Selective and controlled expansion of educated NK cells
11471486 · 2022-10-18
Assignee
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
Abstract
The present invention relates generally to immunotherapy. Disclosed herein are methods for obtaining cytolytic differentiated NKG2A.sup.−NKG2C.sup.+ cells with a given KIR specificity and also compositions comprising these cells as well as the use of these cells for therapy. The NK cell expansion methods provided herein also have non-therapeutic uses.
Claims
1. An in vitro method for expanding NK cells of a given KIR specificity, said method comprising: selecting leukocyte cells from a donor, wherein said leukocyte cells comprise educated NK cells of a given KIR specificity, wherein the given KIR specificity is selected with respect to self HLA class I molecules of a patient in need of a therapy; and contacting the leukocyte cells in vitro with an HLA-E molecule in complex with a leader sequence from an HLA-A, HLA-B, HLA-C, or HLA-G molecule, thereby selectively expanding educated NKG2C+/NKB2A− NK cells of the given KIR specificity within said leukocyte cells to produce an expanded cell population having an increased amount or proportion of NKG2C+/NKG2A− NK cells having said given KIR specificity, when compared to the leukocyte cells prior to expansion.
2. The method of claim 1, wherein said method further comprises isolating or enriching for NK cells of said given KIR specificity from the expanded cell population produced.
3. The method of claim 1, wherein the contacting step further comprises contacting the leukocyte cells with one or more cytokines selected from IL-15, IL-12, IL-18, IL-2, IL-7, IL-21, IFN-alpha, or any combination thereof; and/or one or more ligands for stimulating activating KIR expressed on the NK cells in the leukocyte cells.
4. The method of claim 1, wherein the expanded cell population comprises (i) an increased number and percentage of NKG2C+ NK cells; and/or (ii) a limited or inhibited expansion of NKG2A− NK cells.
5. The method of claim 1, wherein the expanded cell population comprising an increased amount or proportion of NKG2C+/NKG2A− NK cells of said given KIR specificity is produced with a higher efficiency than expanding uneducated NK cells.
6. The method of claim 1, wherein the NK cells of said given KIR specificity in the expanded cell population produced have higher cytolytic potential against HLA-mismatched cells, or cells expressing a low or minimal amount of HLA class I molecules; and are potent in mediating antibody-dependent cellular cytotoxicity (ADCC).
7. The method of claim 1, wherein the leukocyte cells from the donor exhibit an increased amount of educated NK cells expressing KIR specific for donor self HLA class I molecules.
8. The method of claim 1, wherein the donor is cytomegalovirus (CMV) seropositive.
9. The method of claim 1, wherein the leukocyte cells from the donor are homozygous for either HLA-C1, HLA-C2, Bw4 or Bw6, or heterozygous for HLA-C1/C2 or Bw6/Bw4.
10. The method of claim 1, wherein the leukocyte cells from the donor are at least partially mismatched at HLA class I to the patient.
11. The method of claim 10, wherein the leukocyte cells from the donor are partially or fully mismatched to the patient at one or more groups of HLA class I alleles selected from HLA-C1, HLA-C2, Bw4 or Bw6.
12. The method of claim 1, wherein the NK cells of said given KIR specificity are potent in mediating antibody-dependent cellular cytotoxicity (ADCC).
13. The method of claim 1, wherein the HLA-E molecule is (i) exogenous; (ii) expressed by a cell; or (iii) attached to a bead, plate, or bag.
14. The method of claim 1, wherein the leader sequence is derived from HLA-A.
15. The method of claim 1, wherein said KIR specificity is selected from: KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, or combinations thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(8) NK cells expanded according to the present invention from a HLA-C1/C1 donor were stimulated with leukemic blast cells from two patients with distinct HLA types. NK cell degranulation was monitored by surface expression of CD107a.
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DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF
(15) KIR/HLA mismatched donors for cancer treatment has been discussed previously in the setting of allogeneic HSCT or adoptive NK cell therapy, where the patient in most instances, following in vivo expansion of NK cells obtain KIR expression patterns similar to those in the donor and this is not until approximately 6 weeks after transplantation/cell therapy. During such in vivo expansion it is not possible to selectively expand the NK cells in a controlled way as the inventors of the present invention propose to do before NK cells are administered to the patient. An in vivo expansion is not a controlled expansion where it is possible to selectively choose the most appropriate NK cells for best treatment.
