Bifunctional polypeptides
10576162 ยท 2020-03-03
Assignee
Inventors
Cpc classification
C07K2319/33
CHEMISTRY; METALLURGY
A61K47/6425
HUMAN NECESSITIES
C07K16/2809
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C07K2317/73
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
A bifunctional polypeptide comprising a specific binding partner for a peptide-MHC epitope, such as an antibody or T cell receptor, and an immune effector, such as an antibody or a cytokine, the immune effector part being linked to the N-terminus of the peptide-MHC binding part.
Claims
1. A bifunctional molecule, comprising: a single chain T-Cell Receptor (scTCR), the scTCR comprising a TCR or TCR constant region, wherein the scTCR binds specifically to a given pMHC epitope, and an immune effector polypeptide comprising a single chain immunoglobulin (scFv) which specifically binds to CD3, wherein: the N-terminus of the scTCR is linked to the C-terminus of the scFv, and the bifunctional molecule is capable of binding to the given pMHC epitope and CD3, each on a different cell, and thus simultaneously binding to two different cells.
2. The bifunctional molecule of claim 1, wherein the scFv comprises SEQ ID NO: 3.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
(1) These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
DETAILED DESCRIPTION OF THE INVENTION
(19) Accordingly, the present invention provides a bifunctional molecule comprising a polypeptide binding partner specific for a given pMHC epitope, and an immune effector polypeptide, the N-terminus of the pMHC binding partner being linked to the C-terminus of the immune effector polypeptide, PROVIDED THAT the said polypeptide binding partner is not a T-cell receptor comprising the alpha chain SEQ ID No: 7 and the beta chain SEQ ID No: 9.
(20) As mentioned, the polypeptide pMHC binding partner may be an antibody or a TCR. Thus in one embodiment of the invention the pMHC binding partner is a heterodimeric TCR polypeptide pair, or a single chain TCR polypeptide, and the N-terminus of the or chain of the heterodimeric TCR polypeptide pair, or the N-terminus of the scTCR polypeptide, is linked to a C-terminal amino acid of the immune effector polypeptide.
(21) Linkage of the pMHC binding partner and the immune effector polypeptide may be direct, or indirect via linker sequence. Linker sequences are usually flexible, in that they are made up of amino acids such as glycine, alanine and serine which do not have bulky side chains likely to restrict flexibility. Usable or optimum lengths of linker sequences are easily determined in the case of any given pMHC binding partner-immune effector construct. Often the linker sequence will by less than about 12, such as less that 10, or from 5-10 amino acids in length.
(22) In some embodiments of the invention the pMHC binding partner is a heterodimeric TCR polypeptide pair wherein the and polypeptides each have TCR variable and constant regions, but lack TCR transmembrane and cytoplasmic regions. The TCR part in such cases is soluble. In particularly preferred bifunctional molecules of this type, a non-native disulfide bond between residues of the constant regions of TCR and polypeptides is present. In particular the constant regions of the and polypeptides may be linked by a disulfide bond between cysteine residues substituted for Thr 48 of exon 1 of TRAC1 and Ser 57 of exon 1 of TRBC1 or TRBC2, or by the native disulfide bond between Cys4 of exon 2 of TRAC*01 and Cys2 of exon 2 of TRBC1 or TRBC2.
(23) In other embodiments of the invention, the pMHC binding partner is a single chain TCR polypeptide of the V-L-V, V-L-V, V-C-L-V, or V-L-V-C type wherein V and V are TCR and variable regions respectively, C and C are TCR and constant regions respectively, and L is a linker sequence.
(24) Immune effector polypeptides are known. They are molecules which induce or stimulate an immune response, through direct or indirect activation of the humoural or cellular arm of the immune system, such as by activation of T-cells. Examples include: IL-1, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-, IFN-, TNF, Anti-CD2 antibody, Anti-CD3 antibody, Anti-CD4 antibody, Anti-CD8 antibody, Anti-CD44 antibody, Anti-CD45RA antibody, Anti-CD45RB antibody, Anti-CD45RO antibody, Anti-CD49a antibody, Anti-CD49b antibody, Anti-CD49c antibody, Anti-CD49d antibody, Anti-CD49e antibody, Anti-CD49f antibody, Anti-CD16 antibody, Anti-CD28 antibody, Anti-IL-2R antibodies, Viral proteins and peptides, and Bacterial proteins or peptides. Where the immune effector polypeptide is an antibody it may be scFv antibody, one such being an anti-CD3 scFv. Examples of anti-CD3 antibodies include but are not limited to OKT3, UCHT-1, BMA031 and 12F6.
