Reversible MHC Multimer Staining for Functional Purification of Antigen-Specific T Cells

Europäisches Patentamt *EP001227321A1* (19) European Patent Office

Office européen des brevets (11) EP 1 227 321 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.7: G01N 33/50, C12N 5/06 31.07.2002 Bulletin 2002/31

(21) Application number: 00128634.3

(22) Date of filing: 28.12.2000

(84) Designated Contracting States: • Wagner, Hermann, Prof.Dr.med., AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU 82279 Eching am Ammersee (DE) MC NL PT SE TR Designated Extension States: (74) Representative: AL LT LV MK RO SI Weiss, Wolfgang, Dipl.-Chem. Dr. et al Weickmann & Weickmann (71) Applicant: Institut für Bioanalytik GmbH Patentanwälte 37079 Göttingen (DE) Postfach 86 08 20 81635 München (DE) (72) Inventors: • Busch, Dirk H., Dr. med. 81929 München (DE)

(54) Reversible MHC multimer staining for functional purification of antigen-specific T cells

(57) The present invention relates to a new method be substantially maintained after their identification and for reversible staining and functional isolation or char- purification. Thus, this new method is of broad benefit acterization of cells, e.g. antigen-specific T cells. With for basic research and clinical applications. this technique, the original functional status of cells can EP 1 227 321 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 1 227 321 A1 2

Description ral ligand bound to the TCR, placement of cells into in vitro cell culture or adoptive in vivo transfer into recipi- [0001] The present invention relates to a new method ents (situations in which the temperature is shifted to for reversible staining and functional isolation or char- 37°C) results in dramatic alteration of purified T cell pop- acterization of cells, particularly of antigen-specific T 5 ulations (Figs. 3+4), including TCR internalization, acti- cells. With this technique, the original functional status vation, overstimulation, and cell death. of T cells can be substantially maintained after their [0007] Thus, conventional MHC multimer technology identification and purification. Thus, this new method is allows direct visualization and phenotypic analysis of of broad benefit for basic research and clinical applica- epitope-specific T cells. However, subsequent function- tions. 10 al analysis and in vivo transfer of MHC multimer-stained [0002] Identification and purification of antigen-spe- and purified T cells is complicated by the persistence of cific T cells without altering their functional status is of TCR-MHC interactions and subsequently induced sign- great scientific and clinical interest. Methods for direct aling events. Therefore, the development of methods to identification of T cells based on their antigen-specificity remove surface-bound MHC multimers after T cell stain- (ELISPOT assay (1), intracellular cytokine staining (2), 15 ing and purification will be of great interest to the field secretion assay [affinity matrix] (3), MHC multimers (2, of T cell immunology and is essential for the transfer of 4, 5)) have recently been developed. However, most of MHC multimer technologies to clinical applications and these identification techniques require in vitro stimula- functional diagnostics. tion of T cells, which significantly changes the pheno- [0008] Thus, a subject matter of the present invention type and functional status of the cells. To date, only the 20 is a method for reversible staining of cells comprising MHC multimer technology allows the identification and the steps: purification of antigen-specific T cells independent of their phenotype, but unfortunately, also conventional (a) providing a sample comprising a cell having a MHC multimer staining interferes with functional T cell receptor molecule, analysis. 25 (b) contacting said cell with [0003] We here describe the development of a new method for identification and purification of antigen spe- (i) at least one component specifically binding cific T cell populations without substantially altering their to said receptor wherein said at least one com- functional status comprising a reversible staining proce- ponent is conjugated to at least one first partner dure. This approach combines the specificity and sen- 30 of a binding complex, sitivity of MHC multimer staining with preserving the (ii) at least one further partner of said binding functional status of the cells. complex having at least two binding sites for [0004] T cells recognize processed antigen-frag- said first partner and ments (epitopes) in the context of major histocompati- (iii) a detectable label bound to or capable of bility complexes (MHC) (6). The specificity of T cells is 35 binding to (i) and/or (ii), wherein the first partner determined by the structure of their T cell receptor (s) and the further partner(s) of the binding (TCR), which is expressed at high levels on the cell sur- complex are capable of forming a reversible face. Early attempts to use the natural ligand of the TCR bond and wherein an aggregate comprising (i), - the MHC/epitope complex - for direct identification of (ii) and (iii) is bound via the receptor molecule epitope-specific T cells were unsuccessful. The failure 40 to said cell, wherein said cell is stained, of this approach can largely be explained by the low affinity of TCR-MHC/peptide interactions, which are espe- (c) optionally separating said stained cell from other cially characterized by a high dissociation rate (Fig. 1A). components of said sample and [0005] Multimerization of MHC/peptide (e.g. to tetra- (d) optionally removing said staining from said cell meric molecules) increases the relative avidity of the in- 45 by disrupting the reversible bond. teraction with TCRs to an extend which allows stable and epitope-specific binding on the T cell surface (Fig. [0009] A particularly preferred embodiment of the 1B). MHC multimer reagents conjugated with fluoro- present invention is a method for reversible staining of chromes can be used for direct identification of antigen- T cells comprising the steps: specific T cells by flow cytometry. This method has rev- 50 olutionized T cell research over the last few years, al- (a) providing a sample comprising a cell having a T lowing to visualize antigen-specific T cell populations for cell receptor (TCR) molecule, the first time directly ex vivo, in animal models as well (b) contacting said cell with as in humans (7, 8). [0006] As long as MHC multimer staining is performed 55 (i) at least one TCR binding ligand, e.g. a TCR at 4°C, T cells can be identified and purified (FACS, binding peptide and an MHC molecule conju- MACS) without altering their original phenotype. How- gated to at least one first partner of a binding ever, since MHC multimer reagents represent the natu- complex,

