Cytotherapy, 2015; 0: 1e5
Challenges of cancer therapy with natural killer cells
HANS KLINGEMANN Tufts University Medical School and Conkwest Inc, Cambridge, Massachusetts, USA Abstract Background aims. Natural killer (NK) cells from peripheral or cord blood—especially if they are obtained from a human leukocyte antigenemismatched (allogeneic) donor—are increasingly being considered for treatment of malignant diseases and to prevent or treat relapse after stem cell transplant. However, in addition to proving their efficacy, there are some more logistical and technical issues that must be addressed before NK cell infusions will be fully accepted by the medical community. Methods. Issues include (i) the expansion of sufficient numbers of cells under conditions suitable, (ii) cryopreservation and (iii) optimization/standardization of shipping conditions if the cells are used at distant sites. Because the patient’s own autologous cells usually are not fully functional because of inhibition by “self” major histocompatibility complex expression, better methods must be developed to target NK cells to tumor cells and overcome self-inhibition. Results. Tumor-directed NK-cell therapy can be best accomplished through genetic engineering of NK cells expressing receptors for tumor antigens or combination with monoclonal antibodies that preferentially kill tumors through antibody-dependent cellular cytotoxicity. If allogeneic NK cells are used, T-lymphocytes in the cell collections that can cause acute graft-versushost disease in the recipient must be removed. Conclusions. In addition to showing efficacy in clinical trials, the production of NK cells for treatment must be cost-effective to be eligible for reimbursement by third-party players. Key Words: cancer, CAR, cell expansion, cell therapy, cryopreservation, natural killer cells
Introduction For decades, natural killer (NK) NK cells existed as “non-specific” killer cells in the shadow of T cells. Recent discoveries that better explain how NK cells recognize and kill their targets and their ability to produce immune-active cytokines have made them more attractive tools for immunotherapy. NK cells are considered part of the innate immune system, able to respond quickly to “invaders” without a “priming” period as required for T cells. They are operationally defined by their morphology (large granular lymphocytes) and surface marker expression (CD56þ/CD3). Only approximately 10% of all lymphocytes in the peripheral blood are NK cells, which pales in comparison to the number of T-lymphocytes, which is usually in the range of 50e70%. Unfortunately, the cancer patient’s own (autologous) NK cells, even when they are activated with cytokines, are often dysfunctional and compromised by chemotherapy. In addition, we have learned that NK cells are trained to recognize “nonself” histocompatibility antigens (human leukocyte antigen, HLA) on the surface of cells through their
killer cell immunoglobulin-like (KIR) receptors. NK cell activation is blocked through engagement of their KIR receptors when they encounter self (autologous) major histocompatibility complex (MHC) antigens [1]. Unless MHC antigens are mutated or missing on cancer cells, autologous NK cells will not recognize malignant cells. Consequently, clinical trials of infusions of the patient’s own (autologous) NK cells have not shown any clinical benefit [2,3]. The only way to overcome this deficiency of autologous NK cells is to engineer them to express a tumor antigenerecognizing receptor (ie, chimeric antigen receptor [CAR]), which, on engagement, will override inhibitory signals. However, this has proven to be challenging because the transfection efficiency of blood NK cells even with viral vectors is low [4]. Allogeneic KIR-mismatched NK cells, on the other hand, will recognize the discordant HLA antigen pattern on host cells and consequently will not be de-activated. The potential benefit of those KIRmismatched NK cells became evident when the bone marrow transplant team in Perugia analyzed their
Correspondence: Hans Klingemann, MD, PhD, Conkwest Inc at LabCentral, 700 Main Street, Cambridge, MA 02139, USA. E-mail: hans.klingemann@ tufts.edu (Received 15 April 2014; accepted 16 September 2014) http://dx.doi.org/10.1016/j.jcyt.2014.09.007 ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
2
H. Klingemann
Table I. Challenges of cell therapy with NK cells. Unmodified autologous NK cells lack anti-tumor efficacy Yield of NK cells in leukaphereses collections is highly donor-dependent Allogeneic collections require removal of T cells to prevent graft-versus-host-disease Potential risk of hemolysis and EBV: lymphoma if B cells remain and T cells are removed Variable expansion of blood NK cells to clinical scale NK cells are sensitive to cold storage and lose some cytotoxicity after cryopreservation If product is used at distant facility: adhere to defined shipping conditions Need improved transfection efficiency to genetically engineer NK cells Costs of generating the product
