCR and a truncated form of PD-L1, in the presence of peptide-pulsed target cells.

[0072] FIG. 2 shows inhibition of NFAT reporter activity with a bifunctional polypeptide of the invention comprising a soluble TCR and a PD-1 agonist scFv antibody fragment, in the presence of peptide-pulsed target cells.

[0073] FIG. 3 shows inhibition of primary human t cells activation with a bifunctional polypeptide of the invention comprising a soluble TCR and a PD-1 agonist scFv antibody fragment, in the presence of peptide-pulsed target cells.

[0074] FIG. 4 shows inhibition of NFAT reporter activity with a bifunctional polypeptide of the invention comprising one of two soluble TCRs with differing specificity, and a PD-1 agonist scFv antibody fragment, in the presence of peptide-pulsed target cells.

EXAMPLES

Example 1

[0075] The following example demonstrates that a PD-1 agonist fused to a soluble TCR can effectively inhibit T cell activation when targeted to the immune synapse.

[0076] The soluble TCR used in this bifunctional binding polypeptide is an affinity-enhanced version of a native TCR that specifically recognises a HLA-A*02 restricted peptide derived from human pre-pro insulin (such molecules are described in WO2015092362). The PD-1 agonist is a truncated version of the extracellular region of PD-L1 comprising the PD-1 interaction site (Zak et al., Structure 23:2341-2348, 2015). PD-L1 is fused to the N-terminus of the TCR alpha chain via a standard 5 amino acid linker.

[0077] A Jurkat NFAT luciferase PD-1 reporter assay was used for measuring TCR-PD1 agonist fusion molecule-mediated inhibition of T cell NFAT activity in the presence of HEK293T antigen presenting target cells.

Methods

Expression, Refolding and Purification of TCR-PD1 Agonist Fusion Molecules

[0078] Expression of TCR-PD1 agonist fusion molecules was performed using the high-yield transient expression system based on suspension-adapted Chinese Hamster Ovary (CHO) cells (ExpiCHO Expression system, Thermo Fisher). Cells were co-transfected according to the manufacturer's instructions, using mammalian expression plasmids containing the TCR chains fused to a PD-1 agonist. Following the harvest, clarification of cell culture supernatants was done by centrifuging the supernatant at 4000-5000×g for 30 minutes in a refrigerated centrifuge. Supernatants were filtered through a 0.22-μm filter and collected for further purification.

[0079] Alternatively, the expression of TCR-PD1 agonist fusion molecules was carried out using E. coli as the host organism. Expression plasmids containing alpha and beta chain were separately transformed into BL21pLysS E. coli strain and plated onto LB-agar plate containing 100 μg/mL ampicillin. Loopful colonies from each transformation were picked and grown in LB media (with 100 μg/mL ampicillin and 1% glucose) at 37° C. until OD600 reached ˜0.5-1.0. The LB starter culture was then added to autoinduction media (Foremedium) and cells grown for 37° C.˜3 hours followed by 30° C. overnight. Cells were harvested by centrifugation and lysed in Bugbuster (Novagen). Inclusion bodies (IBs) were extracted by performing two Triton wash (50 mM Tris pH 8.1, 100 mM, NaCl, 10 mM EDTA, 0.5% Triton) to remove cell debris and membrane. Each time IBs were harvested by centrifugation @10000 g for 5 minutes. To remove detergent, IBs were washed with 50 mM Tris pH8.1, 100 mM NaCl and 10 mM EDTA. IBs were finally re-suspended in 50 mM Tris pH8.1, 100 mM NaCl and 10 mM EDTA buffer. To measure the protein yield, IBs were solubilized in 8M Urea buffer and concentration determined by absorbance at 280 nM.