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(ii) at least one further partner of a binding com- bond should be capable of being disrupted under con- plex having at least two binding sites for the first ditions suitable for carrying out the claimed method. -2 partner of the binding complex and Preferably the reversible bond has a Kd between 10 (iii) a detectable label bound to or capable of and 10-13 M, more preferably between 10-3 and 10-10 M binding to (i) and/or (ii), 5 and most preferably between 10-5 and 10-8 M as determined under appropriate conditions, e.g. by fluores- wherein the first partner(s) and the further partner cence titration (11). Particularly preferred is a reversible -1 -1 (s) of the binding complex are capable of forming a bond having a high Koff value, e.g. between 10 sec reversible bond and wherein an aggregate compris- and 10-4 sec-1, especially between 10-2 sec-1 and 10-3 ing (i), (ii) and (iii) is bound via the TCR molecule to 10 sec-1. said T cell, wherein said T cell is stained, [0013] It is a further important feature of the present invention that the staining of the T cell and the subse- (c) optionally separating said stained T cell from quent optional steps, namely the isolation and purifica- other components of said sample and tion of the stained T cell and the removal of the staining (d) optionally removing said staining from said T cell 15 may be carried out at low temperatures, i.e. at temper- by disrupting the reversible bond. atures where substantially no activation and/or signal- ling events occur, which might result in an alteration of [0010] The method of the present invention is suitable the T cell phenotype. Preferably the staining and the for reversible staining of cells having a functional recep- subsequent removal of the staining is carried out at a tor molecule, e.g. of T cells, i.e. cells having a functional 20 temperature of ≤ 15°C, more preferably ≤ 10°C and TCR molecule, wherein said receptor molecule is capa- most preferably at about 4°C. ble of binding to a receptor ligand, e.g. a peptide/MHC [0014] The separation of the stained T cell from other complex. The receptor ligand may be any molecule ca- sample components, e.g. unstained T cells may be ef- pable of binding to a receptor molecule present on the fected by conventional methods, e.g. cell sorting, pref- cell to be stained, particularly when the receptor ligand 25 erably by FACS methods using commercially available and the receptor molecule exhibit a low affinity interac- systems (e.g. FACSVantage by Becton Dickinson or -3 tion, e.g. a dissociation constant Kd in the range of 10 Moflo by Cytomation), or by magnetic cell separation (e. to 10-6 M. Further examples of suitable receptor mole- g. MACS by Miltenyi). cules having low affinity interactions towards their lig- [0015] The removal of the staining preferably occurs ands are cell adhesion receptor molecules such as de- 30 by targeted disruption of the reversible bond between scribed in (14) and receptors for costimulatory mole- the first and the second partner of the binding complex. cules such as described in (15). If the receptor ligand This disruption may be achieved by contacting the comprises an MHC specific peptide, it is preferred that stained cell with a free first partner of the binding com- the peptide is a T cell epitope capable of binding to the plex or an analog thereof capable of disrupting the bond TCR receptor of the cell to be analysed and to the MHC 35 between the conjugated first partner (i) and the further molecule. The cell is preferably a mammalian cell, e.g. partner (ii). Preferably, the free first partner is an analog a mammalian T cell, particularly a human cell, e.g. a hu- of the conjugated first partner having a higher affinity to man T cell, particularly a subpopulation of mammalian the further partner than the conjugated first partner. T cells having a predetermined antigen-specifity. More preferably, the free first partner has an affinity [0011] The peptide comprises a T cell epitope capa- 40 which is at least 3 orders of magnitude and particularly ble of binding to a TCR molecule, preferably to a TCR at least 5 orders of magnitude higher than the affinity of molecule having a predetermined antigen-specifity. The the conjugated first partner. Particularly the free first peptide usually has a length of about 8 to about 25 ami- partner has a dissociation constant Kd which is at least no acids and preferably comprises so-called anchor by a factor of 1x10-3 and especially 1x10-5 lower. amino acid residues capable of allele-specific binding to 45 [0016] The label which is used for the detection of a predetermined MHC molecule class, e.g. an MHC stained cells may be any label which is used in diagnos- class I, an MHC class II or a non-classical MHC class. tic and analytical methods. Preferably the label does not The MHC molecule is preferably a recombinant soluble negatively affect the characteristics of the cells in the MHC molecule, which may be prepared in a bacterial method as described. Preferred examples of labels are expression system (16) or in an insect cell expression 50 fluorescent dyes or magnetic labels. The label may be system (17). bound to the ligand, e.g. the peptide and/or the MHC [0012] It is an important feature of the invention that molecule, the first partner and/or the further partner of a binding complex, e.g. a binding pair is selected con- the binding complex. The label may be a direct label, i. sisting of at least one first partner and at least one further e. a label bound to one of the members of the aggregate partner wherein the at least further partner has at least 55 as specified above. Alternatively, the label may be an two binding sites and more preferably at least four bind- indirect label, i.e. a label which is bound to a receptor ing sites for the first partner. The bond between the first which in turn is capable of binding to one of the members and the second partner should be reversible, i.e. the of the aggregate as specified above.