(16) In vitro expanded NK cells have been used for treatment of hyperproliferative disorders, however these cells were not expanded in the presence of feeder cells expressing HLA-E and consequently such expanded cells will be NKG2A.sup.+. Due to the presence of the inhibitory NKG2A receptor such cells will not be as efficient and potent tumor killing cells as the NK cells presented herein.
(17) WO 99/28748 describes the use of HLA-E for detecting, separating and targeting toxins to CD94/NKG2.sup.+ cells. NK cells are believed to have an anti-tumour cell activity and according to the inventors a marker for progress of therapy, or simple prognosis, can be provided by monitoring NK cell numbers and optionally their state of activation. The invention described in WO 99/28748 provides methods of selecting HLA-E binding NK cells from a mixed cell population. The selected cells can be expanded in vitro and returned to the patient. Such treatment may be effective in some serious infections or cancer where a growth deficiency of these cells is associated with poor prognosis.
(18) Igarashi et al. Enhanced cytotoxicity allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells; Blood 2004 104: 170-177 and WO 2006/050270 describe the effects of KIR ligand mismatched NK cell populations against solid tumors. These publications also describe enriched NK cell populations cloned from the blood of cancer patients or healthy donors homozygous for HLA-C alleles in group 1 or group 2. The strategy proposed in Igarashi et al., involves expansion of NK cells on feeder cells expressing given HLA class I molecules.
(19) Siegler et al. Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients Cytotherapy 2010 12: 750-763 expand alloreactive NK cells of single KIR.
(20) However, the strategy taught by Igarashi et al and Siegler et al. differ from the current invention since their strategy will generate cells that express NKG2A and does not consider the education status of the donor.
(21) HLA-E is a ligand for the activating NK cell receptor NKG2C as well as for the inhibitory NK cell receptor NKG2A. It has previously been suggested that HLA-E interaction with NKG2C might contribute to drive the proliferation of NKG2C.sup.+ cells in CMV infected patients. However HLA-E has not previously been used for controlled selective in vitro expansion of NK cells with a given KIR specificity.
(22) HLA-E binding has previously been used to detect NKG2.sup.+ cells, however this has not been selective for different types of NKG2 cells. In WO 99/28748 HLA-E is used in cancer diagnostics by counting numbers of NK cells but gives the reader no clue about how to use HLA-E for selective expansion of NK cells. In WO 99/28748 they have a mixed population of NKG2C and NKG2A, which is not applicable in this invention where it is important to obtain NKG2A.sup.−NKG2C.sup.+ NK cells. NKG2C acts as an activating receptor and it is therefore desirable with NKG2C positive NK cells in the present method, since this will promote tumor cell killing. NKG2A however is an inhibitory receptor and the inventors of the present invention disclose a strategy for how to select NKG2A negative cells. If both NKG2A and NKG2C are present on the NK cells, NKG2A and the inhibitory effect will be dominating. Therefore it is important to only select NK cells that are NKG2C positive and NKG2A negative. This is only a small subpopulation of the NK cells and the inventors disclose a novel and inventive strategy to select these specific cells and enrich them to high numbers.
(23) No one has hitherto thought of selecting this certain subset of NK cells for this type of therapy, previously donors with a large population of mismatched KIR NK-cells have been sought. The invented method opens up a way of generating such cells from any donor and will generate more controlled expansion of potent NK cells for therapy.
(24) The inventors of the present invention have revealed that NK cell education promote the survival and expansion of NKG2C.sup.+ NK cells in vitro, this is contrary to previous findings presented by Lopéz-Vargas et al. who allege such an expansion would be driven by less constrained uneducated NK cells (lacking inhibitory receptors to self).