(25) The principles of the invention are illustrated by the following Examples.
EXAMPLE A.
Preparation of Soluble TCRs Having Effector Polypeptides Fused to the C- or N-Terminus of the TCR Chain
(26) A1. Soluble NY-ESO TCR with Anti-CD3 Antibody as Effector Polypeptide
(27) The soluble NY-ESO TCR of this example has the property of binding to the SLLMWITQV peptide (SEQ ID NO: 16) when presented on an HLA-A2 molecule.
(28) SEQ ID No: 1 (
(29) SEQ ID No: 2 (
(30) SEQ ID No: 3 (
(31)
(32) SEQ ID No: 14 (
(33) SEQ ID No: 15 (
(34) The construct of
(35) Ligation
(36) Synthetic genes encoding (a) the TCR chain SEQ ID No: 1 and (b) the fusion sequence of SEQ ID No: 2 and SEQ ID No: 3, were separately ligated into pGMT7-based expression plasmids, which contain the T7 promoter for high level expression in E. coli strain BL21-DE3(pLysS) (Pan et al., Biotechniques (2000) 29 (6): 1234-8.
(37) Expression
(38) The expression plasmids were transformed separately into E. coli strain BL21 (DES) Rosetta pLysS, and single ampicillin-resistant colonies were grown at 37 C. in TYP (ampicillin 100 g/ml) medium to OD.sub.600 of 0.6-0.8 before inducing protein express with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 ml Bug Buster (NovaGen) in the presence of MgCl.sub.2 and DNase. Inclusion body pellets were recovered by centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three detergent washes were then carried out to remove cell debris and membrane components. Each time the inclusion body pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted by centrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was then removed by a similar wash in the following buffer: 50 mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mg aliquots and frozen at 70 C.
(39) Refolding
(40) Approximately 20 mg of TCR chain and 40 mg of scFv-TCR chain solubilised inclusion bodies were thawed from frozen stocks, diluted into 20 ml of a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 m NaCl, 10 mM EDTA, 20 mM OTT), and incubated in a 37 C. water bath for 30 min-1 hr to ensure complete chain de-naturation. The guanidine solution containing fully reduced an denatured TCR chains was then injected into 1 litre of the following refolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5M Urea. The redox couple (cysteamine hydrochloride and cystamine dihydrochloride (to final concentrations of 16 mM and 1.8 mM, respectively)) were added approximately 5 minutes before addition of the denatured TCR and scFv-TCR chains. The solution was left for 30 minutes. The refolded scFv-TCR was dialysed in dialysis tubing cellulose membrane (Sigma-Aldrich; Product No. D9402) against 10 L H.sub.2O for 18-20 hours. After this time, the dialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5 C.3 C. for another 8 hours. Soluble and correctly folded scFv-TCR was separated from misfolded, degradation products a impurities by a 3-step purification method as described below. The second purification step can either be an ion exchange chromatography or an affinity chromatography, depending on the pl of the soluble. anti-CD3 scFv-TCR fusion.
(41) First Purification Step
(42) The dialysed refold (in 10 mM Tris pH8.1) was loaded onto a POROS 50HQ anion exchange column and the bound protein eluted with a gradient of 0-500 mM NaCl over 6 column volumes using an Akta purifier (GE Healthcare). Peak fractions (eluting at a conductivity 20 mS/cm) were stored at 4 C. Peak fractions were analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled.
(43) Second Purification Step
(44) Ion Exchange Chromatography
(45) Cation Exchange Purification:
(46) The anion exchange pooled fractions were buffer exchanged by dilution with 20 mM MES pH6-6.5, depending on the pl of the scFv-TCR fusion. The soluble and correctly folded scFv-TCR was separated from misfolded, degradation products and impurities by loading the diluted pooled fractions (in 20 mM MES pH6-6.5) onto a POROS 50HS cation exchange column and eluting bound protein with a gradient of 0-500 mM NaCl over 6 column volumes using an Akta purifier (GE Healthcare). Peak fractions (eluting at a conductivity 10 mS/cm) were stored at 4 C.
(47) Alternatively, ion exchange purification using hydroxyapatite matrix can be used a explained below.