outcome data on MHC haplotypeemismatched stem cell transplants for leukemia and noticed that patients with AML whose donors were KIR-mismatched had a lower relapse rate [5]. This finding was confirmed in some larger retrospective analyses [6,7]. Moreover, two additional single-arm trials in Minnesota and Memphis in which allogeneic NK cells were given to AML patients suggested a clinical benefit [8,9]. For allogeneic NK cell therapy to be effective, it is important to select the appropriate donor who is not only mismatched at the KIR loci, but, as recent data have shown, also has the appropriate KIR allelic polymorphism, which may consist of only one amino acid difference. To identify such a “perfect” KIRmismatched donor requires sequencing of the KIR locus. This adds costs to the process, but the group at Memphis has shown that this extra effort seems to be paying off: some patients with active leukemia could be induced into a remission after KIR-selected NK cell infusions (Leung, oral communication, 2013). However, matching for the right KIR on NK cells is not sufficient to guarantee an anti-tumor effect. NK cells also express activating receptors that must “connect” with appropriate ligands on tumor cells that may not always be present or may be mutated. Although is appears attractive to develop allogeneic NK cells for therapeutic infusions, this poses a different problem: the allogeneic T-lymphocytes in the cell collection can cause acute graft-versus-hostdisease that can be responsible for significant morbidity and mortality. Hence, T cells must be removed before the cell product is infused into the recipient. This can be accomplished with the use of anti-CD3 monoclonal antibodies that are conjugated to an iron particle, which is then passed over a magnetic column resulting in the removal of the T cells (CliniMACS, Miltenyi Biotech). Such a T-cell depletion step usually enriches— depending on the donor—NK cells to approximately
20e40% [10,11]. Some investigators may decide to use a second immunomagnetic column to specifically enrich for CD56þ NK cells (positive selection). This can result in some cell loss, and there is some suggestion that these highly enriched NK cell preparations actually may have less activity against cancer because they miss accessory cells such as monocytes that can support NK cell function [11]. However, there are some advantages of further enriching CD56þ NK cells beyond just T-cell depletion. The B-lymphocytes that are left behind after T-cell depletion, especially from blood group Oþ donors, can cause hemolysis in immunecompromised recipients who carry red cells of group A or group B (“passenger lymphocyte syndrome”). There have also been occasional reports on EBVdriven lymphomas originating from B-lymphocytes that (because of T-cell depletion) can proliferate unchecked [11]. Both complications can be prevented either by positive CD56 selection or by an additional B-cell depletion. Expansion of NK cells The yield of blood NK cells from a single-donor leukapheresis that has been T-lymphocyteedepleted is highly variable, and the numbers are not sufficient for therapeutic infusions [11,12]. Hence, expansion and activation of NK cells becomes necessary, a process that usually takes 2e3 weeks of culture of NK cells in the presence of cytokines such as interleukin (IL)-2 that will also activate the NK cells. A feeder layer can optimize the expansion process, and the use of irradiated EBV-transformed lymphoblastic cell line or K562 cells engineered to express IL-15 or IL-21 along with an adhesion molecule [I-BB4] has resulted in more predictable expansion of blood NK cells [13], although the problem of donor variability with respect to cell numbers still remains an issue. Furthermore, these cell collections must be depleted of T cells, which is best done before culture expansion. Because K562 is a malignant cell line, quality control assays before infusion must document complete removal of these cells before infusion into a patient. Expansion of NK cells can be performed in a variety of vessels or bioreactors. In addition to Teflon bags (Vuelife by Afc) or flasks (Wilson Wolf Manufacturing), continuous-flow devices (Zellwerk), stirred-tank bioreactors (Eppendorf) and fully automated devices are under development such as the Miltenyi’s Prodigy system. For any cell therapy to be accepted in the clinical setting, costs must be contained and efforts are directed toward expansion of large numbers of cells on a smaller footprint with less culture medium. This seems to be best accomplished
Challenges of cancer therapy with NK cells with the use of stirred bioreactors that would allow continuous production. Those expanded cells should then be cryopreserved in aliquots with sufficient numbers for treating patients on demand. Another cost factor involves cytokines that may be necessary for the expansion process. For most protocols, IL-2 is required and sufficient, but expansion protocols that use cord blood CD34 progenitor cells may require additional cytokines such as Flt-3, IL-3 and stem cell factor [14]. How many cells are necessary for clinical application? The obvious answer may be “the more the better.” However, it is difficult to perform a dose escalation with human cells because there are technical limitations in expansion of sufficient numbers of NK cells that show dose-limiting side effects. An average human has about 2e3 billion NK cells in the blood. One would think that a cell dose in this range, probably given multiple times, would be sufficient, but there are no hard data to back such a theory.