(25) The present invention presents a new way of selectively expanding highly cytolytic NK cells of a given KIR specificity. This expansion is personalized to fit the HLA class I type of the patient, thereby promoting NK cell-mediated alloreactivity. Cytolytic NK cells are expanded which are reactive with target cells in the patient to be treated. The method for expanding NK cells presented herein is novel and builds on the discovery that education by self-specific inhibitory KIRs promotes the survival and expansion of NK cells stimulated via NKG2C. The invention is partly counterintuitive since 1) there are conflicting data on the expression of KIRs on NKG2C.sup.+ NK cells and 2) since it involves stimulation of bulk NK cells containing a mixture of NKG2A.sup.+ and NKG2C.sup.+ NK cells where the inhibitory interaction is known to dominate when stimulation occurs on the same cell. In scenarios where HLA-E expression is absent or low, in vitro expansion protocols yield high frequencies of NKG2A.sup.+ NK cells. Hence, the presence of HLA-E in the culture serves two purposes at the same time: 1) stimulates the expansion of NKG2C.sup.+ NK cells expressing self-specific KIRs and 2) inhibit the expansion of NKG2A.sup.+ NK cells with a broader KIR repertoire.
(26) This completely new way of expanding NK cells with given specificity enables more specific and potent tumor cell killing. The NK cells of the present invention can effectively be used in therapy, including but not limited to therapy against cancer, including but not limited to refractory lymphoid malignancies such as malignant lymphoma, adult and childhood acute lymphoid leukemia, since they are selected to have a certain KIR specificity and at the same time are selected to be NKG2A negative and will therefore be very potent tumor killers. Further the use of these expanded NK cells in treatment could fill a gap for patients that are refractory to conventional chemotherapy or in those with indication for allogeneic stem cell transplantation but lacking a stem cell donor or are threatening relapse following cord blood transplantation when no donor lymphocytes are available for donor lymphocyte infusion. Another important application of the cells is in the KIR ligand-matched (including autologous and fully HLA-matched) setting when the scope is to target cells that have low or absent levels of the relevant HLA class I molecule (recognized by the selective KIR.sup.+ expansion). Examples of such settings include but are not limited to metastatic melanoma and neuroblastoma.
(27) CMV Skews Human KIR Repertoires
(28) The inventors of the present invention have determined expression patterns on NK cells in a cohort of 204 healthy donors by 14-color flow cytometry (
(29) Because CMV is known to cause dynamic changes in the NK cell compartment in both mice and humans, the inventors stratified the present cohort on the basis of seropositivity for CMV (
(30) These results reveal the potential of high-resolution KIR phenotyping for tracing adaptation of NK cells to viral infection. Intriguingly, in healthy individuals, the occurrence of NK cell expansion correlated strongly with past CMV infection.
(31) CMV Induces Expansion of NK Cells Expressing Self-Specific KIR
(32) Because most of the expanded subsets expressed at least one HLA-C-binding inhibitory KIR, namely KIR2DL1, KIR2DL2/S2 or KIR2DL3 (
(33) NK Cell Education is Essential for Expansion of NKG2.sup.+ NK Cells In Vitro
(34) To delineate the contribution of NK cell expansion to skewing of KIR repertoires, the inventors stratified HCMV-seropositive donors into those with and without evidence of NK cell expansion. This analysis revealed that the bias for expression of self-specific KIR was more pronounced in donors with NK cell expansion, due to an effect of NK cell expansion on the observed KIR repertoire skewing associated with HCMV (
(35) It is known from before that NK cell education via self-specific KIR tunes the NK cell responsiveness to target cells, including cytolysis and cytokine production. In contrast, previous studies suggest that NK cell education does not influence NK cell responsiveness to cytokines, as measured by proliferation or cytokine production. However, the inventors made the surprising observation that a majority of the NK cells that proliferated extensively in response to 721.221 cells transfected with HLA-E (221.AEH) in IL-15 expressed NKG2C in combination with a self-specific KIR (
(36) These results show that expression of self-specific KIR is a necessary requirement for efficient expansion of NKG2C.sup.+ NK cells in vitro.
(37) The invention herein enables one to rapidly generate NK cells with a given specificity in vitro. These cells may be used for NK cell-based therapy guided by the HLA class I of the patient.
(38) In the present invention NK cells are selectively expanded in vitro to gain NK cells of a given KIR specificity. This in vitro expansion as well as the use of these specific NK cells in therapy is novel.
