SSEA-4 BINDING MEMBERS
20220324997 · 2022-10-13
Inventors
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
G01N2400/38
PHYSICS
G01N33/92
PHYSICS
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
G01N33/57492
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/44
CHEMISTRY; METALLURGY
International classification
Abstract
The disclosure relates to the expression of stage-specific embryonic antigen 4 (SSEA-4) on stem memory T-cells (TSCM), which can then be used as a target to isolate, activate and expand this T cell subset both in vivo and in vitro. It also relates to the pharmaceutical antibody composition binding SSEA-4 targeting TSCM, as well as methods for use thereof. The antibody of the disclosure recognises the SSEA-4 glycolipid and induces proliferation of TSCM which could be used to sort this unique population from blood for clinical expansion for adoptive T-cell transfer of T-cell receptor (TCR) transduced, chimeric antigen receptor (CAR)-T transduced or cells for haematopoietic stem cell transplant. Methods of use include, without limitation, in cancer therapies and diagnostics. Examples related to the antibody with the designation F2811.72.
Claims
1. An isolated specific binding member capable of binding specifically to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) and targeting stem memory T-cells (T.sub.SCM).
2. The binding member of claim 1 wherein the binding member is capable of binding SSEA-4 on glycolipids.
3. The binding member of claim 1, wherein the binding member is capable of inducing proliferation of stem memory T-cells (T.sub.SCM).
4. The binding member according to claim 1, wherein the binding member does not bind to SSEA-3.
5. The binding member according to claim 1, wherein the binding member is mAb FG2811.72 or Chimeric FG2811.72 (CH2811/CH2811.72), or a fragment thereof.
6. The binding member according to claim 1, wherein the binding member is bispecific.
7. The binding member according to claim 1, wherein the bispecific binding member is additionally specific for CD3.
8. The binding member according to claim 1, wherein the binding member comprises one or more binding domains selected from the amino acid sequence of residues 27 to 38 (CDRH1), 56-65 (CDRH2) and 105-113 (CDRH3) of
9. The binding member according to claim 1, wherein the binding member comprises one or more binding domains selected from the amino acid sequence of residues 27 to 38 (CDRL1), 56-65 (CDRL2) and 105-113 (CDRL3) of
10. The binding member according to claim 1, wherein the binding member comprises a light chain variable sequence comprising one or more of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises SSVNY, LCDR2 comprises DTS, and LCDR3 comprises FQASGYPLT; and a heavy chain variable sequence comprising one or more of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises GFSLNSYG, HCDR2 comprises IWGDGST, and HCDR3 comprises TKPGSGYAF.
11. The binding member according to claim 1, wherein the binding domain(s) are carried by a human antibody framework.
12. The binding member according to claim 1, wherein the binding member comprises a VH domain comprising residues 1 to 126 of the amino acid sequence of
13. The binding member according to claim 1, wherein the binding member is an antibody, an antibody fragment, Fab, (Fab′)2, scFv, Fv, dAb, Fd or a diabody.
14. The binding member according to claim 1, wherein the binding member is a human, humanized, chimeric or veneered antibody.
15. A binding member according to claim 1, for use in therapy.
16. A method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a binding member according to any of claims claim 1.
17. A method of enhancing a protective immune response against cancer comprising administering a binding member according to claim 1 to a subject in need of thereof.
18. The method of claim 17, wherein the binding member is prepared to be administered with a further immunogenic agent, optionally wherein the immunogenic agent is a cancer vaccine.
19. A nucleic acid comprising a sequence encoding a binding member according to claim 1.
20. A method for diagnosis of cancer comprising using a binding member as claimed in claim 1 to detect the glycans SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) attached to a glycolipid in a sample from an individual.
21. A pharmaceutical composition comprising the binding member according to claim 1, and a pharmaceutically acceptable carrier.
22. A method of inducing proliferation of stem memory T-cells (T.sub.SCM) ex vivo comprising contacting the stem memory T-cells (T.sub.SCM) with a binding member according to claim 1.
23. A cell culture medium for inducing proliferation of stem memory T-cells (T.sub.SCM) comprising a binding member according to claim 1.
24. A method of identifying stem memory T-cells (T.sub.SCM) by detecting the presence of S SEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the cell with a binding member according to claim 1.
25. A method of purifying stem memory T-cells (T.sub.SCM) by detecting the presence of S SEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the cell with a binding member according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0127] As used herein, symbolic, graphic, and text nomenclature for describing glycans and related structures are well established and understood in the art, including, for example “Symbols Nomenclatures for Glycan Representation” by Ajit Varki et al (Varki et al. 2009).
FIGURE LEGENDS
[0128]
[0129]
[0135]
[0136] The binding of FG2811mG3, FG2811mG1, CH2811hG1, CH2811hG2, MC813 (anti-SSEA-4 mAb; mouse IgG1), MC631 (anti-SSEA-3 mAb; rat IgM), FG88.7 (anti-Lewis.sup.a/c/x mAb; mouse IgG3), anti-mouse secondary and tertiary antibody alone, anti-human secondary and tertiary antibody alone and medium alone to SSEA-3/-4-LMTK cells was assessed by flow cytometry. The result was presented as geometric mean (Gm) values.
[0137]
[0142]
[0146]
[0149]
[0154]
[0157]
[0160]
[0164]
[0167]
[0172]
[0174]
[0175] Pure T-cells isolated from 4 healthy donors (BD61, BD2, BD3, BD26) were stimulated with CH2811hG1 (5 μg/ml) at day 0. Unstimulated cells (medium) were included as negative control. Supernatants were collected at day 7, 11 and 14 and assessed for the concentration of IFNγ, TNFα, IL-8, IL-10, IL-2, IL-5, IL-17A, IL-7 and IL-21 (μg/ml). Individual dots represent different donors. Comparative analysis of the cytokine/chemokine results between CH2811hG1stimulated T-cells and unstimulated cells was performed by applying unpaired Student t test with values of P calculated accordingly (***, P<0.0001, **, P<0.01, *, P<0.05; GraphPad Prism 6).
[0176]
[0181]
[0182] HHDII/DP4 mice were euthanised and spleen, mesenteric and inguinal lymph nodes were harvested. i) splenocytes, ii) mesenteric lymph node cells and iii) inguinal lymph node cells were stained with FITC-labelled CH2811hG1 antibody and assessed using flow cytometric analysis.
[0183]
[0188]
[0194]
[0195] Naïve HHDII mice were culled, splenocytes were harvested, pan T cells enriched and CFSE labelled. CFSE labelled T cells were then plated out in wells contained plate bound 2811 mouse IgG1 (5 ug/mL) or Human IgG1 (5 ug/ml) or anti-CD3 (1 ug/ml) and incubated at 37° C. On day 7, 12 and 14, cells were taken as a sample and stained with anti CD4 and anti CD8 analysed by flow cytometry. [0196] (A) At day 12, representative flow cytometry plot of T cells proliferating in response to FG2811mG1 and FG2811hG1. [0197] (B) At day 12, the total percentage of cells proliferating in response to plate bound FG2811mG1, FG2811hG1 or anti CD3. [0198] (C) At day 12, the total percentage of CD8 T cells proliferating in response to plate bound FG2811mG1, FG2811hG1 or anti CD3. [0199] (D) At day 12, the total percentage of CD4 T cells proliferating in response to plate bound FG2811mG1, FG2811hG1 or anti CD3.
[0200]
[0201] Naïve HHDII mice were culled, splenocytes were harvested, pan T cells enriched and CFSE labelled. CFSE labelled T cells were then plated out in wells containing anti CD3 and anti CD28 (1 ug/ml each) and incubated at 37° C. On day 7, 12 and 14, cells were taken as a sample and stained with anti CD4 and anti CD8 analysed by flow cytometry. [0202] (A) At day 11, 15 and 20 cells were taken and stained with CH2811hG2-PeCy7 and/or anti CD3, (i) percentage 2811+ cells of CD3+ cells (ii) percentage CFSElow of CD3+ cells (iii) number of 2811+ cells (×10.sup.4 per mL) (iv) number of CD3+ T cells (×10.sup.5 per mL, (n=2 wells). [0203] (B) Representative flow cytometry plots of splenocytes stained 11 days after CD3/CD28 stimulation, cells were stained with anti CD3, CD44, CD62L, and CH2811hG2-PeCy7 and assessed using flow cytometry analysis. [0204] (C) After 11 days following CD3/CD28 stimulation the total number of 2811+ effector memory, central memory, effector and naive T cells was determined (n=2 wells). [0205] (D) After 11 days following CD3/CD28 stimulation the percentage of 2811.sup.+ effector memory, central memory, effector and naive T cells was determined (n=2 wells).
[0206]
[0207] Naïve HHDII/DP4 mice were culled, splenocytes were harvested, pan T cells enriched and CFSE labelled. CFSE labelled T cells were then plated out in wells containing stimulation with soluble human IgG2 (5 ug/mL) or mouse IgG1 2811 Ab (5 ug/mL) or anti-CD3 (1 μg/ml) and CD28 with and without AKTi, cells were incubated at 37° C. On day 11 and 15, cells were taken as a sample and stained with anti CD3, CD44, CD62L, SCA 1 and CH2811hG2-PeCy7 and assessed using flow cytometry analysis. [0208] (A) At day 11, representative flow cytometry plot of T cells proliferating in response to FG2811mG1 and 2811 hG2 (n=2 wells). [0209] (B) After 11 days following CD3/CD28, FG2811mG1 or 2811 hG2 stimulation the total number of 2811.sup.+ effector memory, central memory, effector and naive T cells was determined (n=2 wells). [0210] (C) After 11 days following CD3/CD28, FG2811mG1 or 2811 hG2 the percentage of 2811.sup.+ effector memory, central memory and effector T cells was determined (n=2 wells).
[0211]
[0212] PBMCs were isolated from Buffy coats, a Pan T cell enrichment was carried out and approximately 2×10.sup.6 cells incubated per well of a 24 well plates, in the presence of anti CD3/CD28 with or without additional cytokines (IL-7 or IL-21) for 20 days. On day 15 and day 20 cells were taken as sample and stained with CD45RA, CD62L, CD122, CD95, CD3, CCR7 and 2811hG Pe-Cy7. [0213] (A) At day 20, representative flow cytometry plot of phenotyping T cells proliferating in response to stimulation with anti CD3/CD28 (n=2 wells). [0214] (B) After 15 and 20 days following CD3/CD28 stimulation the (i) percentage of 2811+ CD3+ T cells, (ii) total number of 2811+ cells (×10.sup.4 per mL, n=2 wells).
[0215]
[0216]
DETAILED DESCRIPTION OF THE INVENTION
[0220] Methods
[0221] Plasma Membrane Glycolipid Extraction
[0222] SSEA-3/-4-LMTK cell pellets (5×10.sup.7 cells) were resuspended in 500 μl of Mannitol/HEPES buffer (50 mM Mannitol, 5 mM HEPES, pH7.2, both Sigma) and passed through 3 needles (23G, 25G, 27G) 30 times each. 5 μl of 1M CaCl.sub.2 was added to the cells and passed through 3 needles 30 times each as above. Sheared cells were incubated on ice for 20 mins then spun at 3,000 g for 15 mins at room temperature. The supernatant was collected and spun at 48,000 g for 30 mins at 4° C. and the supernatant discarded. The pellet was resuspended in 1 ml methanol followed by 1 ml chloroform and incubated with rolling for 30 mins at room temperature. The sample was then spun at 1,200 g for 10 mins to remove precipitated protein. The supernatant, containing plasma membrane glycolipids, was collected and stored at −20° C.