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[0017] The principle of the procedure according to the events occur, preferable at 4°C), the functional status of present invention is explained in the following for a pre- the T cell is not changed upon subsequent transfer to ferred embodiment wherein the binding complex is se- cell culture or adoptive in vivo transfer. lected from: [0021] The generation of prior art MHC multimer rea- 5 gents is based on specific biotinylation of soluble MHC (a) (i) biotin and (ii) a streptavidin or avidin analog complexes. Since streptavidin (SA) has four biotin-bind- capable of reversible binding of biotin, ing sites, incubation of biotinylated MHC molecules with (b) (i) a biotin analog capable of reversible binding (fluorochrome-conjugated) streptavidin at a 4:1 ratio re- to streptavidin or avidin and (ii) streptavidin or avidin sults in the formation of tetrameric MHC reagents (Fig. or a streptavidin or avidin analog capable of revers- 10 1B). The stability of the streptavidin:biotin bond is high -13 -14 ible binding of said biotin analog, and (Kd < 1x10 M, published to be 4x10 M in "Weber, (c) (i) a streptavidin or avidin binding peptide and P.C., Wendoloski, J.J., Pantoliano, M.W. & Salemme, F. (ii) streptavidin or avidin or a streptavidin or avidin R. (1992). Crystallographic and thermodynamic com- analog capable of reversible binding of said strepta- parison of natural and synthetic ligands bound to vidin or avidin binding peptide. 15 streptavidin. J. Am. Chem. Soc. 114, 3197-3200"), so the reagents are very stable. Addition of biotin (tested [0018] In an especially preferred embodiment oligom- up to a concentration of 50 mM) to MHC-streptavidin ers or polymers of streptavidin or of avidin or of analogs multimer stained T cells does not significantly affect the of streptavidin or of avidin may be prepared by the in- stability of the reagents (within a reasonable time win- troduction of carboxyl residues into a polysaccharide, e. 20 dow (≤ 1 day)). g. dextran essentially as described in "Noguchi, A., [0022] In contrast thereto the method of the present Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, invention is a technique allowing fast and targeted dis- Y., Hashida, M. & Sezaki, H. (1992). Preparation and ruption of MHC multimer reagents under physiological properties of the immunoconjugate composed of anti- conditions for functional isolation of antigen-specific T human colon cancer monoclonal antibody and mitomy- 25 cells; particularly a substantially complete and fast dis- cin C dextran conjugate. Bioconjugate Chemistry 3, ruption at low temperatures [preferably at 4°C]. Further, 132-137" in a first step. Then streptavidin or avidin or the procedure is substantially non-toxic to T cells, and analogs thereof are coupled via primary amino groups the substances used are harmless for (clinical) in vivo of internal lysine residues and/or the free N-terminus to applications. the carboxyl groups in the dextran backbone using con- 30 [0023] Peptide sequences (Strep-tags) such as dis- ventional carbodiimide chemistry in a second step. For closed in U.S. patent 5,506,121 demonstrate binding af- the actually especially preferred embodiment the cou- finity for the biotin binding site of streptavidin, e.g. with -4 -5 pling reaction was performed at a molar ratio of about aKdof approx. between 10 and 10 M (9, 10). The 60 moles streptavidin or Strep-Tactin per mole of dex- binding affinity may be further improved by making a tran. It should be noted, however, that oligomers or pol- 35 mutations within the streptavidin molecule. Examples of ymers of streptavidin or avidin may also be obtained by optimized streptavidin muteins (Strep-Tactins) are de- crosslinking via bifunctional linkers such as glutardial- scribed in U.S. patent 6,103,493 or (11), which are here- dehyde or by other methods described in the literature. in incorporated by reference. Preferably, the streptavi- [0019] The streptavidin binding peptide is preferably din muteins are characterized in that at position 44 of selected from the Strep-tag peptides as described in (9), 40 wild-type streptavidin Glu is replaced by a hydrophobic (10) or in U.S. patent 5,506,121 which are herein incor- aliphatic amino acid e.g. Val, Ala, lle or Leu, at position porated by reference. More preferably, the Strep-tag 45 an arbitrary amino acid is present, at position 46 an peptides comprise the amino acid sequence Trp-X-His- aliphatic amino acid and preferably a hydrophobic Pro-Gln-Phe-Y-Z, wherein X is any desired amino acid aliphatic amino acid is present and/or at position 47 Val and Y and Z either both are Gly, or Y is Glu and Z is Arg 45 is replaced by a basic amino acid e.g. Arg or Lys and in or Lys. Especially preferred is the peptide Trp-Ser-His- particular Arg. More preferably, Ala is at position 46 and/ Pro-Gln-Phe-Glu-Lys (Strep-tagII). or Arg is at position 47 and/or Val or lie is at position 44. Most preferably the mutein has a sequence selected Principle of procedure: from Ile44-Gly45-Ala46-Arg47 or Val44-Thr45-Ala46-Arg47 50 ("Strep-Tactin" as described in (11)). The interaction of [0020] MHC multimers increase the "relative binding the Strep-tagII peptide with Strep-Tactin is character- -6 avidity" but not the affinity of monovalent TCR-MHC/ ized by a binding affinity with a Kd of approx. 10 M(11) peptide interactions (Fig. 1A). Since monomeric MHC/ compared to approx. 10-13 M for the biotin-streptavidin peptide complexes do not stably bind to TCRs, a target- interaction. Biotin, which still binds with high affinity to 55 -10 ed disruption of MHC multimers into MHC monomers Strep-Tactin (with a Kd estimated to be between 10 results in rapid dissociation of surface bound TCR lig- M and 10-13 M), competes with Strep-tagII for the bind- ands. After complete dissociation of MHC/peptide com- ing site. plexes (at temperatures where no activation/signaling [0024] According to the present invention, stable

