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Early successes drive the need to overcome safety issues, increase efficacy, and address manufacturing challenges.
Numerous, remarkable results in early clinical trials have driven significant investment in cell therapies, both by large biopharmaceutical companies and startup biotech firms backed by venture capital. Jain PharmaBiotech identified more than 500 companies involved in cell-therapy technology (1).
Adoptive cell transfer (ACT), which uses a patient’s T cells (T lymphocytes) that are harvested and genetically engineered to produce chimeric antigen receptors (CARs) and recognize specific proteins (antigens) on tumor cells, is receiving a good portion of that attention. The CAR-T cells are expanded and then reinfused back into the patient, where they multiply and attack the targeted cancer cells.
There are challenges to ACT using T cells. T cell expansion and the persistence of infused cells can vary significantly from patient to patient, and both directly influence the treatment outcome. Conditioning of the patient in advance of infusion can have an impact, as can the tumor microenvironment. Selection of the most effective, longest-lasting T cells and the right antigen targets is a key focus of research efforts today. On-target, off-tumor toxicity and cytokine release syndrome are important safety issues that must be addressed. Technologies for the manufacture of therapies based on living cells on a commercial scale must also be developed.
T cells are ideal vehicles for immunotherapy because they are central to cell-mediated immunity and are involved in long-term, antigen-specific responses. They have, in fact, been used in the past to treat various viral infections. Once T cells bind to cells expressing the target antigen, they acquire the specific functional properties necessary for eliminating the target cells and generate long-lasting memory T cells that provide a similar response if any target cells reappear. CAR-T cell therapy leverages these natural behaviors of T cells. T cells can also be engineered to express modified T-cell receptors (TCRs) as an alternative type of cell therapy.
Autologous vs. allogeneic
CAR-T cell therapies in clinical development today are largely autologous therapies; the genetically modified cells originate from tissue taken from an individual patient and are returned to that patient once expanded. There is some concern in the industry that the need to perform the same level of extensive quality control and testing on such small-scale product lots as for large-scale production will prevent these treatments from achieving commercial viability. Starting material variability for autologous CAR-T products also presents a challenge; the T cells from each patient differ depending upon the extent of their disease, previous therapies, genetics, and the status of their immune system at the time of cell collection. “These patient-specific issues will always be a challenge to the ability to manufacture a CAR-T product for every patient and to the consistency of performance observed among CAR-T products,” says Derek Jantz, chief scientific officer for gene editing company Precision Biosciences.
Allogeneic cell therapies, in which the T cells are derived from healthy donors that have been screened for desirable characteristics rather than individual patients, have the potential to allow for larger-scale manufacturing and minimize the heterogeneity associated with using raw material from individual patients. “In addition to transduction to introduce the CAR, gene editing using CRISPR/cas9 (EDITAS, Intellia), TALEN (Cellectis), ZFN (Sangamo), or homing endonucleases such as ARCUS (Precision Biosciences) is also necessary to knock out expression of the endogenous T-cell receptor,” says Jantz. “Such gene-edited allogeneic CAR-T cells would have significantly less potential to cause graft-versus-host disease upon adoptive transfer to the patient and can be reproducibly manufactured without the variability inherent in the manufacturing of an autologous CAR-T product,” he adds. These CAR-T products could also be manufactured at large scale and stored frozen, ready to be delivered to the patient when needed.
One downside of allogeneic therapies is the need for the additional gene editing step, which reduces somewhat the economic advantage that may be achieved due to larger-scale manufacturing. In addition, while off-the-shelf allogeneic treatment products would be available for treating patients immediately, given that autologous therapies are now produced in 2-3 weeks, timeliness of delivery is not an issue in most cases, according to Hyam “Hy” Levitsky, executive vice-president and chief scientific officer of Juno Therapeutics. He also notes that the expansion of T cells is limited. “In order to make a quantity of an allogeneic product sufficient to treat large numbers of patients, there is a concern that the extensive expansion needed will rapidly lead to senescence of the allogeneic T cells such that when infused into patients, their ability to further expand would be limited,” he says.
Finally, even though editing the T-cell receptors on allogeneic CAR-T cells will reduce the risk of graft-versus-host disease, they are still foreign cells and prone to be rapidly rejected by the host immune system, which is much less of an issue with autologous therapies. Furthermore, once rejected, there is no possibility of delivering a second dose. The question of how long these cells persist following administration to the patient must be addressed in clinical trials of allogeneic CAR-T products. “Important issues to be determined in clinical trials are whether destruction of allogeneic CAR-T cells occurs in a time frame and to an extent that limits anti-tumor activity and the number of observed complete and durable responses,” Bruce McCreedy, senior vice-president of cell therapy at Precision BioSciences states.
Better cell selection
“Over the past ten years there has been a tremendous increase in our understanding of how T cells function and how the immune system regulates itself. This knowledge has now enabled us to develop highly effective T-cell-based immunotherapies,” Levitsky observes. He adds that one of the key challenges is to identify and select the most effective T-cell subsets to develop into efficacious therapies. “From an efficacy perspective, the main challenges continue to be expansion and persistence of CAR-T cells following administration to the patient and activity of the CAR-T cells within the tumor microenvironment where numerous immunosuppressive factors are at work,” agrees Jantz.
Some companies are moving toward manufacturing schemes in which a defined mix of CD4+ and CD8+ CAR-T cells with naïve and memory phenotypes (i.e., not terminally differentiated and exhausted cells that do not expand well and persist following administration) are represented in the final product.