Cryopreservation The logistics of distributing NK cells for therapy to distant places and in an “on-demand” manner would be greatly facilitated through cryopreservation of expanded NK cells. They could then be thawed at the treatment site, similar to cord blood stem cells that are shipped frozen. The thawing step and the quality control assays (viability, sterility, flow cytometry for CD107 as a marker of function) can be performed by the receiving blood center. In addition to simplifying the shipping process and having the cells available in an “on-demand” manner, batch production would significantly lower costs over the costs of having a continuous expansion protocol running, which will inevitably create unused, leftover cellular product. Quality tests can also be more economically done on batch-expanded cells. In contrast to T cells and many other human cells, NK cells, especially when they have been activated by cytokines, are more sensitive to freezing and thawing. In addition to variable viability on thawing [15], the cytotoxicity is severely reduced immediately after thawing [16]. For the remaining/surviving cells to regain their pre-freeze numbers and cytotoxic activity, it will take several days of culture in IL-2. Although overnight culture in IL-2 can restore cytotoxic activity, this test only reflects the activity of the surviving fraction—usually less than half of the input number of cells. Investigators have tested different conditions and variables such as various concentrations of dimethyl sulfoxide, controlled-rate freezing and different techniques of thawing the cells, but progress has been moderate.
3
Cytokine-activated NK cells are also quite sensitive to exposure to lower temperatures. For example, when the cells are stored overnight in the refrigerator, they may maintain viability, but their cytotoxicity is significantly reduced [15]. In contrast to frozen/ thawed cells though, the cells regain activity rather quickly after culture in IL-2 at 37 C for approximately 12 h (Tonn, oral communication, 2014).
Shipping of NK cells To keep the costs for cellular therapeutics reasonable, it is expected that cells will be prepared in centralized facilities and shipped to the point of service. A recent clinical trial supported by PACT (Production Assistance for Cell Therapy) tested the feasibility of such an approach [12]. Allogeneic blood mononuclear cells were collected by means of leukapheresis in Boston and flown to Minnesota for T-cell depletion, after which the NK celleenriched product was returned to Boston for infusion into recipients of an autologous stem cell transplant. The study confirmed the feasibility of shipping cells safely but also confirmed earlier observations that the NK cell number after CD3 depletion is highly variable and donor-dependent, ranging from 10e40%. Because of the temperature dependence of NK cells, the T-celledepleted product on its way from Minnesota to Boston was kept close to body temperature. Thermal sensors documented that the temperature had dropped only marginally, with no loss of NK cell cytotoxicity. For this particular clinical trial, the NK celleenriched cell product was also placed in IL-2 to maintain NK cell activity. In addition to shipping temperature, the right cell density is important to maintain their activity. At higher cell concentrations, the cells lose their activity, probably because of a more rapid utilization of medium and changes in pH and glucose (Lapteva, oral communication, 2014). Their functional status after shipping can be improved by dilution and replenishment on arrival, with additional medium and IL-2 overnight on arrival. Because in most cases no further manipulation of the cell product should occur at the treatment site to keep the “minimal manipulation” status of a product, shipping at higher density is not a practical option. Other factors that can affect the quality of the product can be difficult to control once the product has left the processing facility (flight delays, custom issues). We recently encountered a small leakage from a bag after an intercontinental flight. We had not considered that the remaining air in the bag can expand when transported in the cargo compartment at an altitude of 35,000 feet.