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MHC multimers for antigen-specific T cell staining are from a sample comprising non-stained T cell population generated based on the interaction of Strep tagII/Strep- and removing said stain, i.e. the functional status of the Tactin. Thus, it is possible to competitively disrupt mul- T cell population before purification is substantially the timers in the presence of relatively low concentrations same as after the purification. This T cell population may of biotin in a rapid manner, preferably less than 2 h, more 5 be obtained by the method as described above. preferably less than 1 h (Fig. 2). [0029] Finally, the present invention relates to a fusion [0025] This system fulfills the required criteria for dis- polypeptide comprising (a) a streptavidin-binding pep- ruption of MHC multimers (see also above): because of tide and (b) an MHC molecule, preferably a soluble MHC the extreme affinity differences between Strep-tagII/ molecule. For example, the streptavin-binding peptide StrepTactin and biotin/StrepTactin, complete and very 10 may be fused to the N-terminus and/or the C-terminus fast competitive binding of biotin (even at low tempera- of the α-chain and/or the β-microglobulin chain of an tures) takes place; biotin (tested up to a concentration MHC molecule. For example, fusion polypeptides com- of 50 mM) is non-toxic for T cells and does not alter T prising at least two streptavidin-binding peptides, e.g. cell function and small amounts of biotin (vitamin H) are fusion polypeptides having a streptavidin-binding pep- harmless for in vivo applications. 15 tide bound both to the C-terminus of the α-chain and the [0026] We have tested the Strep-tagII/StrepTactin β-chain have been shown to be suitable for the purpose system for both, the generation of MHC multimers and of the present invention. Preferred streptavin-binding the targeted disruption of multimers bound to the T cell peptides are Strep-tag peptides as described above, surface by addition of biotin. Alternatively to this ap- particularly Strep-tagII. Further, the invention relates to proach reversible T cell staining can be achieved by 20 nucleic acids encoding a fusion polypeptide as de- substituting Strep-tags by biotin analogs such as ami- scribed above, wherein the nucleic acids are preferably nobiotin, iminobiotin or desthiobiotin having a lower af- located on a recombinant vector, particularly an expres- finity for streptavidin or avidin compared to biotin. A still sion vector allowing expression of the claimed fusion further preferred embodiment relates to the competition polypeptide in a suitable host cell, e.g. a eukaryotic or of free biotin for the binding of biotinylated MHC mole- 25 prokaryotic host cell. cules to Strep-Tactin or other streptavidin analogs hav- [0030] The method of the present invention allows a ing lower affinity for biotin. functional isolation of antigen specific T cell populations [0027] As one key element of the present invention is based on a reversible staining procedure. The original the fact that the reversible bond can be disrupted in a functional status of T cells can be substantially main- targeted manner with the addition of small amounts of 30 tained after the identification and purification. Thus, the the physiological compound biotin (vitamin H) which method of the invention is of broad benefit for basic re- seems neither to be detrimental to living cells nor to alter search and clinical applications. Examples of preferred their functional state, we claim every binding complex applications are as follows: which can be selectively disrupted with that compound or analogs thereof. Thus, apart from the binding com- 35 Basic research: plexes between streptavidin or avidin or analogs thereof and biotin or biotin analogs or streptavidin- or avidin- [0031] binding peptides also other binding complexes are suitable for the purpose of the invention, provided they allow - Direct ex vivo investigation of the functional sta- reversible staining of a cell which can be disrupted by 40 tus of antigen-specific T cell populations. The the addition of compounds such as biotin or biotin ana- functional status of T cell populations in vivo is sug- logs which do not show any detrimental effect to the cell gested to be highly diverse and dependent on spe- to be stained. For example, an antigen/antibody-binding cific in vivo conditions, but because of the lack of pair can be applied, particularly if a polymerized or oli- appropriate investigative tools, these aspects are gomerized antibody is used. Further examples of such 45 only marginally understood. With MHC multimer binding complexes besides streptavidin/avidin and their techniques, epitope-specific T cell populations can analogs and/or antibodies may be so called engineered be identified and purified independent of their func- protein scaffolds for molecular recognition as published tional status. However, the binding of multimer rea- in "Skerra A. (2000). Engineered protein scaffolds for gents interferes with subsequent functional assays. molecular recognition. J. Mol. Recognit. 13, 167-187" 50 Reversible T cell staining e.g. using MHC-Strep- after being engineered to recognize compounds which tagII/Strep-Tactin reagents is the first technology al- may be disrupted in a targeted manner by disruption us- lowing the direct functional ex vivo investigation of ing biotin or biotin analogs. unaltered diverse T cell populations. [0028] Further, the present invention relates to a substantially pure antigen-specific T cell population having 55 - Purification of antigen-specific T cell popula- a functional status which is substantially unaltered by a tions for highly efficient in vitro expansion. The purification procedure comprising staining the desired T characterization of T cell populations obtained ex cell population, isolating the stained T cell population vivo often requires further in vitro expansion to T