(48) Hydroxyapatite Chromatography:
(49) The anion exchange pooled fractions were buffer exchanged by dilution with 10 mM NaH.sub.2PO.sub.4 pH6.0. The soluble and correctly folded scFv-TCR was separated from misfolded, degradation products and impurities by loading the diluted pooled fractions (in 10 mM NaH.sub.2PO.sub.4 pH6.0) onto a hydroxyapatite column and eluting bound protein with a gradient of 10-500 mM NaH.sub.2PO.sub.4/1M NaCl over 6 column volumes using an Akta purifier (GE Healthcare). Peak fractions (eluting at a conductivity 20 mS/cm) were stored at 4 C.
(50) Affinity Chromatography
(51) For some scFv-TCR fusions with a pl close to 6-6.5, the ion exchange step cannot be used but can be replaced by an affinity chromatography step. The protein L affinity chromatography column (Pierce, product number 89928) isolates and purifies certain immunoglobulin classes via their kappa light chains. Protein L can also binds sing chain variable fragments (scFv). The anion exchange pooled fractions were buffer exchanged by dilution with PBS/0.02% sodium azide. The soluble and correctly folded scFv-TCR was separated from misfolded, degradation products and impurities by loading the diluted pooled fractions onto a Protein L column and eluting bound protein with a gradient of 0-25 mM Glycine pH2.3/0.02% sodium azide over 3 column volumes using an Akta purifier (GE Healthcare). The scFv-TCR eluted very late in gradient and the pH of the eluted fractions was neutralized by addition of Tris pH8 1 (100 mM Tris pH8.1 final concentration). The peak fractions were stored at 4 C.
(52) Final Purification Step
(53) Peak fractions from second purification step were analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled. The pooled fractions were then concentrated for the final purification step, when the soluble scFv-TCR was purified and characterised using a Superdex 5200 gel filtration column (GE Healthcare) pre-equilibrated in PBS buffer (Sigma). The peak eluting at a relative molecular weight of approximately 78 kDa was analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled. In a similar manner to that described for the construct of
(54)
(55)
(56)
(57) In a similar manner to that described for the constructs of
(58) A2. Soluble Chimeric TCR with Cytokines as Effector Polypeptides
(59) SEQ ID No: 7 (
(60) SEQ ID No: 9 (
(61) The SEQ ID No: 7 and 9 TCR is a chimeric TCR consisting of an alpha and a beta TCR chain, each comprising a murine variable region and a human constant region. The chimeric version of the TCR was constructed to improve refolding problems encountered with the fully murine TCR; and the chimeric TCR was shown to have the same affinity as the murine TCR for the murine insulin-derived peptide-murine H-2K.sup.d complex.
(62) SEQ ID No: 10 (
(63) SEQ ID No: 11 (
(64)
(65)
(66)
(67)
(68) The constructs of
(69) Ligation
(70) Synthetic genes encoding (a) the TCR chain SEQ ID No: 7 and (b) the fusion sequence of SEQ ID No: 9 and SEQ ID No: 10 or 11, were separately ligated into pGMT7-based expression plasmids, which contain the T7 promoter for high level expression in E. coli strain BL21-DE3(pLysS) (Pan et al., Biotechniques (2000) 29 1234-8).
(71) Expression
(72) The expression plasmids containing the TCR -chain and cytokine--chain respectively were transformed separately into E. coli strain BL21 (DE3) Rosetta pLysS, and single ampicillin-resistant colonies were grown at 37 C. in TYP (ampicillin 100 g/ml) medium to 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 for 30 minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 ml Bug Buster (NovaGen) in the presence of MgCl.sub.2 and DNase. Inclusion body pellets were recovered by centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three detergent washes were then carried out to remove cell debris and membrane components. Each time the inclusion body pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted by centrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was then removed by a similar wash in the following buffer: 50 mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mg aliquots and frozen at 70 C. Inclusion body protein yield was quantified by solubilising with 6M guanidine-HCl and an OD measurement was taken on a Hitachi U-2001 Spectrophotometer. The protein concentration was the calculated using the theoretical extinction coefficient.
(73) Refolding
(74) Approximately 20 mg of TCR chain and 40 mg of cytokine-TCR chain solubilise inclusion bodies were thawed from frozen stocks and diluted into 20 ml of a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 m NaCl, 10 mM EDTA, 10 mM DTT), and incubated in a 37 C. water bath for 30 min-1 hr to ensure complete chain de-naturation. The guanidine solution containing fully reduced and denatured TCR chains was then injected into 1 litre of cold (5-10 C.) refolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5M Urea. The redox couple (cysteamine hydrochloride and cystamine dihydrochloride (to final concentrations 10 mM and 2.5 mM, respectively)) were added approximately 5 minutes before addition of the denatured TCR and cytokine-TCR chains. The solution was left for 30 minutes. The refolded cytokine-TCR was dialysed in dialysis tubing cellulose membrane (Sigma-Aldrich; Product No. 09402) against 10 L H.sub.2O for 18-20 hours. After this time, the dialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5 C.3 C. for another 8 hours.