[0223] Liposome Preparation
[0224] SSEA-3/-4-LMTK plasma membrane (μm) glycolipid extract (5×10.sup.7 cell equivalent) was mixed with a total concentration of 10 mgs of lipids [Cholesterol, dicetylphosphate (DCP),phosphatidylcholine (PC) and α-GalCer] in a round bottom flask at various ratios (Table 2). The lipid mixture was then dried down using a rotary evaporator at 60° C. until the solvent had evaporated, leaving a uniform lipid film on the wall of the flask. The flask was allowed to cool down to room temperature before the addition of 100 μl of sterile PBS. The opening of the flask was covered with parafilm and then immersed in an ultrasonic bath for 10 min to generate liposomes. (All work with chloroform and methanol was carried out in a fume hood).
[0225] Immunisation Protocol
[0226] BALB/c mice were between 6 to 8 weeks old (Charles River, UK). Prior to immunisation, normal mouse serum (NMS) was collected via tail bleed extraction, for use as a negative control, and stored at −20° C. Mice were immunised intraperitoneally (i.p.) with SSEA-3/-4-LMTK cells (1×10.sup.6 cells per immunization per mouse) at two weekly intervals using 1 ml insulin syringe (BD Bioscience, Spain). Seven days after the second immunisation, and every seven days for subsequent, anti-sera was collected via tail bleed extraction and screened for IgG and IgM antibody responses. Once a high titre of IgG response was obtained, the animal was boosted intravenously (i.v.) with SSEA-3/-4-LMTK cells (1×10.sup.5 cells per immunization per mouse) and sacrificed 5 days later.
[0227] mAb Generation
[0228] Isolation of splenocytes—Mice were euthanised and the spleen removed. After washing with 5 ml serum free medium (RPMI 1640) using a 25-gauge needle, the spleen was agitated with sterile forceps gently to harvest splenocytes. 5 ml of splenocytes were collected into a sterile 25 ml universal tube while excess fat and connective tissues was discarded. The total fluid volume containing the splenocytes was increased to 25 ml with serum free medium (RPMI 1640) and centrifuged at 100 g for 10 mins. The supernatant was removed, leaving 1 ml of medium and the splenocytes which were then resuspended in 5 ml serum free medium (RPMI 1640) and counted using a haemocytometer with trypan blue, staining for viability assessment.
[0229] Fusion of splenocytes with NS0 myeloma cells—Washed splenocytes were combined with healthy NS0 myeloma cells in a ratio of 1:10 (NS0: splenocytes; 1×10.sup.7: 1×10.sup.8 cells) in a 25 ml universal tube and centrifuged at 317 g for 5 mins. The supernatant was aspirated and the combined cell pellet was resuspended in 800 μl of polyethylene glycol (PEG) gently and gradually over 1 min. The cell mixture was agitated gently for 1 min prior to the addition of 1 ml of serum free medium (RPMI 1640) over 1 min while continuing to agitate. A further 20 ml of serum free medium (RPMI 1640) was added over 1 min while continuing to agitate. Then the cell mixture was centrifuged at 317 g for 5 mins, the supernatant removed and the cell mixture was resuspended in 15 ml of hybridoma medium [500 ml hybridoma serum free medium (Gibco): 10 ml HT (hypoxanthine thymidine) supplement (50× Hybri-Max; Sigma): 31 μl (31 μg) methotrexate (1 mg/ml; Sigma): 25 ml of Hybridoma cloning factor (Opti-Clone 11; MP): 50 ml of filtered NS0 spent medium]. The cell suspension was spread evenly across a 96 well flat bottom plate and incubated at 37° C. in cell culture incubator (5% CO2).
[0230] CH2811 hG1 Generation
[0231] Total RNA was prepared from 5×10.sup.6 FG2811 hybridoma cells using Trizol (Invitrogen, Paisley, UK), following the manufacturer's protocol. First-strand cDNA was prepared from 3 μg of total RNA using a first-strand cDNA synthesis kit and AMV reverse transcriptase following the manufacturer's protocol (Roche Diagnostic). PCR and sequencing of heavy and light chain variable regions was performed by Syd Labs, Inc (Natick, Mass. 01760, USA) and variable region family usage analysed using the IMGT database (Lefranc et al. 2018). FG2811 variable regions were subsequently cloned into the hIgG1/kappa double expression vector pDCOrig-hIgG1 (Metheringham et al. 2009) and the sequence confirmed by sequencing.
[0232] mAb Characterisation
[0233] mAb isotyping—Spent hybridoma serum free medium (Invitrogen Scotland, UK) was collected and 150 μl diluted in 1/10 dilution in PBS 1% (w/v) BSA and then pipetted into the development tube of the mouse mAb isotyping test kit (AbD Serotec, Kidlington, UK) and incubated at room temperature for 30 seconds. The tube was vortexed briefly to ensure the coloured microparticle solution was completely resuspended. One isotyping strip was placed into the tube, with the solid red end of the strip at the bottom of the tube for 5 to 10 mins. The result was interpreted by checking the blue bands appeared above the letters in one of the class or subclass windows as well as either kappa or lambda window of the strip, indicating the heavy and light chain composition of the mAb.
[0234] Mouse mAb purification— 2 litres of spent hybridoma serum free medium (Invitrogen Scotland, UK) was collected and 0.2% sodium azide (Sigma) added. The spent medium was subsequently filtered through Whatman paper followed by filtration using 0.2 μm steritop filters (Sigma). A HiTrap Protein G HP antibody purification column (GE Healthcare) was used for the purification according to the manufacturer's recommendations. mAb binding buffer consisted of PBS-Tris pH 7.0 and mAb was eluted using Tris-Glycine pH12.0. Fractions containing IgG mAb were pooled, pH-neutralised using 10M HCl and dialysed overnight against PBS, before aliquoting and storing at −80° C.
[0235] Transient mAb production—The FG2811mG1, CH2811hG1 and CH2811hG2 mAbs were obtained following transient transfection of Expi293TM cells using the ExpiFectamine™293 Transfection kit (Gibco, Life Technologies). The HEK293 cells in suspension (100 ml, 2×10.sup.6 cells/ml) were transfected with 100 μg plasmid DNA and conditioned medium harvested at day seven, post transfection.
[0236] Tumour Cell Lines
[0237] Cell lines were maintained by regular replacement of complete culture media and splitting to maintain log phase growth. All cell lines were regularly checked for mycoplasma contamination and authenticated using short tandem repeat (STR) profiling (Table 1).
TABLE-US-00001 TABLE 1 Cancer cell lines. Source Cancer type Media Human cancer cell line U251MG ATCC GBM (adult) EMEM 10% FCS U87MG ATCC GBM (adult) EMEM 10% FCS SF188 ATCC GBM (adult) EMEM 10% FCS KNS42 ATCC GBM (paediatric) EMEM 10% FCS UW2283 ATCC Medulla blastoma EMEM 10% FCS DAOY ATCC Medulla blastoma EMEM 10% FCS T47D ATCC Ductal carcinoma RPMI 10% FCS DU4475 ATCC Breast carcinoma RPMI 10% FCS MCF7 ATCC Breast adenocarcinoma RPMI 10% FCS Colo205 ATCC Colorectal adenocarcinoma RPMI 10% FCS (Duke D) OVCAR3 ATCC Ovarian adenocarcinoma RPMI 10% FCS OVCAR5 ATCC Ovarian adenocarcinoma RPMI 10% FCS IGROV1 ATCC Ovarian adenocarcinoma RPMI 10% FCS Mouse fibroblast cell line LMTK ATCC Mouse fibroblast DMEM 10% FCS SSEA-3/-4-LMTK IJC Josep Carreras SSEA-3/-4-expressing LMTK DMEM 10% FCS Leukemia Research mouse fibroblast cells Institute NS0 ATCC Myeloma cells RPMI 10% FCS GBM: Glioblastoma Multiforme
[0238] Antibody Binding to Cancer Cell Lines and Mouse Fibroblast Cells.
[0239] 1×10.sup.5 cells were incubated with 50 μl of primary antibodies (of various concentrations) at 4° C. for 1 hr. Cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 mins. Supernatant was discarded and 50 μl of FITC-conjugated anti-mouse/anti-human or biotin-conjugated anti-mouse/anti-human IgG/IgM Fc specific antibody (Sigma) diluted 1/100 in RPMI 10% FCS was used as secondary antibody. Cells were incubated in dark for 1 hr at 4° C. Cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 mins. 50 μl of streptavidin-FITC (Sigma) or Strep-PeCy7 (eBioscience) diluted 1/100 in RPMI 10% FCS were used to detect biotinylated secondary antibody. Cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 mins.
[0240] Cells were fixed with 0.4% formaldehyde and analysed on Beckman Coulter Fc-500 flow cytometer (Beckman Coulter, High Wycombe, UK) or MACSQ flow cytometer (Miltenyi Biotech, Bisley, UK).
[0241] Antibody Binding to Whole Blood
[0242] 50 μl of healthy donor whole blood was incubated with 50 μl primary antibody at 4° C. for 1 hr. The blood was washed with 150 μl of RPMI 10% NBCS and spun at 100 g for 5 mins. Supernatant was discarded and 50 μl of FITC conjugated anti-mouse/anti-human or biotin-conjugated anti-mouse/anti-human IgG Fc specific antibody (Sigma; 1/100 in RPMI 10% NBCS) was used as secondary antibody. Cells were incubated at 4° C. in the dark for 1 hr then washed with 150 μl RPMI 10% NBCS and spun at 100 g for 5 mins. 50 μl of streptavidin-FITC (Sigma; 1/100 in RPMI 10% NBCS) or streptavidin-PE-Cy7 (eBioscience; 1/100 in RPMI 10% NBCS) was used to detect biotinylated secondary antibody. Cells were incubated at 4° C. in dark for 1 hr then washed with 200 μl RPMI 10% NBCS and spun at 100 g for 5 mins. After discarding the supernatant, 50 μl/well Cal-Lyse (Invitrogen, Paisley, UK) was used followed by 500 μl/well distilled water to lyse red blood cells. The blood was subsequently spun at 100 g for 5 mins, the supernatant discarded and the cells were resuspended in 500 μl PBS. Samples were analysed on a FC-500 flow cytometer (Beckman Coulter). To analyse and plot raw data, WinMDI 2.9 software was used.
[0243] TLC Analysis of Glycolipid Binding
[0244] LMTK and SSEA-3/-4-LMTK plasma membrane lipid samples were blotted onto silica plates and developed in chloroform (Sigma)/methanol (Sigma)/distilled water (60:30:5 by volume) twice followed by hexane (Sigma):diethyl ether (Sigma):acetic acid (Sigma) (80:20:1.5 by volume) twice. The dried plates were sprayed with 0.1% polyisobutylmethacrylate (Sigma) in acetone. After air drying, the plates were blocked with PBS 2% (w/v) BSA for 1 hr at room temperature and incubated overnight at 4° C. with primary antibodies diluted in PBS 2% (w/v) BSA. The plates were then washed 3 times with PBS and incubated with biotin—conjugated anti-mouse IgG Fc specific secondary antibody (Sigma) diluted 1/1000 in PBS 2% (w/v) BSA for 1 hr at room temperature. The plates were subsequently washed again in PBS before incubating with IRDye 800CW streptavidin (LICOR Biosciences, Cambridge, UK) diluted 1/1000 in PBS 2% (w/v) BSA for 1 hr at room temperature in the dark. The plates were subsequently washed a further 3 times with PBS and air dried in the dark. Lipid bands were visualized using a LICOR Odyssey scanner.