(39) The donor is selected based on their KIR ligand status. Thus, the donor is selected to have NK cells of a desired, or selected, KIR specificity. In particular, the donor is selected to have NK cells which express KIR (specifically inhibitory KIR) which are responsive to (namely which bind) ligands (HLA class I molecules) which are different to the ligands (HLA class I molecules) provided by the target cells in the patient to be treated. Thus, there is an at least partial mismatch at HLA class I between the donor NK cells and the target cells in the patient. Generally this means that there is a mismatch at HLA class I between donor and patient. However, as noted above, a situation where the KIR ligands for the NK cells do not match the HLA class I of the target cells may also be obtained when the target cells do not express HLA class I molecules. Accordingly in one embodiment the donor is chosen to be mismatching to the recipient. In other embodiments the donor may match the recipient. Major KIR ligands are HLA-C1 (ligand for KIR2DL2/3), HLA-C2 (ligand for KIR2DL1), HLA-Bw4 (ligand for KIR3DL1) and HLA-A3/A11 (ligand for KIR3DL2). A nonlimiting list of suitable mismatches is presented in Table 1. Accordingly, in some embodiments, the donor may be at least partially mismatched to the patient at one or more groups of HLA class I alleles, particularly at HLA-C alleles or HLA-B alleles. As discovered by the inventors of the present invention it is essential to have educated NK cells and this must be considered when selecting a donor, therefore HLA-A3/A11 donors are not considered, since KIR3DL2 NK cells are not educated and will therefore not drive an expansion as described in the present invention and can therefore not be used according to the present invention. This does not exclude that KIR3DL2 is co-expressed on the expanded cells and or contribute to the expansion in yet undisclosed ways. Most often, the protocol involves selection of donors that are homozygous for either HLA-C1 or HLA-C2. However it is also possible to generate cells from HLA-C1/C2 donors.
(40) TABLE-US-00001 TABLE 1 Mismatches according to the present invention Donor's HLA-C group Recipient's HLA-C group C1/C1 C2/C2 Cl/C2 C1/C1 or C2/C2 C2/C2 C1/C1 Donor's HLA-B group Recipient's HLA-B group Bw4/Bw4 Bw6/Bw6 Bw6/Bw4 Bw6/Bw6 Bw6/Bw6 none
(41) Once a donor is selected leukocytes are obtained as known in the art. If desired, when the leukocytes are obtained NK cell isolation is performed as known in the art. For example a system for separating NK cells is available from Miltenyi. The cells can also be expanded directly from peripheral blood mononuclear cells (PBMC) obtained as known in the state of the art. In such applications, NK cell isolation can be done at the end of the cultures as known in the art. Thus, in general the method involves separating or isolating leukocytes from the donor, and optionally isolating NK cells therefrom. The method may be performed on isolated NK cells or on a leukocyte cell fraction or preparation containing NK cells. Separation of NK cells is standard and well known in the art and may be performed by negative and/or positive selection for NK cells, for example using cell markers for NK cells, and/or by depleting other (non-NK) cell types from the cell population. Alternatively, if NK cells are not isolated for carrying out the expansion method, they can be isolated or enriched after the expansion step. For example NK cells may be isolated from the expanded cell population using standard techniques as discussed above. Alternatively, T and/or B cells may be depleted from the expanded cell population, again using standard techniques readily available in the art. Generally speaking isolation of or enrichment for NK cells will be necessary when the donor cells are not matched to the patient HLA class I (i.e. where there is an at least partial HLA class I mismatch between donor and recipient).
(42) The expansion step (step (c)) involves contacting the cells with the NKG2C and/or NKG2A stimulator. It will be understood that this step thus involves contacting (or incubating) the cells under conditions which promote or enable cell expansion. Such conditions (namely conditions required for NK cell expansion) are well known in the art and essentially the invention lies in using and modifying such state of the art conditions, by providing a stimulator of the paired NKG2C and NKG2A receptors, to skew the expansion towards educated NKG2C.sup.+NKG2.sup.− cells of given KIR specificity. As is well known in the art, additional signals for NK cell expansion may be provided through a multitude of receptors, including but not limited to NKG2D, NKp46, CD2, 2B4, DNAM-1 and CD137, or a combination thereof. The stimuli for such receptors may be provided by appropriate feeder cells or by complexes of the stimulatory molecules or receptor ligands e.g. on beads. As noted above, techniques for this are well established.