(75) Purification
(76) Soluble cytokine-TCR fusion was separated from degradation products and impurities by a 3-step purification method at RT as described below.
(77) First Purification Step
(78) The dialysed refold was filtered using a Sartopore 0.2 m capsule (Sartorius) prior to column purification. Filtered refold was loaded onto a POROS 50HQ anion exchange column and the bound protein eluted with a linear gradient of 0-500 mM NaCl over 6 column volumes using an Akta purifier (GE Healthcare). Peak fractions eluting at 250 mM NaCl, consisting of correctly folded protein, were stored at 4 C. Peak fractions were analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled.
(79) Second Purification Step
(80) Pooled fractions containing soluble cytokine-TCR were mixed with an equivalent volume of 50 mM Tris/1M (NH.sub.4).sub.2SO.sub.4 pH 8 to give a final concentration of 0.5 M (NH.sub.4).sub.2SO.sub.4 and a conductivity of 75-80 mS/cm at RT. The soluble cytokine-TCR were separated from degradation products and impurities by loading this sample onto pre-equilibrated (50 mM Tris/0.5M (NH.sub.4).sub.2SO.sub.4 pH 8) butyl hydrophobic interaction column (5 ml HiTrap GE Healthcare) and collecting the flow through using an Akta purifier (GE Healthcare). Flow through sample containing soluble cytokine-TCR was analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled and stored at 4 C.
(81) Final Purification Step
(82) Pooled fractions were diluted with an equivalent volume of 10 mM Tris pH8 and concentrated to 10 ml (concentration of 3 mg/ml). The soluble cytokine-TCR as purified using a Superdex S200 gel filtration column (GE Healthcare) pre-equilibrated in PBS buffer (Sigma). The peak eluting at a relative molecular weight of approximately 63 kDa was analysed by Instant Blue Stain (Novexin) stained SDS-PAGE before being pooled.
EXAMPLE B.
Properties of Soluble TCRs Having Effector Polypeptides Fused to the C- or N-Terminus of the TCR Chain
(83) B1. Soluble NY-ESO TCR with Anti-CD3 Antibody as Effector Polypeptide
(84) a. Redirection and Activation of CD8.sup.+ T Cells by the Soluble NY-ESO TCR Fused to an Anti-CD3 Antibody Against NY-ESO Peptide-Presenting Cells
(85) The following assay was carried out to demonstrate the activation of cytotoxic T lymphocytes (CTLs) by an anti-CD3 scFv-TCR fusion via specific peptide-MHC complex. IFN- production, as measured using the ELISPOT assay, was used as a read-out for cytotoxic T lymphocyte (CTL) activation and the evaluation of the potency of the anti-CD3 scFv portion of the fusion.
(86) Reagents
(87) Assay media: 10% FCS (Gibco, Cat#2011-09), 88% RPMI 1640 (Gibco, Cat#42401), 1% glutamine (Gibco Cat#25030) and 1% penicillin/streptomycin (Gibco Cat#15070-063).
(88) Peptide: (SLLMWITQV (SEQ ID NO: 16)) initially dissolved in DMSO (Sigma, cat#D2650) at 4 mg/ml and frozen. T2 cells were pulsed with the described peptide and used as target cells.
(89) Wash buffer: 0.01 M PBS/0.05% Tween 20
(90) PBS (Gibco Cat#10010)
(91) The Human IFN ELISPOT PVDF-Enzymatic kit (Diaclone, France; Cat#856.051.020) contains all other reagents required. (Capture and detection antibodies, skimmed milk powder, BSA, streptavidin-alkaline phosphatase and BCIP/NBT solution as well as the Human IFN- PVDF ELISPOT 96 well plates)
(92) Method
(93) Target Cell Preparation
(94) The target cells used in this method were either (1) natural epitope-presenting cell (such as Mel624 or Mel526 cells) or (2) T2 cells pulsed with the peptide of interest, described in the reagents section. Sufficient target cells (50 000 cells/well) were washed by centrifugation three times at 1200 rpm, 10 min in a Megafuge 1.0 (Heraeus). Cells were then re-suspended in assay media at 10.sup.6 cells/ml.