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cell lines or T cell clones. With MHC multimer tech- pansion, allowing the use of antigen-independent niques, single cells or distinct phenotypic subpopu- stimulation such as mitogens and anti-CD3. With lations within a diverse T cell population can be iso- the invention, e.g. with MHC-Strep-tagll/Strep-Tac- lated, but the binding of the reagents to the TCR tin reagents, antigen-specific T cell populations can interferes with the efficiency of in vitro expansion. 5 be isolated directly ex vivo and expanded in vitro This experimental problem is solved by reversible after dissociation of the reagents. This approach is T cell staining using the reagents of the invention, expected to be much more efficient than purification e.g. MHC-Strep-tagII/Strep-Tactin reagents. using conventional MHC multimer reagents, as the binding of the reagents negatively interferes with - Purification of antigen-specific T cell popula- 10 the efficiency of in vitro T cell expansion. tions for adoptive transfer experiments. Many in vivo experiments in immunological research require - Purification of antigen-specific T cell popula- the adoptive transfer of purified T cells into recipient tions from in vitro expanded cell lines or clones animals. Both, the purity of the transferred T cell for further functional analyses or therapy. In vitro populations and possible changes in the cells that 15 expansion of T cells requires the addition of anti- occur during the isolation procedure are concerns gen-presenting cells or feeder cells to the culture. in these experimental systems. The highest purity For further functional analysis, and especially for of cell populations is achieved by positive selection therapeutic applications (e.g. adoptive transfer), it methods, but the markers (usually antibodies) used would be helpful to remove these contaminating for identification are difficult to remove from the sur- 20 cells. For positive selection procedures (which usu- face of isolated cells and can interfere with the out- ally result in the highest degrees of purity), the se- come of subsequent in vivo experiments. Reversi- lection marker should be removable from the T cell ble T cell staining using the reagents of the inven- surface, as it might interfere with functional assays tion, e.g. MHC-Strep-tagII/Strep-Tactin reagents al- or adoptive transfer. If T cells are used for in vivo lows the combination of positive selection methods 25 applications the selection marker must be further with later removal of the selection marker and might removed if it contains substances that could cause greatly improve adoptive transfer experiments. clinical complications such as allergic reactions. Reversible T cell staining e.g. using MHC-Strep- - TCR-MHC affinity measurements. Diversity of tagII/Strep-Tactin reagents fulfills all these criteria. epitope-specific T cell populations is reflected on 30 the level of TCR-MHC/peptide binding affinities. - Ex vivo purification of antigen-specific T cell While the measurement of TCR-MHC/peptide bind- populations for "direct adoptive immunothera- ing affinities of T cells is still very difficult, several py". The isolation of antigen-specific T cell popula- recent studies indicate that MHC/peptide dissocia- tions directly ex vivo followed by immediate transfer tion rates correlate with relative binding affinities 35 of the cells into recipients (without any further in vit- (12, 13). With the reagents of the invention, e.g. ro propagation) is of special clinical interest. It is ex- MHC-Strep-tagll/Strep-Tactin reagents, MHC/pep- pected that directly isolated cell populations are tide complexes can be accumulated on the cell sur- much more efficient than cultured cells for in vivo face of epitope-specific T cells and MHC dissocia- applications. Extremely high numbers of in vitro ex- tion rates can be determined after fast monomeri- 40 panded T cells are required for effective adoptive zation by addition of biotin. This is possible because transfers, a phenomenon most likely due to the ad- removal of the Strep-Tactin backbone is significant- aptation of T cells to in vitro culture conditions. An ly faster than MHC dissociation rates. example for an important clinical application for this procedure is the parallel purification and adoptive Clinical applications: 45 transfer of EBV-and/or CMV-specific T cell populations during [otherwise] T cell-depleted stem cell [0032] transplantations, a protocol which is likely to dramatically reduce the incidence of EBV and CMV- - Purification of antigen-specific T cell popula- related malignancies in transplant patients. Revers- tions for highly efficient in vitro expansion. Gen- 50 ible T cell staining and isolation e.g. using MHC- eration of human T cell lines or clones (e.g. patho- Strep-tagII/Strep-Tactin reagents could be an ideal gen/tumor-specific or autoreactive T cells) is nec- method for these clinical applications. essary in many areas of clinical research, diagnostics, and immunotherapy. In vitro culture is often lim- - Functional T cell diagnostics. MHC multimer ited by difficulties in standardizing conditions for an- 55 techniques allow quantification and phenotypic tigen-specific stimulation. Improved strategies for characterization of antigen-specific T cells directly the purification of antigen-specific T cell populations ex vivo. However, binding of multimer reagents to could greatly enhance the efficiency of in vitro ex- the TCR complicates the use of purified cells in