(43) Leukocytes (e.g. PBMC) or isolated NK cells are cultured with the NKG2C and/or NKG2A stimulator under the appropriate conditions, as mentioned above. Cytokines may additionally be added, including but not limited to IL-5, IL-15, IL-12, IL-18, IL-2, IL-7, IL-21 or IFN-alpha or a combination with one or more of these cytokines. These cytokines or other agents can be used to stimulate proliferation and/or promote apoptosis. In some embodiments the cells are cultured with IL-15.
(44) The NK cells are expanded in the presence of the stimulator by any suitable technical procedure. The stimulator of NKG2C and/or NKG2A may be any ligand, natural or synthetic, that can bind to and stimulate one or both of these receptors or their constituent monomers. In other words, the stimulator is an agonist of the NKG2C and/or NKG2A receptors. Thus, the stimulator may be a ligand or agonist for CD94, or for NKG2C and/or NKG2. As indicated above, the stimulator is preferably the natural ligand HLA-E. As noted above, HLA-E needs to be provided in a form in which it is able to stimulate the cells, and again this is well understood in the art. Thus the HLA-E may be provided in complex with leader sequence peptides. For example leader sequence peptides can be derived from HLA-A, HLA-B, HLA-C or HLA-G molecules. It is known in the art how to insert a sequence encoding the leader peptide into genetic constructs expressing HLA-E so that the HLA-E is expressed in a complex form capable of binding to and stimulating the NKG2C/NKG2A receptors. Cell lines expressing such constructs are known and available, for example cell line 221AEH used in the Examples below. Such cell lines may be used as feeder cells to provide HLA-E to the donor NK cells.
(45) In other embodiments, HLA-E multimers e.g. tetramers or pentamers, may be used, together with any relevant peptide that provides a signal to CD92/NKG2A and/or CD92/NKG2C. Such multimers may be coated onto to an appropriate solid support e.g. beads, or plastic supports or vessels e.g, plates or bags, to provide the desired combined stimulation/inhibition effects, as described above.
(46) In yet other embodiments the stimulator may be an antibody which binds to one or more of CD94, NKG2C or NKG2A. It will be understood that this will be an agonistic antibody, namely an antibody which is capable of stimulating the receptor. Combinations of such antibodies may be used, for example an antibody which binds to NKG2C may be used together with an antibody that binds to NKG2A. Preferably the antibody is anti-NKG2C, or anti-NKG2C together with anti-NKG2A.
(47) Thus the step of expanding the NK cells may be achieved by methods including but not limited to a) using a feeder cell line engineered by different means to express HLA-E (including but not limited to 721.221.HLA-E or K562-HLA-E, or variants of these cell lines modified to express (or overexpress) agonists stimulating for example IL-15 and/or IL-21 receptors, or ligands for activating receptors such as PVR, CD48 or ICAM-1) or b) plates or bags coated in soluble HLA-E complexes alone or in combination with cytokine receptor complexes or c) plates or bags coated with anti-NKG2C mAbs and/or anti-CD94 and/or anti-NKG2C and/or anti-NKG2A mAbs or d) beads coated with soluble HLA-E complexes and/or anti-CD94 and/or anti-NKG2C and/or anti-NKG2A mAbs. The HLA-E construct used in the embodiments described in a-b uses the generic molecule or a modified construct with improved stability and/or binding to the CD94/NKG2A/C molecules based on changes in the leader sequence peptides. These include but are not limited to HLA molecules coupled to the HLA-A and HLA-G leader and peptide modifications thereof. Leukocyts (PBMC) or isolated NK cells can be cultured under conditions known in the art and as described herein.
(48) The term “antibody” as used herein refers to any type of antibody, or any antibody fragment or derivative. For example, the antibody may be polyclonal or monoclonal. The antibody may be of a single specificity. The antibody may be of any convenient or desired species, class or sub-type. Furthermore, the antibody may be natural, derivatised or synthetic. The term antibody as used herein thus includes all types of antibody molecules and antibody fragments.