(95) Effector Cell Preparation
(96) The effector cells (T cells) used in this method were either CD8+ T cells (obtained by negative selection (using the CD8 Negative Isolation Kit, Dynal, Cat#113.19) from PBL), T cells from an EBV cell line or PBMCs. Effector cells were defrosted and placed in assay media prior to washing by centrifugation at 1200 rpm, 10 min in a Megafuge 1.0 (Heraeus). Cells were then re-suspended in assay media at a 4 the final required concentration.
(97) Reagent/Test Compound Preparation
(98) Varying concentrations of test compounds (the TCR-anti-CD3 fusions; from 10 nM to 0.03 pM) were prepared by dilution into assay media to give 4 final concentration.
(99) ELISPOTs
(100) Plates were prepared as follows: 100 l anti-1FN-y capture antibody was diluted in 10 ml sterile PBS per plate. 100 l of the diluted capture antibody was then aliquoted into each well. The plates were then incubated overnight at 4 C. Following incubation the plates were washed (programme 1, plate type 2, Ultrawash Plus 96-well plate washer; Dynex) to remove the capture antibody. Plates were then blocked by adding 100 l 2% skimmed milk in sterile PBS to each well and incubating the plates at room temperature for two hours. The skimmed milk was then washed from the plates (programme 1, plate type 2, Ultrawash Plus 96-well plate washer, Dynex) and any remaining wash buffer was removed by flicking and tapping the ELISPOT plates on a paper towel.
(101) The constituents of the assay were then added to the ELISPOT plate in the following order: 50 l of target cells 10.sup.6 cells/ml (giving a total of 50 000 target cells/well) 50 l of reagent (the anti-CD3 scFv-TCR fusions; varying concentrations) 50 l media (assay media) 50 l effector cells (between 1000 and 50000 CD8.sup.+ cells/well; between 500 and 1000 EBV cells/well; between 1000 and 50000 PBMC/well).
(102) The plates were then incubated overnight (37 C./5% CO.sub.2). The next day the plates were washed three times (programme 1, plate type 2, Ultrawash Plus 96-well plate washer, Dynex) with wash buffer and tapped on paper towel to rer1 love excess wash buffer. 100 50 l primary detection antibody was then added to each well. The primary detection antibody was prepared by adding 550 l of distilled water to a vial of detection antibody supplied with the Diaclone kit. 100 l of this solution was then diluted in 10 ml PBS/1% BSA (the volume required for a single plate). Plates were then incubated at room temperature for at least 2 hr prior to being washed three times (programme 1, plate type 2, Ultrawash Plus 96-well plate washer, Dynex) with wash buffer, excess wash buffer was removed by tapping the plate on a paper towel. Secondary detection was performed by adding 100 l of diluted streptavidin-Alkaline phosphatase to each well and incubating the plate at room temperature for 1 hour. The streptavidin-Alkaline phosphatase was prepared by addition of 10 l streptavidin-Alkaline phosphatase to 10 ml PBS/1% BSA (the volume required for a single plate). The plates were then washed three times (programme 1, plate type 2, Ultrawash Plus 96-well plate washer, Dynex) with wash buffer and tapped on paper towel to remove excess wash buffer. 100 l of BCIP/NBT solution, as supplied with the Diaclone kit, was then added to each well. During development plates were covered in foil and left for 5-15 min. Developing plates were regularly checked for spots during this period to determine optimal time to terminate the reaction. The plates were washed in a sink full of tap water to terminate the development reaction, and shaken dry prior to their disassembly into their three constituent parts. The plates were then dried at 50 C. for 1 hr prior to counting the spots that have formed on the membrane using an Immunospot Plate reader (CTL; Cellular Technology Limited).
(103) Results
(104) The anti-CD3 scFv-TCR fusion constructs of
(105) From these dose-response curves, the EC.sub.50 values were determined (EC.sub.50 are determined at the concentration of anti-CD3 scFv-TCR fusion that induces 50% of the maximum response).