[0080] For refolding alpha and beta chains were mixed at 1:1 molar ratio and denatured for 30 minutes at 37° C. in 6 M Guanidine-HCl, 50 mM Tris pH8.1, 100 mM NaCl, 10 mM EDTA, 20 mM DTT. The denatured chains were then added to refold buffer consisting of 4 M Urea, 100 mM Tris pH 8.1, 0.4 M L-Arginine, 2 mM EDTA, 1 mM Cystamine and 10 mM Cysteamine and incubated for 10 minutes with constant stirring. The refold buffer containing the denatured chains was dialysed in Spectra/Por 1 membrane against 10× volume of H.sub.2O for ˜16 hours, 10× volume of 10 mM Tris pH 8.1 for ˜7 hours and 10× volume of 10 mM Tris pH8.1 for ˜16 hours.

[0081] Soluble proteins obtained from either mammalian or E. coli expression systems were purified on the AKTA pure (GE healthcare) using a POROS 50 HQ (Thermo Fisher Scientific) anion exchange column using 20 mM Tris pH 8.1 as loading buffer and 20 mM Tris pH8.1 with 1M NaCl as binding and elution buffer. The protein was loaded on the column and eluted with a gradient of 0-50% of elution buffer. Fractions containing the protein were pooled and diluted 20× (volume/volume) in 20 mM MES pH6.0 for second step cation exchange chromatography on POROS 50 HS (Thermos Fisher Scientific) column using 20 mM MES pH6.0 and 20 mM MES pH6.0, 1M NaCl as binding and elution buffer respectively. Bound protein from cation exchange column was eluted using 0-100% gradient of elution buffer. Cation-exchange fractions containing the protein were pooled and further purified on Superdex 200 HR (GE healthcare) gel filtration column using PBS as running buffer. Positive fractions from gel filtration were pooled, concentrated and stored at −80° C. until required.

Jurkat NFAT Luc-PD-1 Reporter Assay

[0082] HLA-A*02 positive HEK293T target cells were transiently transfected with a TCR activator plasmid (BPS Bioscience, Cat no: 60610) and pulsed with the relevant peptide recognised by the TCR-PD1 agonist fusion molecule. Target cells were then incubated with different concentrations of TCR-PD1 agonist fusion molecule to allow binding to cognate peptide-HLA-A2 complex. Jurkat NFAT Luc PD-1 effector cells, which constitutively express PD-1, were added to the target cells and NFAT activity determined after 18-20 h. Experiments were performed with or without washout (post-TCR-PD1 agonist fusion molecule binding). A further control was performed using non-pulsed target cells. TCR Activator/PD-L1 transfected HEK293T A2B2M target cells were included as positive controls.

Results

[0083] The data shown in FIG. 1, demonstrates that dose-dependent inhibition of NFAT reporter activity is observed with TCR-PD1 agonist fusion molecules in the presence of peptide-pulsed target cells, with or without wash out. Crucially, minimal inhibition was observed with non-pulsed target cells indicating that targeting to the immune synapse is critical for PD-1 agonist activity.

Example 2

[0084] The following example provides further evidence that a PD-1 agonist fused to a soluble TCR can effectively inhibit T cell activation when targeted to the immune synapse.

[0085] The experimental system and methods used in this example were the same as those described in Example 1, except that in this case the PD-1 agonist portion of the TCR-PD1 agonist fusion molecule was a scFv antibody fragment, such as described in WO2011110621.

[0086] The Jurkat NFAT luciferase PD-1 reporter assay described in Example 1 was used for measuring TCR-PD1 agonist fusion molecule-mediated inhibition of T cell NFAT activity in the presence of HEK293T antigen presenting target cells.

Results

[0087] As shown in FIG. 2a, substantial inhibition of NFAT activity (>60%) was observed in peptide pulsed cells (labelled +PPI) treated with 100 nM TCR-PD1 agonist fusion molecule; whereas minimal inhibition was seen in non-pulsed target cells (labelled −PPI) treated with the TCR-PD1 agonist fusion molecule. Control experiments, using either the soluble TCR alone, or the PD-1 agonist alone (in both scFv or IgG4 format), showed no inhibition of reporter activity, indicating that targeting of the PD-1 agonist to the immune synapse is required for PD-1 agonist activity. FIG. 2b further shows dose-dependent inhibition of NFAT activity. Again, only the TCR-PD1 agonist fusion molecule format is able to inhibit NFAT activity. Non-targeted PD-1 agonist antibody is not able to inhibit activity.