[0245] Glycan (Coupled to HSA) ELISA
[0246] ELISA plates (Becton Dickinson, Oxford, UK) were coated overnight at 4° C. with 100 ng/well of SSEA-3, SSEA-4, Forssman, Globo-H and Sialyl-Lewis x (SLex) glycan-HSA conjugates, resuspended in PBS (Elicityl, Crolles, France), blocked with 200 μl/well of PBS 5% (w/v) BSA for 1 hr at room temperature, followed by incubation with 50 μl/well of primary antibodies (5 μg/ml). The primary antibodies were detected using biotinylated anti-mouse IgG or anti-rat IgM secondary antibody (Sigma) diluted 1/5000 in PBS 1% (w/v) BSA. After incubation with streptavidin horseradish peroxidase (HRPO) conjugate (Invitrogen) diluted 1/5000 in PBS 1% (w/v) BSA and development with 3,3′,5,5′-Tetramethylbenzidine (TMB; Sigma), plates were read at 450 nm using Tecan Infinite F50.
[0247] Erythrocyte Binding Assay
[0248] Healthy donor erythrocytes were washed thrice in PBS and resuspended in 10 times the packed cell volume of PBS. 50 μl of washed erythrocytes were then incubated with 50 μl of primary antibodies at 37° C. for 1 hr. Cells were washed with 150 μl of PBS and spun at 100 g for 5 mins. Supernatant was discarded and cells resuspended in 50 μl FITC-conjugated anti-mouse IgG Fc specific secondary antibody (Sigma) diluted 1/100 in PBS 1% (w/v) BSA. Cells were incubated at 37° C. in the dark for 1 hr then washed with 150 μl PBS and spun at 100 g for 5 mins. Supernatant was discarded and cells were resuspended in 500 μl PBS. Samples were analysed by FC-500 flow cytometer (Beckman Coulter). To analyse and plot raw data, WinMDI 2.9 software was used.
[0249] Erythrocyte Hemagglutination Assay
[0250] 4 ml of normal donor whole blood was collected into a heparin tube (Becton Dickinson). The whole blood was transferred to a sterile 15 ml conical tube and washed with sterile PBS. The washed blood was centrifuged at 100 g for 5 mins. The supernatant was aspirated. The washing step was repeated twice. After the final wash, the blood cell pellet was diluted with sterile PBS to make a final working concentration of 0.5% erythrocytes. 50 μl of 0.5% erythrocytes was added into each well of a 96 well U bottom plate. On top of erythrocytes, primary antibodies were added at 50 μl/well and incubated at room temperature for 1 hr or until the erythrocytes agglutinated.
[0251] Monoclonal Antibody Affinity Analysis
[0252] The kinetic parameters of the FG2811mG3 mAb binding to SSEA-4-containing liposomes was determined by Surface Plasmon Resonance (SPR, Biacore 3000, GE Healthcare). An L1 sensor chip (GE-healthcare) was preconditioned with 40 mM octyl D-glucoside, followed by coating with SSEA4-containing liposomes (6000RU) and a short pulse of NaOH (10 mM) to remove loosely bound liposomes. The reference flow cell was treated in the same manner with the exception that liposomes devoid of SSEA-4 were used. For both flow cells the degree of coverage was near complete as injection of HSA (0.1 mg/ml) induced a marginal increase in RU (50-60 RU). After stabilisation of the signal from both flow cells, increasing concentrations (0.3 nmol/L-200 nmol/L) of the FG2811mG3 mAb were injected, followed by regeneration (10 mM glycine pH 1.5) after cycle. Binding curves were fitted to a 1:1 (Langmuir) binding model using BIAevaluation 4.1.
[0253] Glycome Analysis of FG2811mG3 (Consortium for Functional Glycomics)
[0254] To determine the fine specificities of the FG2811mG3 antibody, the antibody was FITC-labelled and sent to the Consortium for Functional Glycomics where they were screened against 600 natural and synthetic glycans (core H group, version 5.1). The synthetic and mammalian glycans with amino linkers were printed onto N-hydroxysuccinimide (NHS)-activated glass microscope slides, forming amide linkages. Printed slides were incubated with 5 μg/ml of antibody for 1 hr at room temperature before the binding was detected with Alexa488-conjugated goat anti-mouse IgG. Slides were then dried, scanned and the screening data compared to the Consortium for Functional Glycomics database.
[0255] CSFE T-Cell Proliferation Assay
[0256] PBMC Separation
[0257] Whole blood (buffy coats) were obtained from the national blood service (Sheffield) or was collected from healthy donors in a syringe containing lithium heparin (1000 units/ml; Sigma H0878). Whole blood was diluted with RPMI 1640 at 1:1 ratio and layered on lymphocyte separation medium (Histopaque-1077; Sigma), followed by centrifugation at 800 g, off brake for 25 mins. After centrifugation, plasma was collected from top layer, PBMCs from the buffy coat layer. PBMCs were washed with RPMI 1640 twice and spun at 317 g for 5 mins. Number of PBMCs was counted and the cells were ready for T-cell isolation.
[0258] Pure T-Cell Isolation
[0259] Every 1×10.sup.7 PBMCs was resuspended in 40 μl of cold MACS buffer [PBS 1% (v/v) FCS 1% (v/v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665 and 0.5M pH8 EDTA: Invitrogen). Then 10 μl of Pan T cell Biotin antibody (Miltenyi) was added to every 1×10.sup.7 cells and incubated at 4° C., in dark for 5 mins. 30 μl of cold MACS buffer was added to every 1×10.sup.7 cells followed by 20 μl of Pan T cell microbead (Miltenyi) to every 1×10.sup.7 cells and incubated at 4° C., in dark for 10 mins. Cells were added to LS column (Miltenyi) and the flow through, which contained the CD3 purified T cells were collected. Cells retained in the column were non T cells.
[0260] CSFE Loading
[0261] Purified T cells were washed with RPMI 1640 and the number of cells were counted. Cells were spun at 317 g for 5 mins and supernatant was removed. Every 1×10.sup.7 purified T cells was resuspended in 1 ml of PBS 10% FCS. CSFE was dissolved in 18 μl DMSO (Invitrogen) followed by 1.8 ml of PBS 10% FCS. Then, 110 μl of diluted CSFE was added to every 1×10.sup.7 T cells and incubated in dark at room temperature for 5 mins. CSFE loaded cells were washed with PBS 10% FCS then resuspended at 1×106 cells/ml in complete medium (RPM1640 2% (v/v) Hepes 1% (v/v) L Glutamine 1% (v/v) penicillin streptomycin) 10% donor's plasma. Cells (2×10.sup.6 in 2 mL) were added to each well of a 24 well plate pre coated with CH2811hG1 antibody (5 μg/ml), FG2811mG1 (5 μg/ml), CH2811hG2 antibody (5 μg/ml) or containing anti CD3 antibody (OKT 3; 0.005 μg/ml), anti-CD3e Ab (1 ug/ml, eBioscience, 16-0031-85) and anti-CD28 Ab (1 ug/ml eBioscience 16-0281-85) or medium alone. Cells were harvested at day 7, 11 and 14 and stained with relevant antibodies against CD3 (eBioscience, 17-0031), SCA-1 (Miltenyi, 130-102-343), CD62L (Miltenyi, 130-102-543), CD44 (Miltenyi, 130-116-495), anti-CD4-APC-780 (eBioscience 47-0049), anti-CD8-VioGreen (Miltenyi, 130-102-805, Tim3-PE (eBioscience, 130-118-563) or with CH2811hG2-PeCy7 (in house, 1:50 dilution), followed by analysis using MACSQ flow cytometer (MACSQUANT analyser 10).
[0262] Luminex [Milliplex Map Kit-Human High Sensitivity T-Cell Magnetic Bead Panel (96 Well Plate Assay)]
[0263] Assays in the 9-well format were conducted on filter plates based on the manufacturer's recommendations. In total, 200 μl of wash buffer was added into each well of a 96-well filter plate (Millipore). The plate was sealed and mixed on a plate shaker for 10 mins at room temperature. Wash buffer was removed by inverting the plate and tapping on a paper towel. Then 25 μl of each standard, control and sample (culture supernatant from CSFE proliferation assay) was added into each well, 25 μl of serum matrix was added to each standard and control wells, and 25 μl of assay buffer was added to each sample well. The working bead mix was vortexed immediately before use. Next, 25 μl of the mixed beads was added to each well. The plate was then sealed, wrapped with aluminium foil, and incubated with agitation on a plate shaker (500-800 rpm) for 16-18 hrs at 4° C. After incubation, the plate was rest on a handheld magnet for 60 secs, followed by removing liquid from the plate by inverting the plate and tapping on a paper towel. The plate was washed twice with 200 μl of wash buffer each time. After the second wash, the bottom of the plate was dried by tapping on a paper towel, and 25 μl of detection antibodies was added into each well. The plate was then sealed, wrapped in aluminium foil, and incubated with agitation on a plate shaker for 1 hr at room temperature. Next, 25 μl of streptavidin-phycoerythrin was added to each well containing the 25 μl of detection antibodies. The plate was shaken for an additional 30 mins at room temperature. After incubation, the plate was rest on a handheld magnet for 60 secs, followed by removing liquid from the plate by inverting the plate and tapping on a paper towel. The plate was washed twice with 200 μl of wash buffer each time. Then 150 μl of sheath fluid (Luminex) was added to each well. The beads were resuspended on a plate shaker for 5 mins and read on a Bio-Plex 3D instrument (Bio-Rad, Hercules, Calif.). The instrument was set to collect at least 50 beads per analyte. The raw data were measured as mean fluorescence intensity (MFI).
[0264] Naïve T-Cell Isolation
[0265] Whole blood was collected from normal donor in syringe contained lithium heparin (1000 units/ml; Sigma H0878). Whole blood was diluted with RPMI 1640 at 1:1 ratio and layered on lymphocyte separation medium (Histopaque-1077; Sigma), followed by centrifugation at 800 g, off brake for 25 mins. After centrifugation, plasma was collected from top layer, PBMCs from the buffy coat layer. PBMCs were washed with RPMI 1640 twice and spun at 317 g for 5 mins. Number of PBMCs was counted and the cells were ready for naive T cell isolation. Every 1×10.sup.7 PBMCs was resuspended in 40 μl of cold MACS buffer [PBS 1% (v/v) FCS 1% (v/v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665 and 0.5M pH8 EDTA: Invitrogen). Then 10 μl of Naïve Pan T cell Biotin antibody (Miltenyi) was added to every 1×10.sup.7 cells and incubated at 4° C., in dark for 5 mins. 30 μl of cold MACS buffer was added to every 1×10.sup.7 cells followed by 20 μl of Naïve Pan T cell microbead (Miltenyi) to every 1×10.sup.7 cells and incubated at 4° C., in dark for 10 mins. Cells were added to LS column (Miltenyi) and the flow through, which contained the CD3 purified T cells were collected. Cells retained in the column were non T cells. Naïve T cells were stained with either CH2811hG1 or the combination of CD95/CD122 antibodies for 30 mins. The cells were washed with MACS buffer and proceed to cell sorting. CH2811 hG1.sup.+ and CD95/CD122.sup.+ cells were sorted into RNA protect (QIAGEN) and stored at −80° C.
[0266] Sample Extraction and Quality Control
[0267] 8 T-cell samples were provided in RNA protect reagent. The entire sample volume was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Extracted RNA samples were assessed for quantity and integrity using the NanoDrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany) in conjunction with the Eukaryote RNA Pico Bioanalyser chip, respectively. Samples displayed low levels of degradation with RNA integrity numbers (RIN) between 7.4 and 10, and an average yield of 110 ng.
[0268] cDNA Synthesis
[0269] Full-length cDNA molecules were generated from 1 ng of total RNA per sample using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech, Mountain View, Calif., USA). cDNA quantity was measured using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, Calif., USA), and were checked for quality using the Agilent 2200 Tapestation and high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples displayed good quantities of cDNA, with molecule sizes ranging from 400 to 10,000 bp.