(106) TABLE-US-00001 TABLE 1 Test Construct EC50 EC50 EC50 FIG. 7 C-terminal fusion 5.044e9 1.864e9 2.383e9 FIG. 5 N-terminal fusion 8.502e11 FIG. 4 N-terminal fusion 4.825e11 FIG. 6 N-terminal fusion 3.95e11
(107) These results show that the N-fused constructs of
(108) b. Redirection of CD8+ T Cells by the Soluble NY-ESO TCR Fused to an Anti-CD3 Antibody to Kill the IM9 EBV Transformed B Cell Line (Non-Radioactive Cytotoxicity Assay)
(109) The following assay was carried out to demonstrate: the activation of cytotoxic T lymphocytes (CTLs) by a TCR-anti-CD3 scFv fusion via specific peptide-MHC complex and the evaluation of the potency of the anti-CD3 scFv portion of the fusion to activate the CTLs to kill the IM9 cells. This assay is a colorimetric alternative to .sup.51Cr release cytotoxicity assays and quantitatively measures lactate dehydrogenase (LDH) which is an enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a 30-minute coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of colour formed is proportional to the number of lysed cells. The absorbance data is collected using a standard 96-well plate reader at 490 nm.
(110) Materials
(111) CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) (G1780) contains Substrate Mix, Assay Buffer, Lysis Solution, and Stop Solution Assay media: 10% FCS (heat-inactivated, Gibco, cat#10108-165), 88% RPMI 1640 without phenol red (Invitrogen, cat#32404014), 1% glutamine, 200 mM (Invitrogen, cat#25030024), 1% penicillin/streptomycin (Invitrogen cat#15070063) Nunc microwell round bottom 96 well tissue culture plate (Nunc, cat#163320) Nunc-Immuno plates Maxisorb (Nunc, cat#442404)
Method
Target Cell Preparation
(112) The targets cells used in this assay were the IM9 EBV transformed B cell-line derived from a multiple myeloma patient (HLA-A2.sup.+ NY-ESO). The Mel526 melanoma cell line was used as a control and is HLA-A2.sup.+ NY-ESO. Target cells were prepared in assay medium: target cell concentration was adjusted to 210.sup.5 cells/ml to give 110.sup.4 cells/well in 50 l.
(113) Effector Cell Preparation
(114) The effector cells used in this assay were CD8.sup.+ T cells. The effector to target ratio used was 10:1 (210.sup.6 cells/ml to give 110.sup.5 cells/well in 50 l).
(115) Reagent/Test Compound Preparation
(116) Varying concentrations of the NY-ESO TCR-anti-CD3 fusions, having the TCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ ID No: 14, or having the TCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ ID No: 15, were prepared as described in example A1 and prepared for this assay by dilution (10.sup.13 to 10.sup.8M final concentration) into assay media.
(117) Assay Preparation
(118) The constituents of the assay were added to the plate in the following order: 50 l of target cells, IM9 or Mel526 (prepared as explained previously), to each well 50 l of reagent (prepared as explained previously) to each well. 50 l of effector cells (prepared as explained previously) to each well
(119) Several controls were prepared as explained below: Effector spontaneous release: 50 l effector cells alone. Target cells spontaneous release: 50 l target cells alone. Target maximum release: 50 l target cells plus 80 ug/ml digitonin at the start of the assay to lyse cells Assay medium control: 150 l medium alone.
Experimental wells are prepared in triplicate and control wells in duplicate in a final volume of 150 l.
The plate was centrifuged at 250g for 4 minutes then incubated at 37 C. for 24 hours.
(120) The plate was centrifuged at 250g for 4 minutes. 37.5 l of the supernatant from each well of the assay plate was transferred to the corresponding well of a flat-bottom 96 well Nunc Maxisorb plate. The Substrate Mix was reconstituted using Assay Buffer (12 ml). 37.5 l of the reconstituted Substrate Mix was then added to each well of the plate. The plate was covered with aluminum foil and incubated at room temperature for 30 minutes. 37.5 l of Stop Solution was added to each well the plate to stop the reaction. The absorbance at 490 nm was recorded on an ELIS plate reader within one hour after the addition of Stop Solution.
(121) Calculation of Results
(122) The average of absorbance values of the culture medium background was subtracted from all absorbance values of Experimental, Target Cell Spontaneous Release and Effector Cell Spontaneous Release and Target maximum release.
(123) The corrected values obtained in the first two steps were used in the following formula to compute percent cytotoxicity:
% cytotoxicity=100(ExperimentalEffector SpontaneousTarget Spontaneous)/(Target Maximum ReleaseTarget Spontaneous)
Results
(124) The NY-ESO TCR-anti-CD3 scFv fusion constructs having (i) the TCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ ID No: 14 (C-terminal fusion) or (ii) the TCR alpha chain SEQ ID No: 1 and the TCR beta chain anti-CD3 scFv fusion SEQ ID No: 15 (N-terminal fusion) were tested by LDH release assay (as described above). The % cytotoxicity observed in each well was plotted against the concentration of the test construct using Prism (Graph Pad). From these dose-response curves, the EC50 values were determined (EC50 are determined at the concentration of TCR fusion that induces 50% of the maximum response).