[0088] Taken together, these results demonstrate that targeting the PD-1 agonist to the immune synapse is critical for PD-1 agonist activity.

Example 3

[0089] The following example provides further evidence that a PD-1 agonist fused to a soluble TCR can effectively inhibit T cell activation when targeted to the immune synapse.

[0090] The TCR-PD1 agonist fusion molecule used in this example was the same as described in Example 2, in which the PD1 agonist is a scFv antibody fragment.

[0091] In this case an alternative assay was used to assess the effect of TCR-PD1 agonist fusion molecules on primary human T cell function.

Method

Primary Human T Cell Assay

[0092] Primary human T cells were isolated from freshly prepared PBMCs using a pan-T cell isolation kit (Miltenyi, cat no: 130-096-535). HLA-A*02 positive Raji B cells (Raji A2B2M) were pre-loaded with staphylococcal enterotoxin B (SEB, 100 ng/ml, Sigma S4881) for 1 h and then irradiated with 33Gy. For pre-activation, primary human T cells were incubated with SEB-loaded Raji A2B2M target cells at a 1:1 ratio, using 1×10E6 cells/ml of each cell type in 24-well cell culture plates. Primary human T cells were incubated for 10 days with SEB-loaded Raji A2B2M cells, with IL-2 (50 U/ml) added at d 3 and d 7. On day 10 pre-activated T cells were washed and re-suspended in fresh media. Fresh Raji A2B2M cells were pulsed with 20 μM of the relevant peptide recognised by TCR-PD1 agonist fusion molecules, or left non-pulsed for 2 h. Raji A2B2M cells were loaded with SEB (10 ng/ml) for the final 1 h of peptide pulsing and then irradiated with 33Gy. Raji A2B2M cells were plated into 96-well cell culture plates at 1×10E5 cells/well and then pre-incubated with TCR-PD1 agonist fusion molecules titrations for 1 h. Pre-activated T cells were added to the Raji A2B2M target cells at 1×10E5 cells/well and incubated for 48 h. Supernatants were collected and IL-2 levels were determined using an MSD ELISA.

Results

[0093] The data shown in FIG. 3 demonstrate that TCR-PD1 agonist fusion molecules dose-dependently inhibits primary human T cell IL-2 production in the presence of peptide pulsed target cells, whereas non-targeted TCR-PD1 agonist fusion molecules (i.e. with non-pulsed target cells) or the PD-1 agonist scFv alone do not. These data demonstrate that targeting PD-1 agonist to the immune synapse leads to PD-1 agonist activity in primary cells

Example 4

[0094] The following example demonstrates the same technical effect is observed using TCRs that recognise alternative antigens.

[0095] The experimental system and methods used in this example were the same as those described in Example 2. In this case a PD-1 agonist antibody was fused to two different soluble TCRs.

[0096] The Jurkat NFAT luciferase PD-1 reporter assay described in Example 1 was used for measuring TCR-PD1 agonist fusion molecule-mediated inhibition of T cell NFAT activity in the presence of HEK293T antigen presenting target cells.

Results

[0097] As shown in FIG. 4, potent and dose dependent inhibition was observed with two TCR-PD1 agonist fusion molecules (comprising a PD-1 agonist scFv antibody fragment fused to either TCR 1 or TCR 2) administered in the presence of target cells pulsed with their respective peptides (peptides 1 or 2). For both TCR-PD1 agonist fusion molecules, minimal activity was observed when the study was conducted without the presence of targeting peptide.

[0098] These results demonstrate that TCR-PD1 agonist fusion molecules can be directed to different tissues using soluble TCRs with specificities for different pMHC and facilitate targeted inhibition of T cell activity.