(49) More particularly the “antibody” may be:
(50) (a) any of the various classes or subclasses of immunoglobulin e.g. IgG, IgA, IgM, IgD or IgE derived from any animal e.g. any of the animals conventionally used e.g. sheep, rabbits, goats, or mice or egg yolk
(51) (b) monoclonal or polyclonal antibodies
(52) (c) intact antibodies or fragments of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody e.g. fragments devoid of the Fc portion (e.g. Fab, Fab', F(ab′)2, Fv), the so called “half molecule” fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody. Fv may be defined as a fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
(53) (d) antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, fragments of antibodies, humanised antibodies, chimeric antibodies, or synthetically made or altered antibody-like structures. Also included are functional derivatives or “equivalents” of antibodies e.g. single chain antibodies. A single chain antibody may be defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a fused single chain molecule. Also included are single chain (Sv) intrabodies.
(54) Methods of making such antibody fragments and synthetic and derivatised antibodies are well known in the art. Also included are antibody fragments containing the complementarity-determining regions (CDRs) or hypervariable regions of the antibodies. These may be defined as the region comprising the amino acid sequences on the light and heavy chains of an antibody which form the three dimensional loop structure that contributes to the formation of the antigen binding site. CDRs may be used to generate CDR-grafted antibodies. As used herein “CDR grafted” defines an antibody having an amino acid sequence in which at least parts of one or more sequences in the light and/or variable domains have been replaced by analogous parts of CDR sequences from an antibody having a different binding specificity for a given antigen. One of skill in the art can readily produce such CDR grafted antibodies using methods well known in the art.
(55) A chimeric antibody may be prepared by combining the variable domain of an antibody of one species with the constant regions of an antibody derived from a different species. The expanded cells can then be infused to patients to be treated e.g. patients with cancer, infectious diseases, immunodeficient patients or patients with autoimmune diseases and since the donors are selected in such way that the transfer of NK cells are performed over HLA barriers this will trigger alloreactivity. The cells can also be infused in HLA-matched settings provided that the relevant HLA class I allele is low or absent. Cells can be infused using any suitable method known in the art or as described herein.
(56) Making the transfer in conjunction with therapeutic mAbs can further enhance the desired cellular specificity. Suitable mAbs include but are not limited to Rituximab (anti-CD20), GA 101 (anti-CD20), mabcampath (anti-CD52), mylotarg, and cetuximab. Genetically modifying the expanded cell product can further enhance the desired specificity. Suitable modifications include but are not limited to expression of chemokine receptors such as CXCR5, CCR5 and CCR2 and chimeric antigen receptors such as CD19 linked to CD3zeta and 41-BB and TcRs specific for tumor antigens including but not limited to CD20.
(57) Another embodiment involves the co-expression of activating KIRs on the expanded NK cells. The inventors have successfully expanded NK cells expressing for example KIR2DL3 (recognizing HLA-C1) together with the activating KIR2DS1 (recognizing HLA-C2). These cells can be generated with the here described invention and display highly potent killing of HLA mismatched HLA-C2/C2 leukemic cells. In one embodiment, the protocol is combined with ligands for the activating KIR (including but not limited to HLA-C2) to further stimulate expansion of such cells.
(58) In one embodiment such NK cells expressing a desired combination of inhibitory and activating KIRs are infused to patients patients with cancer, infectious diseases, immunodeficient patients or patients with autoimmune diseases and since the donors are selected in such way that the transfer of NK cells are performed over HLA barriers this will trigger alloreactivity.
(59) The NK cells obtained by the present method have a customized KIR specificity, based on the selected donor, and the majority of the cells lack the NKG2A inhibitory receptor (NKG2A.sup.−NKG2C.sup.+). When the in vitro expanded NK cells are infused to a patient they will be highly cytolytic since the cells have a customized KIR specificity, are NKG2C positive, lack the inhibitory receptor NKG2A and are differentiated.
(60) When stimulating NK cells with HLA-E this will generate a proliferation that promotes cells lacking NKG2A and at the same time only educated, potent tumor killing, NK cells will proliferate. This is a new way of expanding effective NK cells.
EXAMPLE 1
In Vitro Expansion of NK Cells
(61) Isolated NK cells obtained from donor are cultured together with IL-15 and the feeder cell line 721.221 cells transfected with a hybrid HLA-E containing the HLA-A2 signal sequence (221.AEH) (Lee et al., 1998). NK cells are cultured at a density of 2-10×10.sup.6 cells/ml for 12-14 days in X-vivo 15 (BioWhittaker) supplemented with IL-15 and 5% human AB sera at 5% CO.sub.2, 37° C. (35-38° C.).