(125) TABLE-US-00002 TABLE 2 Test Construct EC50 C-terminal fusion 1.2e.sup.9 (SEQ ID No: 1 and SEQ ID No: 14) N-terminal fusion 3.2e.sup.11 (SEQ ID No: 1 and SEQ ID No: 15)
(126) These results show that the N-terminal fusion comprising the TCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ ID No: 15 was at least 2-fold more potent in its ability to redirect cytotoxic T lymphocytes to kill the target cells than the C-terminal fusion construct comprising the TCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ ID No: 14.
(127) B2. Soluble Chimeric TCR with Cytokines as Effector Polypeptides
(128) a. Murine IL-4 Cytokine as Effector Polypeptide
(129) The following assay was used to test the biological activity of the cytokine portion of the murine IL-4-TCR fusion constructs of
(130) Materials
(131) CTLL-2 cells, Promega CellTiter-Glo luminescent cell viability assay (Cat# G7572) including CellTiter-Glo Buffer and CellTiter-Glo Substrate (lyophilized) Assay media: RPMI supplemented with 10% heat inactivated foetal bovine serum (Gibco, cat#10108-165), 88% RPMI 1640 (Gibco, cat#42401-018), 1% glutamine (Gibco, cat#25030-024), 1% penicillin/streptomycin (Gibco, cat#15070-063).
(132) CTLL-2 cells were harvested, washed once in assay media (centrifuged at 1200 rpm for 5 mins), counted, and viability was assessed using Trypan blue solution. If viability was less than 80% a ficoll gradient was performed to remove the dead cells (800g for 15 mins with brake off). Cells were washed a further two times and the volume was adjusted to give 110.sup.5 cells/ml final. CTLL-2 cells were added to a Nunc white flat-bottomed 96-well plate (5000 cells/well), followed by 50 l titrated concentrations of standard murine IL-4 (Peprotech), or murine IL-4-chimeric TCR fusion constructs of
(133) Results
(134) TABLE-US-00003 TABLE 3 Test construct EC50 EC50 EC50 m-IL4 4.984e13 3.767e13 5.148e13 FIG. 13 7.464e12 C-term fusion FIG. 12 5.913e13 8.897e13 N-term fusion
(135) These results show that the N-fused construct of
(136) b. Murine IL-13 Cytokine as Effector Polypeptide
(137) The following assay was used to test the biological activity of the cytokine portion of the murine IL-13-TCR fusion constructs of
(138) This assay was carried out to demonstrate the activity of the cytokine portion from a cytokine-TCR fusion, i.e. the inhibition of the production of IL-1 by human monocytes. This assay can be used to test cytokine-TCR fusions where the cytokine is murine IL-13.
(139) Materials
(140) Monocytes Derived from Buffy Coats (Buffy Coats from NBS Bristol Transfusion Service)
(141) Dynal Dynabeads MyPure Monocyte Kit 2 for untouched human cells (113.35) Assay media: 10% foetal bovine serum (heat-inactivated, Gibco, cat#10108-165), 88% RPMI 1640 (Gibco, cat#42401-018), 1% glutamine (Gibco, cat#25030-024), 1% penicillin/streptomycin (Gibco, cat#15070-063)
(142) Wash buffer: 0.01M PBS/0.05% Tween 20 (1 sachet of Phosphate buffered saline with Tween 20, pH7.4 from Sigma, cat# P-3563 dissolved in 1 Litre distilled water gives final composition 0.01M PBS, 0.138M NaCl, 0.0027M KCl, 0.05% Tween 20) PBS (Gibco, cat#10010-015).
(143) HBS.sup.+S Ca.sup.+2 and.sup.+2 Free (Gibco, cat#1018-165)
(144) Cytokine Eli-pair ELISA kits: IL-1 (Diaclone cat# DC-851.610.020) these kits contain all other reagents required i.e. capture antibody, detection biotinylated antibody, streptavidin-HRP, IL-1 standards, ready-to-use TMB. The following method is based on the instructions supplied with each kit.
(145) Nunc-Immuno plates Maxisorb (Nunc, cat#442404).