4
H. Klingemann
Immuno-engineering of NK-92 Although NK cells have spontaneous cytotoxicity against malignant cell lines, it is well known that primary malignant cells from cancer patients can be resistant to their cytotoxicity even in the KIR-mismatched allogeneic situation, which can have different etiologies. One way to overcome this resistance is to engineer the NK effector cells with a specific receptor that recognizes and binds directly to a cancer surface antigen. These CARs have been successfully transfected into T-lymphocytes with some remarkable responses in patients with advanced leukemia [17,18]. However, in contrast to T cells, blood-derived NK cells are more difficult to transfect. Physical methods such as electroporation and lipid-based methods usually result in
Challenges of cancer therapy with natural killer cells
HANS KLINGEMANN Tufts University Medical School and Conkwest Inc, Cambridge, Massachusetts, USA Abstract Background aims. Natural killer (NK) cells from peripheral or cord blood—especially if they are obtained from a human leukocyte antigenemismatched (allogeneic) donor—are increasingly being considered for treatment of malignant diseases and to prevent or treat relapse after stem cell transplant. However, in addition to proving their efficacy, there are some more logistical and technical issues that must be addressed before NK cell infusions will be fully accepted by the medical community. Methods. Issues include (i) the expansion of sufficient numbers of cells under conditions suitable, (ii) cryopreservation and (iii) optimization/standardization of shipping conditions if the cells are used at distant sites. Because the patient’s own autologous cells usually are not fully functional because of inhibition by “self” major histocompatibility complex expression, better methods must be developed to target NK cells to tumor cells and overcome self-inhibition. Results. Tumor-directed NK-cell therapy can be best accomplished through genetic engineering of NK cells expressing receptors for tumor antigens or combination with monoclonal antibodies that preferentially kill tumors through antibody-dependent cellular cytotoxicity. If allogeneic NK cells are used, T-lymphocytes in the cell collections that can cause acute graft-versushost disease in the recipient must be removed. Conclusions. In addition to showing efficacy in clinical trials, the production of NK cells for treatment must be cost-effective to be eligible for reimbursement by third-party players. Key Words: cancer, CAR, cell expansion, cell therapy, cryopreservation, natural killer cells
Introduction For decades, natural killer (NK) NK cells existed as “non-specific” killer cells in the shadow of T cells. Recent discoveries that better explain how NK cells recognize and kill their targets and their ability to produce immune-active cytokines have made them more attractive tools for immunotherapy. NK cells are considered part of the innate immune system, able to respond quickly to “invaders” without a “priming” period as required for T cells. They are operationally defined by their morphology (large granular lymphocytes) and surface marker expression (CD56þ/CD3). Only approximately 10% of all lymphocytes in the peripheral blood are NK cells, which pales in comparison to the number of T-lymphocytes, which is usually in the range of 50e70%. Unfortunately, the cancer patient’s own (autologous) NK cells, even when they are activated with cytokines, are often dysfunctional and compromised by chemotherapy. In addition, we have learned that NK cells are trained to recognize “nonself” histocompatibility antigens (human leukocyte antigen, HLA) on the surface of cells through their
killer cell immunoglobulin-like (KIR) receptors. NK cell activation is blocked through engagement of their KIR receptors when they encounter self (autologous) major histocompatibility complex (MHC) antigens [1]. Unless MHC antigens are mutated or missing on cancer cells, autologous NK cells will not recognize malignant cells. Consequently, clinical trials of infusions of the patient’s own (autologous) NK cells have not shown any clinical benefit [2,3]. The only way to overcome this deficiency of autologous NK cells is to engineer them to express a tumor antigenerecognizing receptor (ie, chimeric antigen receptor [CAR]), which, on engagement, will override inhibitory signals. However, this has proven to be challenging because the transfection efficiency of blood NK cells even with viral vectors is low [4]. Allogeneic KIR-mismatched NK cells, on the other hand, will recognize the discordant HLA antigen pattern on host cells and consequently will not be de-activated. The potential benefit of those KIRmismatched NK cells became evident when the bone marrow transplant team in Perugia analyzed their
Correspondence: Hans Klingemann, MD, PhD, Conkwest Inc at LabCentral, 700 Main Street, Cambridge, MA 02139, USA. E-mail: hans.klingemann@ tufts.edu (Received 15 April 2014; accepted 16 September 2014) http://dx.doi.org/10.1016/j.jcyt.2014.09.007 ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
2
H. Klingemann
Table I. Challenges of cell therapy with NK cells. Unmodified autologous NK cells lack anti-tumor efficacy Yield of NK cells in leukaphereses collections is highly donor-dependent Allogeneic collections require removal of T cells to prevent graft-versus-host-disease Potential risk of hemolysis and EBV: lymphoma if B cells remain and T cells are removed Variable expansion of blood NK cells to clinical scale NK cells are sensitive to cold storage and lose some cytotoxicity after cryopreservation If product is used at distant facility: adhere to defined shipping conditions Need improved transfection efficiency to genetically engineer NK cells Costs of generating the product