EXAMPLE 2
The Specificity of the Expanded Cells
(62) In the example shown in
EXAMPLE 3
Expanded Cells Display a Differentiated Phenotype and Have High Cytolytic Potential
(63) Expansion of NK cells according to example 1 is associated with NK cell differentiation, a process during which the cells accumulate effector molecules such as Perforin and Granzyme B, become more responsive to target cell stimulation and highly cytolytic against numerous tumor targets. The cells also become very potent in mediating ADCC against antibody-coated cancer cells (
EXAMPLE 4
Expansion of NK Cells under GMP Conditions Yield High Number of Cells that Recognize Mismatched Leukemic Blasts
(64) Apheresis of the donor is performed to obtain large number of leukocytes. NK cell isolation is performed in a GMP laboratory using kit and device from Miltenyi. The isolated cells are treated as in example 1. The expanded NKG2C.sup.+ KIR2DL3 single positive NK cells display specific killing against mismatched HLA-C2/C2 blasts but not against matched HLA-C1/C1 blasts (
EXAMPLE 5
(65) Cells expanded according to example 1 are formulated in physiological salt solution supplemented with 5% human serum albumin and filled in a transfer bag for delivery to the clinic immediately upon release. The cells are infused to a patient suffering from acute lymphoid leukemia. The NKG2A.sup.−NKG2C.sup.+ NK cells with given KIR specificity enhance tumor specificity in this treatment.
EXAMPLE 6
Selective and Controlled Expansion of Educated NK Cells
(66) NK cells were isolated and co-cultured with irradiated 721.221 cells, transfected with HLA-E and the HLA-A leader sequence (221.AEH) in IL-15 for 14 days. Representative FACS plots showing the KIR repertor in NK cells from (A) a C1/C1 donor and (B) a C2/C2 donor at day 0 and day 14 are shown in
(67) This Figure (in particular the middle panel in (B)) illustrates how the protocol generates NK cells with self-specific KIRs. Such cells will be highly specific against HLA-mismatched targets or targets lacking HLA class I.
EXAMPLE 7
Specificity and Efficacy of the Expanded NK Cells
(68) NK cell-mediated killing was assessed in a flow cytometry-based assay by monitoring caspase-3 activity and staining of a live/dead cell marker (DCM) in PHA blasts following co-incubation with NK cells at E:T ratio 5:1 for 4 hours.
(69) This Figure illustrates the specificity of the expanded NK cells. Relative to other strategies to expand NK cells, the invented protocol generates cells that display high specificity against matched targets. Due to the unique moulding of the KIR repertoire during culture in combination with the preferential expression of the activating NKG2C rather than the inhibitory NKG2A, the generated cells are highly effective against mismatched targets with high expression of both HLA class I and HLA-E.
EXAMPLE 8
Selectively Expanded NK Cells Efficiency Lyse Primary Blasts from Children with Acute Lymphoblastoid Leukemia
(70) The expanded NK cells were tested against primary ALL blasts from 24 children in a FACS-based killing assay at an E:T ratio of 5:1 for 4 hours.
(71) This Figure illustrates the efficacy of the expanded NK cells against primary lymphoid leukemia blasts. This is particularly relevant, since ALL cells have been shown to be resistant to NK cells in general and to NK92 cells (the cells sold by CoNKwest) in particular.
EXAMPLE 9
Targeting of High-Risk Myeloid Malignancies
(72) NK cell-mediated killing was assessed in a flow cytometry-based assay by monitoring caspase-3 activity and staining of a live/dead cell marker (DCM) in CD34+ blasts following co-incubation with NK cells at E:T ratio 5:1 for 4 hours.
EXAMPLE 10
Targeting of Malignant Melanoma
(73) Expanded NK cells with two distinct specificities were tested in a standard Cr51-release assay against a range of melanoma cell lines obtained from ATCC (see
EXAMPLE 11
Selective Targeting of HLA-C2/C2 Targets by KIR2DL3+KIR2DS1+ NK Cells
(74) NK cell-mediated killing was assessed in a flow cytometry-based assay by monitoring caspase-3 activity and staining of a live/dead cell marker (DCM) in ALL blasts following co-incubation with NK cells at E:T ratio 5:1 for 4 hours.