[0270] Library Generation and RNA-Sequencing Sequencing libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina
[0271] Inc., Cambridge, UK) with an input of 150 μg of cDNA per sample. 11 cycles of final PCR amplification were carried out. Final libraries were quantified and qualified using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, Calif., USA), and the Agilent 2200 Tapestation with a high-sensitivity D1000 screentape (Agilent Technologies, Waldbronn, Germany). Equimolar amounts of each sample library were pooled together for sequencing which was carried out using the Ilumina NextSeq®500 Mid-output kit to generate 75 bp paired-end reads.
[0272] Differential Expression Analysis
[0273] After quality check using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), 75-bp paired-end reads were aligned to the Homosapiens reference genome hg19 using STAR (version 2.6.1d). Mapping was run with default parameters, and reads were counted with GeneCounts. Differential expression analysis (DE) of the 2811- and similarly CD95/CD122-enriched T-cells, was performed using datasets from CD4 and CD8 naïve T-cells from GSE114765 (Pilipow et al. JCI insight 2018) using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple testing correction to estimate the FDR (FDR <0.05). Common genes between the DE sets (two for CD8 and two for CD4) T-cells were identified using Venny 2.1 and used as input in the StemChecker database to identify a ‘sternness’ signature (Pinto et al. 2015).
[0274] Transcriptional Profiling Using RNAseq
[0275] Eight T-cell samples (four CH2811.sup.+, four CD122/CD95.sup.+) were sorted in RNA protect reagent. The entire sample volume was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Extracted RNA samples were assessed for quantity and integrity using the NanoDrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany) in conjunction with the Eukaryote RNA Pico Bioanalyser chip, respectively. Samples displayed low levels of degradation with RNA integrity numbers (RIN) between 7.4 and 10, and an average yield of 110 ng. Full-length cDNA molecules were generated from 1 ng of total RNA per sample using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech, Mountain View, Calif., USA). cDNA quantity was measured using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, Calif., USA), and were checked for quality using the Agilent 2200 Tapestation and high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples displayed good quantities of cDNA, with molecule sizes ranging from 400 to 10,000 bp. Sequencing libraries were prepared using the IIlumina Nextera XT Sample Preparation Kit (IIlumina Inc., Cambridge, UK) with an input of 150 μg of cDNA per sample. 11 cycles of final PCR amplification were carried out. Final libraries were quantified and qualified using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, Calif., USA), and the Agilent 2200 Tapestation with a high-sensitivity D1000 screentape (Agilent Technologies, Waldbronn, Germany). Equimolar amounts of each sample library were pooled together for sequencing which was carried out using the Ilumina NextSeq®500 Mid-output kit to generate 75 bp paired-end reads. After quality check using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), 75-bp paired-end reads were aligned to the Homosapiens reference genome (Ensembl assembly GRCh37 (hg19) using STAR (version 2.5.1b). Mapping was run with default parameters, and reads were counted with GeneCounts. Differential expression analysis (DE) of the CH2811- and similarly CD95/CD122-enriched transcription profiles, was performed using datasets from CD4 and CD8 naïve T-cells from GSE83808 (Hosokawa et al. 2017) using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple testing correction to estimate the FDR (FDR <0.05). Common genes between the DE sets (two for CD8 and two for CD4) T-cells were identified using Venny 2.1 and used as input in the StemChecker database to identify ‘sternness’ signatures and overlap with genesets associated with haematopoetic stem cells (HSC) and embryonic stem cells (ESC) (Pinto et al. 2015). The distribution of the significantly enriched genes was displayed via heatmap analysis (https://software.broadinstitute.org/morpheus/). Concurrently, the distribution of T.sub.SCM- and effector T differentiation associated genes, for the CH2811- and CD95/CD122-enriched profiles compared to published naïve and memory CD4 and CD8 T-cells (GSE23321) as well as activated naïve CD8 T-cells (GSE114765) was visualised using http://bioinformatics.sdstate.edu/idep/.
[0276] Mouse Study
[0277] C57BL/6J mice (Charles River), HHDII/HLA-DP4 (DP*0401) mice (EM:02221, European Mouse Mutant Archive), HHDII mice (Pasteur Institute), aged between 8 to 12 weeks were used. All work was carried out under a Home Office approved project license. Six mice were randomised into two groups (Group A and B) and not blinded to the investigators. Endotoxin free FG2811mG1 mAb were immunised into group A mice (250 μg/mouse) via intraperitoneal route (i.p.) at day 0. Group B mice were used as unimmunised control group. Spleens were harvested for analysis at day 16, followed by pooling splenocytes together within same group and restimulated in the presence or absence of plate bound FG2811mG1 antibody (5 μg/ml). Splenocytes were harvested from culture at day 24, 27 and 30 for analysis using anti-CD3, CD4, CD8, CD44, CD62L, SCA-1 and CH2811hG1 antibodies.
[0278] Staining Murine Tissues
[0279] Naive HHDII DP4 mice were used. All work was carried out under a Home Office approved project license. Spleens, mesenteric lymph nodes, inguinal lymph nodes, bone marrow and blood samples were from naive mice were harvested for analysis. Tissues were incubated with CH2811hG2-PeCy7 (in house, 1:50 dilution), anti CD3 (eBioscience, 17-0031), SCA-1 (Miltenyi, 130-102-343), CD62L (Miltenyi, 130-102-543), CD44 (Miltenyi, 130-116-495), anti-CD4-APC-780 (eBioscience 47-0049), anti-CD8-VioGreen (Miltenyi, 130-102-805) and Tim3-PE (eBioscience, 130-118-563).
EXAMPLES
[0280] The present invention will now be described further with reference to the following examples and the accompanying drawings.
Example 1. Generation of FG2811.72
[0281] Generation and Characterisation of FG2811 mAb
[0282] BALB/c mice were immunised intraperitoneally (i.p.), and boosted intravenously (i.v.) over a period of 3 months with the SSEA-3/-4 expressing cell line (SSEA-3/-4-LMTK). This cell line was produced by transducing wild type LMTK mouse fibroblast cells with α-1-4-galactosyltransferase (A4GALT), β-1-3-N-acetylgalactosaminyltransferase (B3GALNT1) and β-1-3-galactosyltransferase (B3GALT5) genes (Cid et al. 2013). The cell line has endogenous sialyl-transferases, which adds sialic acid at the terminal end of SSEA-3 glycan, producing the SSEA-4 glycan (
[0283] To generate the anti-SSEA-4 specific mAb, splenocytes of immunised mice were fused with myeloma NS0 cells. After repeated rounds of screening and limiting dilution cloning, the anti-SSEA-4 mAb, FG2811mG3 was obtained.
[0284] It is known that SSEA-3 and SSEA-4 are globoseries glycolipids. To confirm that FG2811mG3 mAb recognises cell surface glycolipid antigens on SSEA-3/-4-LMTK cells, high performance thin layer chromatography (HPTLC) analysis of SSEA-3/-4-LMTK plasma membrane lipid extracts and immunostaining with FG2811mG3 mAb was performed (
[0285] SSEA-3/-4-LMTK plasma membrane lipid antigen binding kinetics of FG2811mG3 mAb was examined using surface plasmon resonance (SPR; Biacore X). Fitting of the binding curves revealed strong apparent functional affinity (K.sub.d˜2×10.sup.−9 M) with fast association (˜10.sup.5 1/Ms) and slow dissociation (˜10.sup.−4 1/s) rates for the 2811 mAb (
Example 2. FG2811 Antibody Sequence
[0286] DNA sequencing revealed that FG2811mG3 mAb belonged to the IGHV2-3*01 heavy chain and IGKV4-63*01 light gene families (
[0287] FG2811 heavy and light chain variable regions were cloned into mouse IgG1, human IgG2 and IgG1 expression vectors (
Example 3. 2811 Binding to a Panel of Human Cancer Cell Lines
[0288] The overexpression of SSEA-4 have been reported on glioblastoma cancer cell lines, as defined by MC813 mAb. Thus, a panel of brain cancer cell lines were assessed for SSEA-4 expression, using both FG2811mG3 and MC813 mAbs at 5 μg/ml by flow cytometry analysis (
Example 4. Cytotoxicity of 2811 mAb
[0289] The ability of FG2811mG3 mAb to induce tumour cell death through ADCC was investigated (
Example 5. 2811 Staining of Erythrocytes
[0290] Both SSEA-3 and SSEA-4 were reported to be expressed on the erythrocytes of the majority of people. Therefore, binding of FG2811mG3 mAb at a range of concentrations (10 μg/ml) to erythrocytes from 5 donors was assessed by flow cytometry (
Example 6. 2811 Binding to Stem Memory T-Cells (T.SUB.SCM.)
[0291] The discovery of T.sub.SCM cells and the fact that SSEA-4 is a stem cell marker leads us to hypothesise that 2811 mAb may recognise T.sub.SCM cells. Whole blood samples were collected from seven healthy donors (BD3, BD13, BD18, BD27, BD38, BD96, BD31) and stained with FG2811mG1 mAb (
TABLE-US-00002 TABLE 2 PBMCs phenotyping. PBMCs were isolated from two healthy donors (BD13 and BD38) and stained with a panel of antibodies (CD3, FG2811, CD45RA, CD45RO, CD62L, CD95 and CCR-7). Phenotype of the PBMCs were determined using flow cytometry, and results were presented as the percentage of positive cells. Donor Percentage (+) cells BD13 BD38 CD3+2811+ 0.41 0.32 CD3+2811+CD45RA+ 38.6 37.5 CD3+2811+CD45RA+RO+ 12.7 23.1 CD3+2811+CD45RO+ 47.8 38 CD3+2811+CD45RA+CD62L+ 90 88.7 CD3+2811+CD45RA+RO+CD62L+ 79.5 89.6 CD3+2811+CD45RO+CD62L+ 64.5 67.1 CD3+2811+CD45RA+CD95+ 27.6 59.7 CD3+2811+CD45RA+RO+CD95+ 29.5 85.7 CD3+2811+CD45RO+CD95+ 86.1 81.2 CD3+2811+CD45RA+CCR-7+ 56.1 78.9 CD3+2811+CD45RA+RO+CCR-7+ 51.4 78.1 CD3+2811+CD45RO+CCR-7+ 61.2 53.7
[0292] In the hierarchical model of human T-cell differentiation, after antigen priming, naïve T-cells (T.sub.N) progressively differentiate into stem memory T-cells (T.sub.SCM), central memory T-cells (T.sub.CM), effector memory T-cells (T.sub.EM) and ultimately into terminally differentiated effector T-cells (T.sub.TE/TEMRA). These T-cell subsets are distinguished by the combinatorial expression of different markers (Table 3) (Gattinoni et al. 2017).