outcome data on MHC haplotypeemismatched stem cell transplants for leukemia and noticed that patients with AML whose donors were KIR-mismatched had a lower relapse rate [5]. This finding was confirmed in some larger retrospective analyses [6,7]. Moreover, two additional single-arm trials in Minnesota and Memphis in which allogeneic NK cells were given to AML patients suggested a clinical benefit [8,9]. For allogeneic NK cell therapy to be effective, it is important to select the appropriate donor who is not only mismatched at the KIR loci, but, as recent data have shown, also has the appropriate KIR allelic polymorphism, which may consist of only one amino acid difference. To identify such a “perfect” KIRmismatched donor requires sequencing of the KIR locus. This adds costs to the process, but the group at Memphis has shown that this extra effort seems to be paying off: some patients with active leukemia could be induced into a remission after KIR-selected NK cell infusions (Leung, oral communication, 2013). However, matching for the right KIR on NK cells is not sufficient to guarantee an anti-tumor effect. NK cells also express activating receptors that must “connect” with appropriate ligands on tumor cells that may not always be present or may be mutated. Although is appears attractive to develop allogeneic NK cells for therapeutic infusions, this poses a different problem: the allogeneic T-lymphocytes in the cell collection can cause acute graft-versus-hostdisease that can be responsible for significant morbidity and mortality. Hence, T cells must be removed before the cell product is infused into the recipient. This can be accomplished with the use of anti-CD3 monoclonal antibodies that are conjugated to an iron particle, which is then passed over a magnetic column resulting in the removal of the T cells (CliniMACS, Miltenyi Biotech). Such a T-cell depletion step usually enriches— depending on the donor—NK cells to approximately
20e40% [10,11]. Some investigators may decide to use a second immunomagnetic column to specifically enrich for CD56þ NK cells (positive selection). This can result in some cell loss, and there is some suggestion that these highly enriched NK cell preparations actually may have less activity against cancer because they miss accessory cells such as monocytes that can support NK cell function [11]. However, there are some advantages of further enriching CD56þ NK cells beyond just T-cell depletion. The B-lymphocytes that are left behind after T-cell depletion, especially from blood group Oþ donors, can cause hemolysis in immunecompromised recipients who carry red cells of group A or group B (“passenger lymphocyte syndrome”). There have also been occasional reports on EBVdriven lymphomas originating from B-lymphocytes that (because of T-cell depletion) can proliferate unchecked [11]. Both complications can be prevented either by positive CD56 selection or by an additional B-cell depletion. Expansion of NK cells The yield of blood NK cells from a single-donor leukapheresis that has been T-lymphocyteedepleted is highly variable, and the numbers are not sufficient for therapeutic infusions [11,12]. Hence, expansion and activation of NK cells becomes necessary, a process that usually takes 2e3 weeks of culture of NK cells in the presence of cytokines such as interleukin (IL)-2 that will also activate the NK cells. A feeder layer can optimize the expansion process, and the use of irradiated EBV-transformed lymphoblastic cell line or K562 cells engineered to express IL-15 or IL-21 along with an adhesion molecule [I-BB4] has resulted in more predictable expansion of blood NK cells [13], although the problem of donor variability with respect to cell numbers still remains an issue. Furthermore, these cell collections must be depleted of T cells, which is best done before culture expansion. Because K562 is a malignant cell line, quality control assays before infusion must document complete removal of these cells before infusion into a patient. Expansion of NK cells can be performed in a variety of vessels or bioreactors. In addition to Teflon bags (Vuelife by Afc) or flasks (Wilson Wolf Manufacturing), continuous-flow devices (Zellwerk), stirred-tank bioreactors (Eppendorf) and fully automated devices are under development such as the Miltenyi’s Prodigy system. For any cell therapy to be accepted in the clinical setting, costs must be contained and efforts are directed toward expansion of large numbers of cells on a smaller footprint with less culture medium. This seems to be best accomplished
Challenges of cancer therapy with NK cells with the use of stirred bioreactors that would allow continuous production. Those expanded cells should then be cryopreserved in aliquots with sufficient numbers for treating patients on demand. Another cost factor involves cytokines that may be necessary for the expansion process. For most protocols, IL-2 is required and sufficient, but expansion protocols that use cord blood CD34 progenitor cells may require additional cytokines such as Flt-3, IL-3 and stem cell factor [14]. How many cells are necessary for clinical application? The obvious answer may be “the more the better.” However, it is difficult to perform a dose escalation with human cells because there are technical limitations in expansion of sufficient numbers of NK cells that show dose-limiting side effects. An average human has about 2e3 billion NK cells in the blood. One would think that a cell dose in this range, probably given multiple times, would be sufficient, but there are no hard data to back such a theory.