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functional assays (e.g. chronic virus infections [HIV, of protection against Listeria infection was determined CMV, EBV, HBV, HCV], tumor-specific T cell popu- by counting the number of viable bacteria 48 h after in- lations). Reversible T cell staining and isolation e. fection with 5 x LD50 Listeria monocytogenes. g. using MHC-Strep-tagII/Strep-Tactin reagents opens the door for powerful evaluation of antigen- 5 Examples specific T cell status in many clinical situations. 1. Materials and Methods [0033] Further, the present invention is explained in more detail by the following figures and examples. Generation of H2-Kd Strep-tagll fusion proteins [0034] Fig. 1: The binding of monomeric and mul- 10 timeric peptide/MHC conjugates to the TCR of T cells. [0040] The pET3a/H2-Kd expression vector (5) was Due to the binding of the peptide/MHC complex to the mutated by standard PCR techniques to exchange the TCR, the functional status of the T cells is dramatically C-terminal biotinylation site to the Strep-tagII sequence. altered under physiological conditions (prior art). Protein expression was induced in the expression host [0035] Fig. 2: By targeted disruption of peptide/MHC 15 BL21(DE3) by addition of IPTG and subsequently, inclu- multimer binding to the TCR molecules the staining is sion bodies were purified as described before (5). removed resulting in a purified T cell population having substantially unaltered characteristics (invention). In vitro refolding and generation of multimeric MHC [0036] Fig. 3: MHC multimer binding interferes with Strep-tagII reagents 20 functional T cell assays. Listeria (LLO91-99) specific T d cells were incubated with binding (H2-K sStrep-tagII/ [0041] H2-Kd Strep-TagII and mouse β2-microglobu- d LLO91-99/Strep-Tactin polymer) or non binding (H2-K / lin (containing a HSV epitope tag at the C-terminus) in- p60217-225/Strep-Tactin polymer) multimer reagents for clusion bodies were dissolved in 8 M urea and thereafter 60 min at 4°C; for reversible staining cells were subse- refolded by rapid dilution into arginine-rich buffer in the quently incubated and washed in 2 mM d-biotin (2 h at 25 presence of high amounts of synthetic MHC binding 4°C). Subsequently, T cells were tested for [A] cytotox- peptide (here: 0.5 mg/ml LLO91-99, GYKDGNEYI or 3 icity (peptide titration/LLO91-99)or[B] proliferation ( H- p60217-225, KYGVSVQDI, respectively) as described thymidine incorporation; 5 days in the presence of an- previously (5). Soluble MHC complexes were further pu- ti-CD3, antigen-specific, or no (p60217-225) stimulus. rified by gel filtration (Superdex 200HR, Pharmacia) and [0037] Fig. 4: MHC multimer binding interferes with 30 aliquots were stored in liquid nitrogen. For multimeriza- in vivo function of T cells. Listeria (LLO91-99) specific T tion Strep-Tactin polymers were incubated overnight d cells were incubated with binding (H2-K /LLO91-99/ with soluble H2-Kd Strep-tagII/LLO91-99 complexes at a d streptavidin; red) or non binding (H2-K /p60217-225/ molar ratio of 2:1 (2 MHC molecules per 1 Strep-tag streptavidin; black) MHC multimer reagents for 60 min binding site). at 4°C and 1 x 107 cells were adoptively tranferred into 35 naïve recipient BALB/c mice, respectively (no cell trans- Generation and staining of T cell lines, functional assays fer control: white column). The degree of protection against Listeria infection was determined by counting [0042] LLO91-99 specific T cell lines were expanded in the number of viable bacteria 48 h after infection with 5 vitro as described before (18). MHC-multimer staining 40 x LD50 Listeria monocytogenes. was performed by incubation of approximately 2 µg [0038] Fig. 5: Reversible T cell staining using H2-Kd Strep-tagII multimer reagent per 1 x 106 cells at MHC-Strep-tagII Strep-Tactin multimers. Listeria 4°C for 30 min. For epitope-tag staining cells were (LLO91-99) specific T cells were stained with convention- washed, fixed briefly in 1% para formaldehyde, and sub- d al MHC-biotin multimers (H2-K /LLO91-99/streptavidin; sequently stained for anti-HSV tag using the unconju- red) or with MHC-Strep-tagll-Strep-Tactin(polymer) re- 45 gated primary mAb (Novagen) and a secondary anti- agents (left histogram, surface accumulation of MHC is rat-PE antibody. Flow cytometry was performed using a detected by staining for an epitope-tag (HSV) attached FACSCalibur and FlowJo software. For dissociation ex- to the recombinant β2m). The right histogramm shows periments stained cells were washed several times the same cells 30 min after incubation and washing in (usually 10 times) in 1 mM biotin buffer (PBS, 5% FCS). the presence of 1 mM biotin (all done at 4°C). 50 Conventional 51Cr-release and 3H-thymidine incorpo- [0039] Fig. 6: Reversible T cell staining using MHC- ration assays were performed as described before (18). Strep-tagll-Strep-Tactin multimers preserves protective capacity in vivo. Listeria (LLO91-99) specific T cells were Adoptive transfer experiments stained with conventional MHC-Strep-tagII/Strep-Tactin 55 (polymer) reagents and subsequently washed in the [0043] LLO91-99 specific T cells were stained with presence of biotin (1 mM) or buffer alone, respectively H2-Kd Strep-tagII multimer reagents and subsequent (all done at 4°C). 1 x 107 cells were adoptively trans- dissociation by addition of biotin were done as described ferred per recipient (naïve BALB/c mice) and the degree above (control cells were treated equally in the absence

7 13 EP 1 227 321 A1 14 of biotin). Adoptive cell transfer was performed by injec- form tetrameric reagents for antigen-specific T cell tion of 1 x 107 cells - per mouse per group - into the tail staining. Addition of biotin (2-5 mM) results in dis- vein of naive BALB/c mice. 48h after i.v. injection with 5 ruption of MHC-biotin/Strep-Tactin multimers (T1/2 xLD50 Listeria monocytogenes the number of bacteria approx. 10 min at 4°C) and the remaining MHC per organ (spleen and liver) was measured by plating 5 monomers dissociate from the cell surface. This ap- out serial dilutions of tissue homogenate. proach represents an alternative method to MHC-Strep-tagll/Strep-Tactin-polymer reagents for 2. Results reversible T cell staining.