TABLE-US-00003 TABLE 3 Hierarchical model of human T-cell differentiation. Progressive T-cell differentiation model T.sub.N T.sub.SCM T.sub.CM T.sub.EM T.sub.TE/TEMRA CD45RA + + − − + CD45RO − − + + − CCR-7 + + + − − CD62L + + + − − CD28 + + + +/− − CD27 + + + +/− − IL-7Rα + + + +/− − CXCR3 − + + − − CD95 − + + + + CD11a − + + + + IL-2Rβ − + + + + CD58 − + + + + CD57 − − − +/− +
Example 7. RNA Sequencing of 2811 Positive T-Cells
[0293] By transcriptome analysis, we investigated the degree of relatedness between putative T.sub.SCM cells (Gattinoni et al. 2017) and 2811.sup.+ T-cells. The CD95 and CD122 (IL-2Rβ) markers discriminate T.sub.N cells to T.sub.SCM cells; the CD45RO marker distinguishes other memory T-cell subtypes from T.sub.SCM cells. Thus, naïve T-cells were isolated from four healthy donors using Pan naïve human T-cell isolation kit (Miltenyi), which contained a cocktail of biotinylated antibody for the depletion of memory T-cells and non-T-cells. The purified naïve T-cells (CD45RA.sup.+) were subsequently stained with CH2811hG1 or the combination of CD95/CD122 to isolate 2811.sup.+ and putative T.sub.SCM cells, respectively. RNA sequencing on CH2811hG1- and CD95/CD122-enriched T-cells and differential gene expression (DE) analysis using a data set from CD8 native T-cells showed that of the 5,036 genes that were significantly up or down regulated in the SSEA-4 positive (CH2811) cells, 2227 (44%) were common with the up or down regulated DE genes in CD95/CD122 positive T-cells suggesting there was a substantial overlap genes between these two populations (
Example 8. T-Cell Proliferation and Expansion
[0294] According to the paradigm of co-stimulation, T.sub.N cells require the engagement of both T-cell receptor (TCR) signal 1 and costimulatory signal 2 for complete activation leading to proliferation and differentiation. However, a subclass of CD28 specific antibodies known as CD28 superagonists, which unlike conventional CD28 antibodies, are capable of fully activating T-cells without additional stimulation of TCR. We investigated whether CH2811hG1 mAb is capable of inducing CD4 and CD8 T-cell proliferations. Initially, PBMCs were isolated from two healthy donors (BD3 and BD18) and CSFE labelled followed by antibody stimulation using plate bound CH2811hG1 mAb at 5 μg/ml, which showed proliferation of both CD4 (13-20%) and CD8 (2-31%) T-cells at day 11 (
[0295] To obviate that this was due to Fc activation of antigen presenting cells, purified T-cells (96% purity;
Example 10. Assessment of TCR Repertoire Clonotype
[0296] The clonality of the CH2811IgG1 stimulated T cells was assessed from 2 donors (BD3 and BD26), The TCR repertoire was determined, a fully automated multiplex PCR was performed to generate TCRα (TRA) and TCRβ (TRB) chain libraries for next generation sequencing (NGS) analysis of unique CDR3s (uCDR3). A tree plot analysis (
Example 11. Dynamic of Individual Cytokine/Chemokine Responses
[0297] T.sub.SCM cells have been shown to have high proliferative capacity and are both self-renewing and multi-potent, in which they can further differentiate into other T-cell subsets. We hypothesised that FG2811.sup.+ T.sub.SCM cells could proliferate and self-renew in vitro in the absence of any supplemental cytokines. We first aimed to identify the cytokines released by FG2811.sup.+ T.sub.SCM cells following CH2811hG1 antibody stimulation, then design a method that can be used to expand and maintain the stemness of putative FG2811.sup.+ T.sub.SCM cells. T-cells were purified from four healthy donors and stimulated with plate bound CH2811hG1 mAb, supernatant was collected at day 7, 11 and 14 and assessed for cytokines or chemokines release. Unstimulated cells (media only) were used as negative control. Secretion of nine cytokines/chemokines (IFNγ, IL-10, IL-17A, IL-2, IL-21, IL-5, IL-7, IL-8 and TNFα) was assayed using a multiplexed cytokine assay (Luminex technology) (
[0298] The unstimulated and the anti-CD3 stimulated T-cells did not survive in culture beyond day 14, only CH2811hG1 stimulated T-cells survived beyond 14 days (
Example 12. Identification of FG2811.SUP.+ T.SUB.SCM .Cells in Mice
[0299] Next, we investigated the expression of SSEA-4 on mouse splenocytes, mesenteric and inguinal lymph node cells using the CH2811hG1 antibody (
Example 13. FG2811 (Mouse IgG1) Induces Phenotypic T.SUB.SCM .Cells in C57B/6J Mice
[0300] To determine the T-cell agonistic effect of FG2811mG1 in vivo, a group of 3 mice (Group A) were immunised i.p. with FG2811 at 250 μg at day 0 (Group A). Three unimmunised mice were included as control group (Group B). At day 16, mice from both groups were euthanised and spleens were harvested. The total cell number of splenocytes from Group A was higher compared to Group B mice, ranged from 7×10.sup.7 to 1×10.sup.8 cells and 3.9×10.sup.7 to 6.2×10.sup.7 cells, respectively (
TABLE-US-00004 TABLE 4 Summary of the frequencies of different immune cell subsets in Group A and B splenocytes at day 16. Marker 2811 immunised (Group A) Control (Group B) 2811 0.97-1.2% 1.62-1.74% SCA-1 37.23-55.06% 42.22-61.73% CD62L 5.51-10.83% 17.39-19.2% CD62L, CD44 8.74-15.03% 18.4-27.34%
[0301] Splenocytes from each group were pooled together and then cultured in the presence (A+2811 and B+2811) or absence (A-2811 and B-2811) of 5 μg/ml of plate bound FG2811mG1 mAb. Subsequently, at day 24, 27 and 30, these cells were harvested and stained with FG2811, CD3, CD4, CD8, CD44, CD62L, SCA-1, CD11b, F4/80 and CD19 antibodies and analysed by multiparameter flow cytometry (
[0302] In the hierarchical model of mouse T-cell differentiation, after antigen priming, naïve T-cells (T.sub.N) progressively differentiate into stem memory T-cells (T.sub.SCM), central memory T-cells (T.sub.CM), effector memory T-cells (T.sub.EM). These T-cell subsets are distinguished by the combinatorial expression of different markers (Table 5).
TABLE-US-00005 TABLE 5 Phenotypic markers of murine T-cell populations CD3+ T.sub.N T.sub.SCM T.sub.CM T.sub.EM CD44 − −/+ + + CD62L + + + −
[0303] Phenotypic analysis revealed that the CD3.sup.mo-hi population in A+/−2811 cultures mainly consists of T-cells with CD44.sup.−CD62L.sup.+(T.sub.N and/or T.sub.SCM; 28.8-32.49%) and CD44.sup.+CD62L.sup.+(T.sub.CM; 37.92-41.08%) phenotypes, followed by CD44.sup.+CD62L.sup.− (T.sub.EF/T.sub.EM; —27.8%) phenotype and a small fraction of cells with CD44.sup.−CD62L.sup.− phenotype (1.72-2.26%). In contrast, the CD3.sup.lo-mo population in all cultures mainly consists of CD44.sup.+CD62L.sup.− (T.sub.EF/T.sub.EM; 66.09-70.83%) phenotype followed by CD44.sup.−CD62L.sup.− phenotype (26.77-32.17%). The percentages of T.sub.N and/or T.sub.SCM cells and T.sub.CM cells were between 0.08-0.24% and 1.5-2.29%, respectively. In addition to T-cells, the large-sized cell population also contained CD19.sup.hi, CD62L.sup.+ and CD62L.sup.+SCA-1.sup.+ cells, which were all absence from the small-sized cell population. Only CD19.sup.lo cells were detected in the small-sized cell population. Interestingly, the percentage of CD11b.sup.+F4/80.sup.+ macrophage population was significantly reduced in the A+/−2811 groups. FG2811mG1 antibody stimulation in vitro of splenocytes from unimmunised mice splenocytes B+2811 culture did not form these large-sized population even by day 30, indicating that the generation of this cell population was an in vivo FG2811 antibody immunisation effect.
Example 14. Identification of T.SUB.SCM .Cells in HHDII and HHDII Transgenic Mice
[0304] To determine the frequency of T.sub.SCM cells in HHDII (
[0305] In HHDII/DP4 mice 12.01% of cells were 2811+CD3+ cells this translated into cells per 0.91×10.sup.5 ml, in addition 6.98% of the CD3+ population was also 2811+(
[0306] A more detailed phenotypic analysis was performed on the T cell populations from HHDII/DP4 mice, this analysis looked at the expression of 2811 in the CD4 and CD8 T cell subsets but also looked at the expression of the exhaustion marker, Tim3. The percentage of CD4+ T cells in the HHDII/DP4 mice was 14.30%, however, the percentage of CD8+ T cells was very low with only 0.50% CD8+ cells (
Example 15. Plate Bound Human (IgG1) and Mouse (IgG1) 2811 Induced Ex Vivo Proliferation of Mouse Splenocytes
[0307] We investigated whether plate bound CH2811hG1 and FG2811mG1 mAb are capable of inducing CD4 and CD8 T cell proliferation. Splenocytes were harvested from HHDII naive mice, pan T cells enriched (CD3+) and labelled with CFSE, followed by antibody stimulation using plate bound CH2811hG1 mAb or FG2811mG1, anti CD3 was used as a positive control and media as a negative control (
Example 16. Anti CD3 and CD28 Induces the Ex Vivo Expansion of 2811+ Cells from HHDII Mice
[0308] We investigated whether anti CD3 and anti CD28 could induce the proliferation of 2811+ cells isolated from HHDII mice. Splenocytes were harvested from HHDII naive mice, pan T cells enriched (CD3+) and labelled with CFSE, followed by stimulation with anti CD3 and anti CD28 (1 μg/mL). The proliferative responses of the 2811+ population was determined on days 11, 15 and day 20 using CH2811hG2-PeCy7 mAb (
[0309] Phenotypic analysis was performed on the 2811+ cells that had expanded following stimulation with anti CD3 and anti CD28. Staining was performed on day 11 (
[0310] These results show that anti CD3 and anti CD28 induces the ex vivo expansion of 2811+ cells from HHDII mice. Stimulation with anti CD3 and anti CD28 led to an increase in the number and percentage of 2811+ cells 11-15 days post stimulation. The total number of 2811+ T cells within each subset increased (T.sub.CM, T.sub.N, T.sub.EM, T.sub.EFF), however, the stimulation did push T cells into a more effector T cell phenotype.
Example 17. Human (IgG2) and Mouse (IgG1) 2811 Induced Ex Vivo Proliferation of Splenocytes from HHDII/DP4 Mice
[0311] We investigated whether plate bound CH2811hG2 and FG2811mG1 mAb are capable of inducing CD4 and CD8 T cell proliferation. Splenocytes were harvested from HHDII naive mice, pan T cells enriched (CD3+) and labelled with CFSE, followed by antibody stimulation using plate bound CH2811hG2, FG2811mG1, anti CD3/CD28 (+/− AKTi) was used as a positive control, media as a negative control. The proliferative responses of the CD3 T cell population was determined on days 11, 15 and 20 (
[0312] Phenotypic analysis was then performed on the 2811+ cells that had expanded following stimulation with anti CD3/CD28 (+/−AKTi), CH2811hG2 and FG2811mG1 mAbs. Staining was performed on day 11 (
[0313] These results show that splenocytes from HHDII/DP4 mice proliferate ex vivo in response to plate bound CH2811hG2 and FG2811mG1 mAb, this leads to an increase in the total number of 2811+CD3+ T cells in addition to increases in the number of 2811+ effector memory, central memory, effector and naive T cells. The magnitude of the proliferative ex vivo response to CH2811hG2 was larger when compared to the response to FG2811mG1 thus leading to a higher number of 2811+ cells.
Example 18. Anti CD3 and CD28 Induces the Ex Vivo Expansion of 2811+ Cells from Healthy Donors
[0314] TSCM cells have been shown to have high proliferative capacity and are both self-renewing and multi-potent, in which they can further differentiate into other T-cell subsets. We investigated if anti CD3/CD28 stimulation or the addition of different cytokines could induce the ex vivo proliferation of Tscm cells isolated from healthy donors. PBMCs were isolated from 4 healthy donors (buffy coats), a pan T cell enrichment was performed, T cells were cultured in the presence of anti CD3/CD28, IL-7, IL-15 or IL-21. Phenotypic analysis was performed on days 15 and 20 using anti CD3, CD45RA, CD45RO, CD62L, CD95, CD122 and CCR7, the expression of different makers used to identify T cell populations are listed in table 6.