Cryopreservation The logistics of distributing NK cells for therapy to distant places and in an “on-demand” manner would be greatly facilitated through cryopreservation of expanded NK cells. They could then be thawed at the treatment site, similar to cord blood stem cells that are shipped frozen. The thawing step and the quality control assays (viability, sterility, flow cytometry for CD107 as a marker of function) can be performed by the receiving blood center. In addition to simplifying the shipping process and having the cells available in an “on-demand” manner, batch production would significantly lower costs over the costs of having a continuous expansion protocol running, which will inevitably create unused, leftover cellular product. Quality tests can also be more economically done on batch-expanded cells. In contrast to T cells and many other human cells, NK cells, especially when they have been activated by cytokines, are more sensitive to freezing and thawing. In addition to variable viability on thawing [15], the cytotoxicity is severely reduced immediately after thawing [16]. For the remaining/surviving cells to regain their pre-freeze numbers and cytotoxic activity, it will take several days of culture in IL-2. Although overnight culture in IL-2 can restore cytotoxic activity, this test only reflects the activity of the surviving fraction—usually less than half of the input number of cells. Investigators have tested different conditions and variables such as various concentrations of dimethyl sulfoxide, controlled-rate freezing and different techniques of thawing the cells, but progress has been moderate.
3
Cytokine-activated NK cells are also quite sensitive to exposure to lower temperatures. For example, when the cells are stored overnight in the refrigerator, they may maintain viability, but their cytotoxicity is significantly reduced [15]. In contrast to frozen/ thawed cells though, the cells regain activity rather quickly after culture in IL-2 at 37 C for approximately 12 h (Tonn, oral communication, 2014).
Shipping of NK cells To keep the costs for cellular therapeutics reasonable, it is expected that cells will be prepared in centralized facilities and shipped to the point of service. A recent clinical trial supported by PACT (Production Assistance for Cell Therapy) tested the feasibility of such an approach [12]. Allogeneic blood mononuclear cells were collected by means of leukapheresis in Boston and flown to Minnesota for T-cell depletion, after which the NK celleenriched product was returned to Boston for infusion into recipients of an autologous stem cell transplant. The study confirmed the feasibility of shipping cells safely but also confirmed earlier observations that the NK cell number after CD3 depletion is highly variable and donor-dependent, ranging from 10e40%. Because of the temperature dependence of NK cells, the T-celledepleted product on its way from Minnesota to Boston was kept close to body temperature. Thermal sensors documented that the temperature had dropped only marginally, with no loss of NK cell cytotoxicity. For this particular clinical trial, the NK celleenriched cell product was also placed in IL-2 to maintain NK cell activity. In addition to shipping temperature, the right cell density is important to maintain their activity. At higher cell concentrations, the cells lose their activity, probably because of a more rapid utilization of medium and changes in pH and glucose (Lapteva, oral communication, 2014). Their functional status after shipping can be improved by dilution and replenishment on arrival, with additional medium and IL-2 overnight on arrival. Because in most cases no further manipulation of the cell product should occur at the treatment site to keep the “minimal manipulation” status of a product, shipping at higher density is not a practical option. Other factors that can affect the quality of the product can be difficult to control once the product has left the processing facility (flight delays, custom issues). We recently encountered a small leakage from a bag after an intercontinental flight. We had not considered that the remaining air in the bag can expand when transported in the cargo compartment at an altitude of 35,000 feet.
4
H. Klingemann
Immuno-engineering of NK-92 Although NK cells have spontaneous cytotoxicity against malignant cell lines, it is well known that primary malignant cells from cancer patients can be resistant to their cytotoxicity even in the KIR-mismatched allogeneic situation, which can have different etiologies. One way to overcome this resistance is to engineer the NK effector cells with a specific receptor that recognizes and binds directly to a cancer surface antigen. These CARs have been successfully transfected into T-lymphocytes with some remarkable responses in patients with advanced leukemia [17,18]. However, in contrast to T cells, blood-derived NK cells are more difficult to transfect. Physical methods such as electroporation and lipid-based methods usually result in