[0044] 10 References

- Conventional prior art MHC multimers bound to the [0045] TCR at 37°C dramatically alter the phenotype and functional status of T cells in vitro (characterized by 1. Miyahira, Y., K. Murata, D. Rodriguez, J.R. Rod- diminished maximum target cell lysis, decreased 15 riguez, M. Esteban, M.M. Rodriguez, and F. Zavala. peptide sensitivity, and proliferation, Fig. 3) and in 1995. Quantification of antigen specific CD8 + T vivo (almost complete loss of the protective capacity cells using an ELISPOT assay. J. Immunol. Meth- of multimer-stained pathogen-specific T cells after ods 181:45-54. adoptive cell transfer Fig. 4). 20 2. Murali-Krishna, K., J.D. Altman, M. Suresh, D.J. - Recombinant MHC-Strep-tagII fusion proteins can D. Sourdive, A.J. Zajac, J.D. Miller, J. Slansky, and be expressed and refolded in vitro. R. Ahmet. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during - Multimeric MHC-Strep-tagII complexes form in the viral infection. Immunity 8:177-187. presence of Strep-Tactin. 25 3. Manz, R., M. Assenmacher, E. Pfluger, S. - Multimeric MHC-Strep-tagII/Step-Tactin molecules Miltenyi, and A. Radbruch. 1995. Analysis and sort- stain T cells, and binding can be disrupted by addi- ing of live cells to secreted molecules, relocated to tion of biotin. a cell-surface affinity matrix. PNAS 92:1921-1925. 30 - With the use of Strep-Tactin-polymers (approx. 56 4. Altman, J.D., P.A.H. Moss, P.J.R. Goulder, D.H. Strep-tagII-biding sites per polymer, obtained from Barouch, M.G. McHeyzer-Williams, J.I. Bell, A.J. IBA (Institut für Bioanalytik/Göttingen/Germany), McMichael, and M.M. Davis. 1996. Phenotypic higher forms of multimeric MHC/peptide complexes analysis of antigen specific T lymphocytes. Science can be generated. These reagents allow epitope- 35 274:94-96. specific T cell staining with intensities comparable to conventional multimers (Fig. 5). Addition of biotin 5. Busch, D.H., I.M. Pilip, S. Vijh, and E.G. Pamer. results in rapid (<5 min) and complete disruption of 1998. Coordinate regulation of complex T cell pop- multimers (performed at 4°C by in the presence of ulations responding to bacterial infection. Immunity 1mM biotin, Fig. 5), and the remaining MHC mono- 40 8:353-362. mers dissociate from the cell surface with slower kinetics (T1/2 approximately 5 min). 6. Pamer, E., and P. Cresswell. 1998. Mechanisms of MHC class I-restricted antigen processing. Annu. - After targeted disruption of surface-bound multim- Rev. Immunol. 16:323-358. ers in the presence of biotin (based on the Strep- 45 TagII/Strep-Tactin interaction) and subsequent dis- 7. McMichael, A., and C. O'Callaghan. 1998. A new sociation of monomers at 4°C, multimer stained T look at T cells. J Exp Med 187:1367-1371. cells are phenotypically and functionally indistin- guishable from untreated T cells in vitro (Figs. 3 + 8. Doherty, P. 1998. The numbers game for virus- 4). It is assumed that disruption and dissociation of 50 specific CD8+ T cells. Science 280:227. surface-bound MHC multimers from T cells also preserve their in vivo functional characteristics after 9. Schmidt, T., and A. Skerra. 1993. The random adoptive transfer; preliminary data indicate that peptide library-assisted engeneering of a C-termi- pathogen-specific T cells are protective after target- nal affinity peptide, useful for the detection and pu- ed disruption and dissociation of MHC multimers 55 rification of a functional Ig Fv fragment. Protein Eng (Fig. 6). 6:109-122.

- Biotinylated MHC molecules bound to Strep-Tactin 10. Schmidt, T., J. Koepke, R. Frank, and A. Skerra.

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1996. Molecular interaction between the strep-tag (i) at least one component specifically bind- affinity peptide and its cognate target, streptavidin. ing to said receptor wherein said at least J Mol Biol 9:753-766. one component is conjugated to at least one first partner of a binding complex, 11. Voss, S., and A. Skerra. 1997. Mutagenesis of 5 (ii) at least one further partner of said bind- a flexible loop in streptavidin leads to higher affinity ing complex having at least two binding for the Strep-tag II peptide and improved perform- sites for said first partner and ance in recombinant protein purification. Protein (iii) a detectable label bound to or capable Eng 10:975-982. of binding to (i) and/or (ii), wherein the first 10 partner(s) and the further partner(s) of the 12. Savage, P.A., J.J. Boniface, and M.M. Davis. binding complex are capable of forming a 1999. A kinetic basis for the T cel receptor repertoire reversible bond and wherein an aggregate selection during an immune response. Immunity 10: comprising (i), (ii) and (iii) is bound via the 485-492. receptor molecule to said cell, wherein said 15 cell is stained, 13. Busch, D.H., and E.G. Pamer. 1998. T cell affinity maturation by selective expansion during in- (c) optionally separating said stained cell from fection. J Exp Med 189:701-709. other components of said sample and (d) optionally removing said staining from said 14. Van der Merwe, P.A., M.H. Brown, S.J. Davis, 20 cell by disrupting the reversible bond. and A.N. Barclay. 1993. Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat 2. A method for reversible staining of T cells compris- CD2 and CD48. EMBO J 12:4945-54. ing the steps:

15. AI-Shamkhani A., S. Mallett, M.H. Brown, W. 25 (a) providing a sample comprising a cell having James, and A.N. Barclay. 1997. Affinity and kinetics a T cell receptor (TCR) molecule, of the interaction between soluble trimeric OX40 lig- (b) contacting said T cell with and, a member of the tumor necrosis family, and its receptor OX40 on activated T cells. J Biol Chem (i) at least one TCR binding ligand compris- 272:5275-82. 30 ing a TCR binding peptide and an MHC molecule conjugated to at least one first 16. Garboczi D.N., D.T. Hung and D.C. Wiley. 1992. partner of a binding complex, and HLA-A2-peptide complexes: refolding and crystalli- (ii) at least one further partner of a binding zation of molecules expressed in Escherichia coli complex having at least two binding sites and complexed with single antigenic peptides. Proc 35 for the first partner of the binding complex Nat/Acad Sci USA 89:3429-3433. and (iii) a detectable label bound to or capable 17. Scott C.A., K.C. Garcia, F.R. Carbone, I.A. Wil- of binding to (i) and/or (ii), wherein the first son, L. Teyton. 1996. Role of chair pairing for the partner(s) and the further partner(s) of the production of functional soluble IA major histocom- 40 binding complex are capable of forming a patibility complex class II molecules. J Exp Med reversible bond and wherein an aggregate 183:2087-2095. comprising (i), (ii) and (iii) is bound via the TCR molecule to said T cell, wherein said 18. Busch D.H., and E.G. Pamer. 1998. MHC class T cell is stained, I/peptide stability: implications for immunodomi- 45 nance, in-vitro proliferation, and diversity of re- (c) optionally separating said stained T cell sponding CTL. J Immunol 160: 4441-4448. from other components of said sample, and (d) optionally removing said staining from the T cell by disrupting the reversible bond. Claims 50 3. The method of claim 1 or 2 wherein step (b), step 1. A method for reversible staining of cells comprising (c) and step (d) are carried out at a temperature of the steps: ≤ 15°C.

(a) providing a sample comprising a cell having 55 4. The method of claim 3 wherein the temperature is a receptor molecule, about 4°C. (b) contacting said cell with 5. The method of any one of claims 1-4 wherein said

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cell is a mammalian T cell. 16. The use of claim 12 for T cell diagnostics.

6. The method of any one of claims 2-5 wherein said 17. Reagent kit for reversible staining of cells compris- MHC molecule is a soluble mammalian MHC mol- ing ecule. 5 (i) at least one component specifically binding 7. The method of any one of claims 1-6 wherein said to a receptor molecule on a cell wherein said at staining is removed by contacting said stained cell least one component is conjugated to at least with a free first partner of said binding complex or one first partner of a binding complex, an analog of said first partner capable of disrupting 10 (ii) at least one further partner of said complex the bond between the conjugated first partner (i) having at least two binding sites for said first and the further partner (ii). partner, and (iii) a detectable lable bound or capable of bind- 8. The method of any one of claims 1-7 wherein the ing to (i) and/or (ii), wherein the first partner(s) first free partner is a compound which is not detri- 15 and the second partner(s) of the binding com- mental to the cell to be stained. plex are capable of forming a reversible bond.

9. The method of any one of claims 1-8 wherein said 18. Reagent kit for reversible staining of T cells com- binding complex is selected from prising 20 (a) (i) biotin and (ii) a streptavidin or avidin an- (i) at least one TCR binding ligand comprising alog capable of reversible binding of biotin, at least an MHC molecule conjugated to at least (b) (i) a biotin analog capable of reversible bind- one first partner of a binding complex, and ing to streptavidin or avidin and (ii) streptavidin (ii) at least one further partner of a binding com- or avidin or a streptavidin or avidin analog ca- 25 plex having at least two binding sites for the first pable of reversible binding of said biotin analog, partner of the binding complex and and (iii) a detectable label bound or capable of bind- (c) (i) a streptavidin or avidin binding peptide ing to (i) and/or (ii), wherein the first partner(s) and (ii) streptavidin or avidin or a streptavidin and the further partner(s) of the binding com- or avidin analog capable of reversible binding 30 plex are capable of forming a high affinity re- of said streptavidin or avidin binding peptide. versible bond.

10. The method of claim 9 wherein said binding com- 19. The kit of claim 18 wherein the TCR binding ligand plex is selected from (i) the streptavidin-binding further comprises a TCR binding peptide. peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys and (ii) 35 the streptavidin-analogs streptavidin Val44-Thr45- 20. The kit of claim 18 wherein a TCR binding peptide Ala46-Arg47) and/or streptavidin Ile44-Gly45-Ala46- having a desired specificity is provided as a sepa- Arg47. rate part.

11. Use of the method of any one of claims 1-10 for the 40 21. The kit of any of claims 17-20 further comprising a isolation of a specific cell population without sub- free first partner of said binding complex or an an- stantially altering the original functional status alog of said first partner capable of disrupting the thereof. bond between the conjugated first partner (i) and the further partner (ii). 12. The use of claim 11 wherein said cell population is 45 an antigen-specific T cell population. 22. Substantially pure antigen-specific T cell population having a functional status which is substantially un- 13. The use of claim 12 for the determination of the altered by the purification. functional status of an antigen-specific T cell population. 50 23. The T cell population of claim 22 obtainable by the method of any one of claims 1-10. 14. The use of claim 12 for the purification of an antigen- specific T cell population for in vitro expansion. 24. A fusion polypeptide comprising (a) at least one streptavidin-binding peptide and (b) an MHC mole- 15. The use of claim 12 for the purification of an antigen- 55 cule. specific T cell population for adoptive transfer or immunotherapy.

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