TABLE-US-00006 TABLE 6 Phenotypic markers of human T-cell populations CD3+ CD45RA CD45RO CD62L CD95 CD122 CCR7 T.sub.N + + + T.sub.SCM + (+) + + + + T.sub.CM + + + + + T.sub.EM + + − T.sub.EMRA + + + −
[0315] Phenotypic analysis was performed on the CD3+ T cells that had expanded following stimulation with anti CD3/CD28 or with the addition of IL-7, IL-15 and IL-21 added in a range of combinations. Staining was performed on day 15 and day 20 (
[0316] Further and more detailed phenotypic analysis was performed on T cells from 2 donors to identify Tscm cells in T cells cultured in the presence of CD3/CD28 alone or in combination with IL-7, IL-15 and IL-21. The frequency of Tscm cells in humans is low, the percentage of Tscm in four healthy donors ranged from 0.64% to 3.48%. We investigated if Tscm could be expanded in the presence of CD3/CD28 alone or in combination with IL-7, IL-15 and IL-21 (
[0317] These results show that stimulation with CD3/CD28 induced the ex vivo expansion of 2811+ cells isolated form healthy human donors. The stimulation of T cells with anti CD3/CD28 increased the frequency of 2811+ cells, this expansion was increased further when IL-7, IL-15 and IL-21 where all added to the culture, this expansion peaked 15 days after stimulation. The stimulation of T cells with anti CD3/CD28 in combination expanded the Tscm population, this expansion resulted in a 3 to 9-fold expansion of these cells.
Example 19. T Cell Stimulated with Soluble FG2811mG1 Stimulate CD4 and CD8 T Cell Proliferation Via Fc Cross Linking
[0318] We next investigated if soluble FG2811mG1 could stimulate CD4 and CD8 T cells when cultured in the presence or absence of splenocytes. The addition of splenocytes should allow Fc crosslinking and stimulate a T cell response. Splenocytes were isolated from HHDII and HHDII/DP4 mice, pan T cells enriched (CD3+) from the HHDII splenocytes, both the HHDII T cells and HHDII/DP4 splenocytes were labelled with CFSE. HHDII T cells were then cultured with or without HHDII/DP4 splenocytes in addition to FG2811mG1, LPS or media alone. On day 15 the CD4 and CD8 proliferative responses were determined. In the absence of co culture with splenocytes only 0.14% CD4 T proliferated (CFSE.sup.low) in the presence of soluble FG2811mG1, however, this was not above the media only control (0.16% CFSE.sup.low) and therefore just background levels. In the absence of co culture with splenocytes only 0.02% CD8 T proliferated (CFSE.sup.low) in the presence of soluble FG2811mG1, this was very similar to the media only control (0% CFSE.sup.low) and therefore just background levels. Both the CD4 and CD8 T cells showed a good proliferative response to LPS (3.34%, 39.56% respectively). In the presence of co culture with splenocytes 15.2% CD4 T proliferated (CFSE.sup.low) in the presence of soluble FG2811mG1 and 2.33% CD8 T proliferated (CFSE.sup.low) in the presence of soluble FG2811mG1, Both the CD4 and CD8 T cells showed a good proliferative response to LPS which was enhanced in the presence of PBMCs (59.88%, 54.10% respectively).
[0319] These results show that soluble FG2811mG1 can stimulate CD4 and a CD8 proliferative response when cocultured in the presence of splenocytes (
Embodiments
[0320] Further embodiments of the invention are described below:
[0321] 1. An isolated specific binding member capable of binding to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc).
[0322] 2. The binding member of embodiment 1 wherein the binding member is capable of binding SSEA-4 on glycolipids.
[0323] 3. The binding member of any preceding embodiment wherein the binding member is capable of targeting stem memory T-cells (T.sub.SCM).
[0324] 4. The binding member of any preceding embodiment wherein the binding member is capable of inducing proliferation of stem memory T-cells (T.sub.SCM).
[0325] 5. The binding member according to any preceding embodiment, wherein the binding member does not bind to SSEA-3.
[0326] 6. The binding member according to any preceding embodiment, wherein the binding member is mAb FG2811.72 or Chimeric FG2811.72 (CH2811/CH2811.72), or a fragment thereof.
[0327] 7. The binding member according to any preceding embodiment, wherein the binding member is bispecific.
[0328] 8. The binding member according to embodiment 7, wherein the bispecific binding member is additionally specific for CD3.
[0329] 9. The binding member according to any preceding embodiment, wherein the binding member comprises one or more binding domains selected from the amino acid sequence of residues 27 to 38 (CDRH1), 56-65 (CDRH2) and 105-113 (CDRH3) of
[0330] 10. The binding member according to any preceding embodiment, wherein the binding member comprises one or more binding domains selected from the amino acid sequence of residues 27 to 38 (CDRL1), 56-65 (CDRL2) and 105-113 (CDRL3) of
[0331] 11. The binding member according to any preceding embodiment, wherein the binding member comprises a light chain variable sequence comprising one or more of LCDR1, LCDR2 and LCDR3, wherein [0332] LCDR1 comprises SSVNY, [0333] LCDR2 comprises DTS, and [0334] LCDR3 comprises FQASGYPLT; and
[0335] a heavy chain variable sequence comprising one or more of HCDR1, HCDR2 and HCDR3, wherein [0336] HCDR1 comprises GFSLNSYG, [0337] HCDR2 comprises IWGDGST, and [0338] HCDR3 comprises TKPGSGYAF.
[0339] 12. The binding member according to any preceding embodiment, wherein the binding domain(s) are carried by a human antibody framework.
[0340] 13. The binding member according to any preceding embodiment, wherein the binding member comprises a VH domain comprising residues 1 to 126 of the amino acid sequence of
[0341] 14. The binding member according to any preceding embodiment, wherein the binding member comprises a human antibody constant region.
[0342] 15. The binding member according to any preceding embodiment, wherein the binding member is an antibody, an antibody fragment, Fab, (Fab′)2, scFv, Fv, dAb, Fd or a diabody.
[0343] 16. The binding member according to any preceding embodiment, wherein the binding member is an scFv comprising, in the following order, 1) leader sequence, 2) heavy chain variable region, 3) 3×GGGGS spacer, 4) light chain variable region, and 5) poly-Ala and a 6×His tag for purification.
[0344] 17. The binding member according to any of embodiments 1 to 15, wherein the binding member is an scFv comprising, in the following order, 1) leader sequence, 2) light chain variable region, 3) 3×GGGGS spacer, and 4) heavy chain variable region, optionally further comprising either 5′ or 3′ purification tags.
[0345] 18. The binding member according to any preceding embodiment, wherein the binding member is provided in the form of a chimeric antigen receptor (CAR).
[0346] 19. The binding member according to embodiment 18, wherein the binding member is an scFv provided in the form of a chimeric antigen receptor (CAR) either in the heavy chain-light chain orientation or the light chain-heavy chain orientation.
[0347] 20. The binding member according to any of embodiments 1 to 17, wherein the binding member is provided in the form of an agonist (IgG2) monoclonal antibody.
[0348] 21. The binding member according to any of embodiments 1 to 17, wherein the binding member is provided in the form of an antagonist monoclonal antibody.
[0349] 22. The binding member according to any preceding embodiment, wherein the binding member is monoclonal, such as a monoclonal antibody.
[0350] 23. The binding member according to any preceding embodiment, wherein the binding member is a human, humanized, chimeric or veneered antibody.
[0351] 24. An isolated specific binding member capable of binding specifically to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc), which competes with an isolated specific binding member as embodimented in any one of embodiments 1 to 23.
[0352] 25. A binding member according to any preceding embodiment for use in therapy.
[0353] 26. A binding member according to any of embodiments 1 to 24 for use in a method of the preventing, treating or diagnosing cancer.
[0354] 27. A binding member according to any of embodiments 1 to 24, for use in a method of treating chronically virally infected patients.
[0355] 28. A binding member according to any of embodiments 1 to 24, for use in a method of treating an autoimmune disease, HIV, adult T-cell leukaemia or graft versus host disease.
[0356] 29. A method of treating or preventing cancer, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need of thereof.
[0357] 30. A method of treating or preventing chronically virally infected patients, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need of thereof.
[0358] 31. A method of treating or preventing an autoimmune disease, HIV, adult T-cell leukaemia or graft versus host disease, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need of thereof.
[0359] 32. A method of enhancing a protective immune response against cancer comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need of thereof.
[0360] 33. The method of embodiment 32, wherein the binding member is prepared to be administered with a further immunogenic agent, optionally wherein the immunogenic agent is a cancer vaccine.
[0361] 34. The method of embodiment 33, wherein the binding member and the further immunogenic agent are prepared to be administered simultaneously or sequentially.
[0362] 35. The binding member for use of embodiments 25 or 26, or the method of embodiment 29, wherein the cancer is pancreatic, gastric, colorectal, ovarian or lung cancer.
[0363] 36. The binding member for use of embodiments 25, 26 or 35, or the method of embodiment 28 or embodiment 31, wherein the binding member is administered, or prepared to be administered, alone or in combination with other treatments.
[0364] 37. A nucleic acid comprising a sequence encoding a binding member according to any of embodiments 1-24.
[0365] 38. The nucleic acid according to embodiment 37, wherein the nucleic acid is a construct in the form of a plasmid, vector, transcription or expression cassette.
[0366] 39. A recombinant host cell which comprises the nucleic acid according to embodiment 37 or embodiment 38.
[0367] 40. A method for diagnosis of cancer comprising using a binding member as embodimented in any of embodiments 1 to 24 to detect the glycans SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) attached to a glycolipid in a sample from an individual.
[0368] 41. The method according to embodiment 40, wherein the pattern of glycans detected by the binding member is used to stratify therapy options for the individual.
[0369] 42. A pharmaceutical composition comprising the binding member according to any of embodiments 1 to 24, and a pharmaceutically acceptable carrier.
[0370] 43. The pharmaceutical composition according to embodiment 42, further comprising at least one or other pharmaceutical active.
[0371] 44. The pharmaceutical composition according to embodiment 42 or embodiment 43, for use in the treatment of cancer.
[0372] 45. The pharmaceutical composition according to embodiment 42 or embodiment 43, for use in the treatment of chronically virally infected patients.
[0373] 46. The pharmaceutical composition according to embodiment 42 or embodiment 43, for use in the treatment of autoimmune disease, HIV, adult T-cell leukaemia or graft versus host disease.
[0374] 47. A method of inducing proliferation of stem memory T-cells (T.sub.SCM) ex vivo comprising contacting the stem memory T-cells (T.sub.SCM) with a binding member according to any of embodiments 1 to 24.
[0375] 48. A cell culture medium for inducing proliferation of stem memory T-cells (T.sub.SCM) comprising a binding member according to any of embodiments 1 to 24.
[0376] 49. A method of inducing proliferation of stem memory T-cells (T.sub.SCM) in vivo comprising administering a subject with a binding member according to any of embodiments 1 to 24.
[0377] 50. A method of identifying stem memory T-cells (T.sub.SCM) by detecting the presence of SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the cell with a binding member according to any of embodiments 1 to 24.
[0378] 51. A method of purifying stem memory T-cells (T.sub.SCM) by detecting the presence of SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the cell with a binding member according to any of embodiments 1 to 24.
[0379] 52. The method of embodiment 50 or 51 wherein the identifying or purifying is conducted in vivo or ex vivo.
[0380] 53. The method of embodiment 51 or 52 wherein the binding member is used to label the stem memory T-cells (T.sub.SCM) for purification.
[0381] 54. A binding member substantially as described herein, optionally with reference to the accompanying figures.