(146) Nunc microwell round bottom 96 well tissue culture plate (Nunc, cat#163320)
(147) BSA (Sigma, cat# A3059)
(148) H2SO4 (Sigma cat# S1526)
(149) Trypan blue (Sigma cat# T8154)
(150) Lipopolysaccharides (LPS) derived from E. coli 0111:84 (Sigma, cat# L4391)
(151) Recombinant murine IL-13 (Peprotech, cat#210-13) standard used when murine IL-13-TCR fusion reagents tested.
(152) Monocyte Isolation
(153) PBMCs were isolated from buffy coats: a buffy coat was diluted 1 in 2 with HBSS (Ca.sup.2+ and Mg.sup.2+ free), diluted blood was layered onto lymphoprep (up to 35 ml blood over 15 ml lymphoprep) and centrifuged 15 min at 800g (room temp) with the brake off; cells at the interface were removed and washed four times with HBSS and centrifuged at 1200 rpm for 10 min. After the final wash, cells were resuspended 50 ml assay media counted and viability was assessed using Trypan blue solution. Dynal Dynabeads MyPure Monocyte Kit 2 was used to isolate the monocytes. The PBMC were resuspended in PBS/0.1% BSA in 100 l buffer per 10.sup.7 cells, 20 l of Blocking Reagent per 10.sup.7 cells and 20 l Antibody Mix per 10.sup.7 cells were added and cells were incubated for 20 min at 4 C. The cells were washed and resuspended in 0.9 ml PBS/0.1% BSA per 10.sup.7 cells. Pre-washed beads were added (100 l per 10.sup.7 cells), mixed and incubated for a further 15 min at 20 C. with gentle rotation. Rose were resuspended by careful pipetting and 1 ml PBS/0.1% BSA per 10.sup.7 cells were added. The tube was placed in the Dynal magnet for 2 minutes. Supernatant containing negatively isolated cells was transferred to a fresh tube and counted. Cells were either used immediately or frozen down in 90% FCS/10% DMSO for future use.
(154) Cell Assay Preparation
(155) The ELISA plate was coated with 100 l/well IL-1 capture antibody in PBS and left at 4 C. overnight. Monocytes were thawed, washed twice in assay media and resuspended at 510.sup.5 cells/ml. The monocytes were plated out into a round bottomed 96 well plate (100 l per well, i.e. 510.sup.4 per well). LPS, Peprotech recombinant cytokine and test cytokine-TCR fusion proteins were prepared by dilution into assay media to give 4 final concentration. LPS was added in each well (long/ml final) followed by 50 l of titrated concentrations (6 points of 1 in 10 serial dilutions) of Peprotech recombinant IL-13 (10.sup.8 to 10.sup.13M final) or test cytokine-TCR fusion proteins (10.sup.7 to 10.sup.13M final) in triplicate wells. The plate was incubated at 37 C., 5% CO2 overnight.
(156) IL-1 ELISA
(157) The antibody coated IL-1 ELISA plate was washed three times in wash buffer and blocked with 250 l PBS/5% BSA/well for at least 2 hours at room temperature (or overnight at 4 C.). The ELISA plate was washed three times in wash buffer and tapped dry. The IL-1 standards were diluted in PBS/1% BSA. The plate containing the cells was centrifuged at 1200 rpm for 5 mins. The supernatant from each well was then transferred to the pre-coated IL-1 ELISA plate. 100 l of cell supernatant (diluted 1 in 3 with PBS/1% BSA) or standard were added to the relevant wells and 50 l detection antibody/well (dilution as per kit instructions) were added. The plate was incubated for 2 hours at room temperature. Plates were washed three times in wash buffer. 100 l of streptavidin-HRP were added per well (dilution as per kit instructions) and plates were incubated at room temp for 20 min. Plates were washed three times in wash buffer. 100 l of ready-to-use TMB per well were added and plates let to develop for 5-20 min (depending on signal strength) in the dark (under foil). Reaction was stopped by adding 100 l/well 1M H.sub.2 SO.sub.4.
(158) Plates absorbance was read on microplate reader at 450 nm and a reference filter set to 650 nm. The amount of inhibition for each titration point is calculated as a percentage of the sample containing monocytes and LPS without cytokineTCR fusion protein which gives the maximum signal thus producing a dose-response curve.
(159) Results
(160) TABLE-US-00004 TABLE 4 Test construct EC50 EC50 m-IL13 1.535e10 9.534e11 FIG. 15 6.21e10 C-term fusion FIG. 14 1.493e10 N-term fusion
(161) These results show that the N-fused construct of