REFERENCES
[0382] Akinleye, A., P. Avvaru, M. Furqan, Y. Song, and D. Liu. 2013. ‘Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics’, J Hematol Oncol, 6: 88. [0383] Akinleye, A., Y. Chen, N. Mukhi, Y. Song, and D. Liu. 2013. ‘Ibrutinib and novel BTK inhibitors in clinical development’, J Hematol Oncol, 6: 59. [0384] Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. ‘Basic local alignment search tool’, J Mol Biol, 215: 403-10. [0385] Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. ‘Gapped BLAST and PSI-BLAST: a new generation of protein database search programs’, Nucleic Acids Res, 25: 3389-402. [0386] Ausubel, F M. 1992. Short protocols in molecular biology (John Wiley & Sons). [0387] Barbas, C. F., 3rd, D. Hu, N. Dunlop, L. Sawyer, D. Cababa, R. M. Hendry, P. L. Nara, and D. R. Burton. 1994. ‘In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity’, Proc Natl Acad Sci USA, 91:3809-13. [0388] Beers, S. A., M. J. Glennie, and A. L. White. 2016. ‘Influence of immunoglobulin isotype on therapeutic antibody function’, Blood, 127: 1097-101. [0389] Bird, R. E., K. D. Hardman, J. W. Jacobson, S. Johnson, B. M. Kaufman, S. M. Lee, T. Lee, S. H. Pope, G. S. Riordan, and M. Whitlow. 1988. ‘Single-chain antigen-binding proteins’, Science, 242: 423-6. [0390] Bodanzsky, M., and A. Bodanzsky. 1984. The practice of peptide synthesis (Springer Verlag: New York). [0391] Breton, C. S., A. Nahimana, D. Aubry, J. Macoin, P. Moretti, M. Bertschinger, S. Hou, M. A. Duchosal, and J. Back. 2014. ‘A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies’, J Hematol Oncol, 7: 33. [0392] Cahan, L. D., R. F. Irie, R. Singh, A. Cassidenti, and J. C. Paulson. 1982. ‘Identification of a human neuroectodermal tumor antigen (OFA-I-2) as ganglioside GD2’, Proc Natl Acad Sci USA, 79: 7629-33. [0393] Chahroudi, A., G. Silvestri, and M. Lichterfeld. 2015. ‘T memory stem cells and HIV: a long-term relationship’, Curr HIV/AIDS Rep, 12: 33-40. [0394] Chang, W. W., C. H. Lee, P. Lee, J. Lin, C. W. Hsu, J. T. Hung, J. J. Lin, J. C. Yu, L. E. Shao, J. Yu, C. H. Wong, and A. L. Yu. 2008. ‘Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis’, Proc Natl Acad Sci USA, 105: 11667-72. [0395] Christiansen, M. N., J. Chik, L. Lee, M. Anugraham, J. L. Abrahams, and N. H. Packer. 2014. ‘Cell surface protein glycosylation in cancer’, Proteomics, 14: 525-46. [0396] Cid, E., M. Yamamoto, M. Buschbeck, and F. Yamamoto. 2013. ‘Murine cell glycolipids customization by modular expression of glycosyltransferases’, PLoS One, 8: e64728. [0397] Cieri, N., B. Camisa, F. Cocchiarella, M. Forcato, G. Oliveira, E. Provasi, A. Bondanza, C. Bordignon, J. Peccatori, F. Ciceri, M. T. Lupo-Stanghellini, F. Mavilio, A. Mondino, S. Bicciato, A. Recchia, and C. Bonini. 2013. ‘IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors’, Blood, 121: 573-84. [0398] Cooling, L., and D. Hwang. 2005. ‘Monoclonal antibody B2, a marker of neuroendocrine sympathoadrenal precursors, recognizes the Luke (LKE) antigen’, Transfusion, 45: 709-16. [0399] Coulie, P. G., B. J. Van den Eynde, P. van der Bruggen, and T. Boon. 2014. ‘Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy’, Nat Rev Cancer, 14: 135-46. [0400] Dalziel, M., M. Crispin, C. N. Scanlan, N. Zitzmann, and R. A. Dwek. 2014. ‘Emerging principles for the therapeutic exploitation of glycosylation’, Science, 343: 1235681. [0401] Daniotti, J. L., A. A. Vilcaes, V. Torres Demichelis, F. M. Ruggiero, and M. Rodriguez-Walker. 2013. ‘Glycosylation of glycolipids in cancer: basis for development of novel therapeutic approaches’, Front Oncol, 3: 306. [0402] Darlak, K. A., Y. Wang, J. M. Li, W. A. Harris, C. R. Giver, C. Huang, and E. K. Waller. 2014. ‘Host bone marrow-derived IL-12 enhances donor T cell engraftment in a mouse model of bone marrow transplantation’, J Hematol Oncol, 7: 16. [0403] Di Benedetto, S., E. Derhovanessian, E. Steinhagen-Thiessen, D. Goldeck, L. Muller, and G. Pawelec. 2015. ‘Impact of age, sex and CMV-infection on peripheral T cell phenotypes: results from the Berlin BASE-II Study’, Biogerontology, 16: 631-43. [0404] Eppstein, D. A., Y. V. Marsh, M. van der Pas, P. L. Feigner, and A. B. Schreiber. 1985. ‘Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor’, Proc Natl Acad Sci USA, 82: 3688-92. [0405] Fuertes Marraco, S. A., C. Soneson, L. Cagnon, P. O. Gannon, M. Allard, S. Abed Maillard, N. Montandon, N. Rufer, S. Waldvogel, M. Delorenzi, and D. E. Speiser. 2015. ‘Long-lasting stem cell-like memory CD8.sup.+ T cells with a naive-like profile upon yellow fever vaccination’, Sci Transl Med, 7: 282ra48. [0406] Fuster, M. M., and J. D. Esko. 2005. ‘The sweet and sour of cancer: glycans as novel therapeutic targets’, Nat Rev Cancer, 5: 526-42. [0407] Gang, E. J., D. Bosnakovski, C. A. Figueiredo, J. W. Visser, and R. C. Perlingeiro. 2007. ‘SSEA-4 identifies mesenchymal stem cells from bone marrow’, Blood, 109: 1743-51. [0408] Garrido, F., T. Cabrera, A. Concha, S. Glew, F. Ruiz-Cabello, and P. L. Stern. 1993. ‘Natural history of HLA expression during tumour development’, Immunol Today, 14: 491-9. [0409] Gattinoni, L., E. Lugli, Y. Ji, Z. Pos, C. M. Paulos, M. F. Quigley, J. R. Almeida, E. Gostick, Z. Yu, C. Carpenito, E. Wang, D. C. Douek, D. A. Price, C. H. June, F. M. Marincola, M. Roederer, and N. P. Restifo. 2011. ‘A human memory T cell subset with stem cell-like properties’, Nat Med, 17: 1290-7. [0410] Gattinoni, L., and N. P. Restifo. 2013. ‘Moving T memory stem cells to the clinic’, Blood, 121: 567-8. Gattinoni, L., D. E. Speiser, M. Lichterfeld, and C. Bonini. 2017. ‘T memory stem cells in health and disease’, Nat Med, 23: 18-27. [0411] Gottschling, S., K. Jensen, A. Warth, F. J. Herth, M. Thomas, P. A. Schnabel, and E. Herpel. 2013. ‘Stage-specific embryonic antigen-4 is expressed in basaloid lung cancer and associated with poor prognosis’, Eur Respir J, 41: 656-63. [0412] Gram, H., L. A. Marconi, C. F. Barbas, 3rd, T. A. Collet, R. A. Lerner, and A. S. Kang. 1992. ‘In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library’, Proc Natl Acad Sci USA, 89: 3576-80. [0413] Hakomori, S. 2002. ‘Glycosylation defining cancer malignancy: new wine in an old bottle’, Proc Natl Acad Sci USA, 99: 10231-3. [0414] Hakomori, S. I. 2008. ‘Structure and function of glycosphingolipids and sphingolipids: recollections and future trends’, Biochim Biophys Acta, 1780: 325-46. [0415] Hakomori, S., and Y. Zhang. 1997. ‘Glycosphingolipid antigens and cancer therapy, Chem Biol, 4: 97-104. [0416] Han, E. Q., X. L. Li, C. R. Wang, T. F. Li, and S. Y. Han. 2013. ‘Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges’, J Hematol Oncol, 6: 47. [0417] Harichandan, A., K. Sivasubramaniyan, and H. J. Buhring. 2013. ‘Prospective isolation and characterization of human bone marrow-derived MSCs’, Adv Biochem Eng Biotechnol, 129: 1-17. [0418] Henderson, J. K., J. S. Draper, H. S. Baillie, S. Fishel, J. A. Thomson, H. Moore, and P. W. Andrews. 2002. ‘Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens’, Stem Cells, 20: 329-37. [0419] Holliger, P., T. Prospero, and G. Winter. 1993. ‘“Diabodies”: small bivalent and bispecific antibody fragments’, Proc Natl Acad Sci USA, 90: 6444-8. [0420] Holliger, P., and G. Winter. 1993. ‘Engineering bispecific antibodies’, Curr Opin Biotechnol, 4: 446-9. Hosokawa, T., T. Kimura, S. Nada, T. Okuno, D. Ito, S. Kang, S. Nojima, K. Yamashita, T. Nakatani, Y. Hayama, Y. Kato, Y. Kinehara, M. Nishide, N. Mikami, S. Koyama, H. Takamatsu, D. Okuzaki, N. Ohkura, S. Sakaguchi, M. Okada, and A. Kumanogoh. 2017. ‘Lamtor1 Is Critically Required for CD4(+) T Cell Proliferation and Regulatory T Cell Suppressive Function’, J Immunol, 199: 2008-19. [0421] Huang, Y. L., J. T. Hung, S. K. Cheung, H. Y. Lee, K. C. Chu, S. T. Li, Y. C. Lin, C. T. Ren, T. J. Cheng, T. L. Hsu, A. L. Yu, C. Y. Wu, and C. H. Wong. 2013. ‘Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer’, Proc Natl Acad Sci USA, 110: 2517-22. [0422] Huston, J. S., D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotny, M. N. Margolies, R. J. Ridge, R. E. Bruccoleri, E. Haber, R. Crea, and et al. 1988. ‘Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli’, Proc Natl Acad Sci USA, 85: 5879-83. [0423] Hwang, K. J., K. F. Luk, and P. L. Beaumier. 1980. ‘Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study’, Proc Natl Acad Sci USA, 77: 4030-4. [0424] Jespers, L. S., A. Roberts, S. M. Mahler, G. Winter, and H. R. Hoogenboom. 1994. ‘Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen’, Biotechnology (N Y), 12: 899-903. [0425] Junghans, R. P., T. A. Waldmann, N. F. Landolfi, N. M. Avdalovic, W. P. Schneider, and C. Queen. 1990. ‘Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders’, Cancer Res, 50: 1495-502. [0426] Kannagi, R., N. A. Cochran, F. Ishigami, S. Hakomori, P. W. Andrews, B. B. Knowles, and D. Solter. 1983. ‘Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells’, EMBO J, 2: 2355-61. [0427] Karlin, S., and S. F. Altschul. 1990. ‘Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes’, Proc Natl Acad Sci USA, 87: 2264-8. 1993. ‘Applications and statistics for multiple high-scoring segments in molecular sequences’, Proc Natl Acad Sci USA, 90: 5873-7. [0428] Klebanoff, C. A., L. Gattinoni, and N. P. Restifo. 2012. ‘Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy?’, J Immunother, 35: 651-60. [0429] Lau, K. S., and J. W. Dennis. 2008. ‘N-Glycans in cancer progression’, Glycobiology, 18: 750-60. [0430] Lefranc, M. P., F. Ehrenmann, S. Kossida, V. Giudicelli, and P. Duroux. 2018. ‘Use of IMGT(®) Databases and Tools for Antibody Engineering and Humanization’, Methods Mol Biol, 1827: 35-69. [0431] Li, G., Q. Yang, Y. Zhu, H. R. Wang, X. Chen, X. Zhang, and B. Lu. 2013. ‘T-Bet and Eomes Regulate the Balance between the Effector/Central Memory T Cells versus Memory Stem Like T Cells’, PLoS One, 8: e67401. [0432] Lou, Y. W., P. Y. Wang, S. C. Yeh, P. K. Chuang, S. T. Li, C. Y. Wu, K. H. Khoo, M. Hsiao, T. L. Hsu, and C. H. Wong. 2014. ‘Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers’, Proc Natl Acad Sci USA, 111: 2482-7. [0433] Lugli, E., M. H. Dominguez, L. Gattinoni, P. K. Chattopadhyay, D. L. Bolton, K. Song, N. R. Klatt, J. M. Brenchley, M. Vaccari, E. Gostick, D. A. Price, T. A. Waldmann, N. P. Restifo, G. Franchini, and M. Roederer. 2013. ‘Superior T memory stem cell persistence supports long-lived T cell memory’, J Clin Invest, 123: 594-9. [0434] Lugli, E., L. Gattinoni, A. Roberto, D. Mavilio, D. A. Price, N. P. Restifo, and M. Roederer. 2013. ‘Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells’, Nat Protoc, 8: 33-42. [0435] Mahnke, Y. D., T. M. Brodie, F. Sallusto, M. Roederer, and E. Lugli. 2013. ‘The who's who of T-cell differentiation: human memory T-cell subsets’, Eur J Immunol, 43: 2797-809. [0436] Marks, J. D., A. D. Griffiths, M. Malmqvist, T. P. Clackson, J. M. Bye, and G. Winter. 1992. ‘By-passing immunization: building high affinity human antibodies by chain shuffling’, Biotechnology (N Y), 10: 779-83. [0437] Martin, P. J. 2014. ‘Reversing CD8+ T-cell exhaustion with DLI’, Blood, 123: 1289-90. [0438] Mateus, J., P. Lasso, P. Pavia, F. Rosas, N. Roa, C. A. Valencia-Hernandez, J. M. Gonzalez, C. J. Puerta, and A. Cuellar. 2015. ‘Low frequency of circulating CD8.sup.+ T stem cell memory cells in chronic chagasic patients with severe forms of the disease’, PLoS Negl Trop Dis, 9: e3432. [0439] Meezan, E., H. C. Wu, P. H. Black, and P. W. Robbins. 1969. ‘Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by sephadex chromatography’, Biochemistry, 8: 2518-24. [0440] Metheringham, R. L., V. A. Pudney, B. Gunn, M. Towey, I. Spendlove, and L. G. Durrant. 2009. ‘Antibodies designed as effective cancer vaccines’, MAbs, 1: 71-85. [0441] Mezzanzanica, D., S. Canevari, A. Mazzoni, M. Figini, M. I. Colnaghi, T. Waks, D. G. Schindler, and Z. Eshhar. 1998. ‘Transfer of chimeric receptor gene made of variable regions of tumor-specific antibody confers anticarbohydrate specificity on T cells’, Cancer Gene Ther, 5: 401-7. [0442] Myers, E. W., and W. Miller. 1989. ‘Approximate matching of regular expressions’, Bull Math Biol, 51: 5-37. [0443] Nilsson, O., F. T. Brezicka, J. Holmgren, S. Sorenson, L. Svennerholm, F. Yngvason, and L. Lindholm. 1986. ‘Detection of a ganglioside antigen associated with small cell lung carcinomas using monoclonal antibodies directed against fucosyl-GM1’, Cancer Res, 46: 1403-7. [0444] Noto, Z., T. Yoshida, M. Okabe, C. Koike, M. Fathy, H. Tsuno, K. Tomihara, N. Arai, M. Noguchi, and T. Nikaido. 2013. ‘CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics’, Oral Oncol, 49: 787-95. [0445] Novero, A., P. M. Ravella, Y. Chen, G. Dous, and D. Liu. 2014. ‘Ibrutinib for B cell malignancies’, Exp Hematol Oncol, 3: 4. [0446] Nudelman, E., S. Hakomori, R. Kannagi, S. Levery, M. Y. Yeh, K. E. Hellstrom, and I. Hellstrom. 1982. ‘Characterization of a human melanoma-associated ganglioside antigen defined by a monoclonal antibody, 4.2’, J Biol Chem, 257: 12752-6. [0447] Pearson, W. R., and D. J. Lipman. 1988. ‘Improved tools for biological sequence comparison’, Proc Natl Acad Sci USA, 85: 2444-8. [0448] Pilipow, K., E. Scamardella, S. Puccio, S. Gautam, F. De Paoli, E. M. Mazza, G. De Simone, S. Polletti, M. Buccilli, V. Zanon, P. Di Lucia, M. lannacone, L. Gattinoni, and E. Lugli. 2018. ‘Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity’, JCI Insight, 3. [0449] Pinto, J. P., R. K. Kalathur, D. V. Oliveira, T. Barata, R. S. Machado, S. Machado, I. Pacheco-Leyva, I. Duarte, and M. E. Futschik. 2015. ‘StemChecker: a web-based tool to discover and explore stemness signatures in gene sets’, Nucleic Acids Res, 43: W72-7. [0450] Pluckthun, A. 1991. ‘Antibody engineering: advances from the use of Escherichia coli expression systems’, Biotechnology (N Y), 9: 545-51. [0451] Raphael, I., S. Nalawade, T. N. Eagar, and T. G. Forsthuber. 2015. ‘T cell subsets and their signature cytokines in autoimmune and inflammatory diseases’, Cytokine, 74: 5-17. [0452] Reff, M. E. 1993. ‘High-level production of recombinant immunoglobulins in mammalian cells’, Curr Opin Biotechnol, 4: 573-6. [0453] Remington, R P. 1980. Remington's pharmaceutical sciences (Mack Pub. Co.). [0454] Restifo, N. P., and L. Gattinoni. 2013. ‘Lineage relationship of effector and memory T cells’, Curr Opin Immunol, 25: 556-63. [0455] Saito, S., H. Aoki, A. Ito, S. Ueno, T. Wada, K. Mitsuzuka, M. Satoh, Y. Arai, and T. Miyagi. 2003. ‘Human alpha2,3-sialyltransferase (ST3Gal II) is a stage-specific embryonic antigen-4 synthase’, J Biol Chem, 278: 26474-9. [0456] Saito, S., S. Orikasa, M. Satoh, C. Ohyama, A. Ito, and T. Takahashi. 1997. ‘Expression of globo-series gangliosides in human renal cell carcinoma’, Jpn J Cancer Res, 88: 652-9. [0457] Sambrook, J. 1989. Molecular cloning: A laboratory manual (Cold Spring Harbor Laboratory Press). [0458] Sandstedt, J., M. Jonsson, K. Vukusic, G. Dellgren, A. Lindahl, A. Jeppsson, and J. Asp. 2014. ‘SSEA-4.sup.+CD34− cells in the adult human heart show the molecular characteristics of a novel cardiomyocyte progenitor population’, Cells Tissues Organs, 199: 10.sup.3-16. [0459] Schier, R., A. McCall, G. P. Adams, K. W. Marshall, H. Merritt, M. Yim, R. S. Crawford, L. M. Weiner, C. Marks, and J. D. Marks. 1996. ‘Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site’, J Mol Biol, 263: 551-67. [0460] Schmueck-Henneresse, M., R. Sharaf, K. Vogt, B. J. Weist, S. Landwehr-Kenzel, H. Fuehrer, A. Jurisch, N. Babel, C. M. Rooney, P. Reinke, and H. D. Volk. 2015. ‘Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency’, J Immunol, 194: 5559-67. [0461] Sell, S. 1990. ‘Cancer-associated carbohydrates identified by monoclonal antibodies’, Hum Pathol, 21: 1003-19. [0462] Shevinsky, L. H., B. B. Knowles, I. Damjanov, and D. Solter. 1982. ‘Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells’, Cell, 30: 697-705. [0463] Sidman, K. R., W. D. Steber, A. D. Schwope, and G. R. Schnaper. 1983. ‘Controlled release of macromolecules and pharmaceuticals from synthetic polypeptides based on glutamic acid’, Biopolymers, 22: 547-56. [0464] Stemmer, W. P. 1994. ‘Rapid evolution of a protein in vitro by DNA shuffling’, Nature, 370: 389-91. [0465] Stewart, J M., and J D. Young. 1984. Solid phase peptide synthesis (Pierce Chemical Company: Rockford, Ill.). [0466] Suresh, T., L. X. Lee, J. Joshi, and S. K. Barta. 2014. ‘New antibody approaches to lymphoma therapy’, J Hematol Oncol, 7: 58. [0467] Suzuki, Y., N. Haraguchi, H. Takahashi, M. Uemura, J. Nishimura, T. Hata, I. Takemasa, T. Mizushima, H. Ishii, Y. Doki, M. Mori, and H. Yamamoto. 2013. ‘SSEA-3 as a novel amplifying cancer cell surface marker in colorectal cancers’, Int J Oncol, 42: 161-7. [0468] Takeshita, M., K. Suzuki, Y. Kassai, M. Takiguchi, Y. Nakayama, Y. Otomo, R. Morita, T. Miyazaki, A. Yoshimura, and T. Takeuchi. 2015. ‘Polarization diversity of human CD4+ stem cell memory T cells’, Clin Immunol, 159: 107-17. [0469] Taylor-Papadimitriou, J., and A. A. Epenetos. 1994. ‘Exploiting altered glycosylation patterns in cancer: progress and challenges in diagnosis and therapy’, Trends Biotechnol, 12: 227-33. [0470] Tondeur, S., S. Assou, L. Nadal, S. Hamamah, and J. De Vos. 2008. ‘[Biology and potential of human embryonic stem cells]’, Ann Biol Clin (Paris), 66: 241-7. [0471] Torelli, A., and C. A. Robotti. 1994. ‘ADVANCE and ADAM: two algorithms for the analysis of global similarity between homologous informational sequences’, Comput Appl Biosci, 10: 3-5. [0472] Traunecker, A., A. Lanzavecchia, and K. Karjalainen. 1991. ‘Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells’, EMBO J, 10: 3655-9. [0473] Trill, J. J., A. R. Shatzman, and S. Ganguly. 1995. ‘Production of monoclonal antibodies in COS and CHO cells’, Curr Opin Biotechnol, 6: 553-60. [0474] van Beek, W. P., L. A. Smets, and P. Emmelot. 1973. ‘Increased sialic acid density in surface glycoprotein of transformed and malignant cells—a general phenomenon?’, Cancer Res, 33: 2913-22. [0475] Varki, A., R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, J. D. Marth, C. R. Bertozzi, G. W. Hart, and M. E. Etzler. 2009. ‘Symbol nomenclature for glycan representation’, Proteomics, 9: 5398-9. [0476] Ward, E. S., D. Gussow, A. D. Griffiths, P. T. Jones, and G. Winter. 1989. ‘Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli’, Nature, 341: 544-6. [0477] Wright, A. J., and P. W. Andrews. 2009. ‘Surface marker antigens in the characterization of human embryonic stem cells’, Stem Cell Res, 3: 3-11. [0478] Ye, F., Y. Li, Y. Hu, C. Zhou, Y. Hu, and H. Chen. 2010. ‘Stage-specific embryonic antigen 4 expression in epithelial ovarian carcinoma’, Int J Gynecol Cancer, 20: 958-64. [0479] Yvon, E., M. Del Vecchio, B. Savoldo, V. Hoyos, A. Dutour, A. Anichini, G. Dotti, and M. K. Brenner. 2009. ‘Immunotherapy of metastatic melanoma using genetically engineered GD2-specific T cells’, Clin Cancer Res, 15: 5852-60. [0480] Zhang, Y., G. Joe, E. Hexner, J. Zhu, and S. G. Emerson. 2005. ‘Host-reactive CD8+ memory stem cells in graft-versus-host disease’, Nat Med, 11: 1299-305.