Strep-tag technology (2) developed by researchers at the Fred Hutchinson Cancer Research Center, Technical University of Munich, and San Raffaele Scientific Institute in Milan looks like a promising approach to the problem. The small protein tag can be used to separate out T cells carrying a CAR protein to yield highly pure samples that can then be expanded to provide more potent therapies with high regenerative potential in less time than is needed for mixed cell samples. In addition, the researchers have shown that by using a special antibody that binds the Strep-tag, engineered cells can be rapidly and repeatedly expanded. The Strep-tag when used in combination with a different antibody may also serve as a “kill switch” if cytokine release syndrome (CRS) or other toxic events occur. Once infused into patients, T cells with the Strep-tag can also be tracked using a fluorescent antibody specific for the tag.
Juno Therapeutics, which funded the work at the Hutchinson Center, has an exclusive license to the tag technology for uses related to oncology (as well as a non-exclusive license for other purposes). “We have a significant program focused on the development of technology for the physical selection of specific cells in order to generate defined cell products. These investments are allowing us to select and steer cells at the early manufacturing stage,” Levitsky states.
Another approach involves the engineering of “armored” CAR-T cells that are genetically modified to express a pro-inflammatory cytokine (e.g., interleukin 12, IL-12) in addition to the CAR. The localized secretion of IL-12 recruits help from other immune cells and supports the activity of CAR-T cells within the immunosuppressive tumor microenvironment, according to McCreedy. In addition, replacement of the murine scFv (tumor targeting portion of the CAR that is exposed on the outer surface of the cell) with human sequences that do not negatively impact the binding affinity of the scFv is expected to reduce the frequency of patient immune responses directed against CAR-T cells and hopefully improve their persistence. Gene-editing technologies are also being employed to genetically modify CAR-T cells in ways that render them more capable of trafficking to tumor sites and make them less susceptible to immunosuppression within the tumor microenvironment.
Manufacturing of CAR-T cell therapies involves multiple steps, including collection of the raw material, separation of the T cells, transduction with a viral vector (typically gammaretrovirus or lentivirus) to introduce the CAR receptor and other genetic modifications, expansion of the engineered cells, cryopreservation, and eventual infusion into the patient. While effective small-scale bioprocessing methods have been developed to meet the product needs for early-phase clinical trials, because these treatments are based on living cells (and thus the cells must be isolated as the product, not a recombinant protein), larger-scale manufacturing presents unique challenges. “An incredibly high level of organization and standardization of processes are both essential,” Levitsky notes.
In a poster presented at the American Society of Hematology Annual Meeting in December 2015, Novartis reported on how it has successfully transferred cell processing technology from the University of Pennsylvania to the company’s cell manufacturing center in Morris Plains, NJ (3). Novartis was the first healthcare company to initiate Phase II CAR-T therapy trials in the United States, Europe, Canada, and Australia, and the manufacturing facility now supports their global clinical trial program, according to a company spokesperson.
Commercial-scale cell-therapy production processes must be designed as cost-effective, closed manufacturing systems that are flexible, yet meet cGMP manufacturing requirements, and allow the use of simple techniques for cell recovery on a large scale. Cell expansion is particularly challenging at larger scales because cell culture must be achieved while maintaining the phenotype and function of the cells. At small scale, 2D culture processes are widely used and understood. They are not suitable, however, for the production of trillions of cells, which may be the typical lot size for allogeneic therapies. For autologous treatments, however, planar technologies using adherent 2D culture flasks, multilayer vessels, or multiplate bioreactors may be sufficient. Advances in the automation of these systems can be advantageous as well.
Suspension on microcarriers using 3D culture in typical bioreactors is the most likely way forward for the large-scale expansion of allogeneic CAR-T cells. The challenge is to choose a microcarrier with the appropriate surface characteristics and to establish the optimum microcarrier concentration, cell seeding density, media, and shear conditions for each cell system. The use of microcarriers is attractive at large scale because they provide greater surface area to volume for higher cell densities, and because the expansion can be performed in traditional bioreactors, control of various process parameters is possible.
Harvesting of the cells from the microcarriers is typically achieved via treatment with an enzyme, although some microcarriers are being developed that allow non-enzymatic removal. Once harvested, a volume reduction step is performed, followed by product filling. Development of effective methods for the reduction of larger volumes (< 5-10 liters) is a focus area for many companies, with tangential flow filtration (TFF) and single-use fluidized-bed centrifugation two technologies of interest.
In fact, disposable systems are highly preferred for CAR-T and other cell-therapy production processes due to the need for low-cost, flexible, closed systems that minimize contamination. McCreedy notes that several systems are in development that can separate desired cells (e.g., via elutriation or magnetic beads coated with antibodies), electroporate and/or transduce cells, wash, resuspend, and culture large numbers of cells, including removal of spent media and addition of fresh media that is designed to stimulate the proliferation and expansion of CAR-T cells with specific desired phenotypes. “Such instrumentation to automate the process and minimize the space required in a manufacturing facility should have a positive impact by increasing the consistency and reducing the cost of GMP manufacture of cell therapies,” he states.
The logistics involved in autologous CAR-T cell therapies are often raised as an important issue, but Levitsky believes they are an engineering problem that is not without technical challenges, but certainly not the biggest challenge facing developers of these next-generation treatments. Eventually, he believes it may even be possible to have CAR-T cell therapies produced at the hospital using automated instrumentation that can perform all of the necessary steps. “Such a solution is not out of the realm of possibility; there is nothing to indicate it can’t be done,” Levitsky asserts.
It is also important to note that CAR-T products require the separate manufacture of viral vectors for delivery of the CAR transgene in addition to cell expansion and harvesting. “Challenges associated with process development and validation include establishment of transduction conditions that reproducibly result in an acceptable percentage of T cells that express the CAR at defined levels on the T cell surface,” says McCreedy.
Product Characterization and Release Testing Issues
Product characterization and release testing present additional challenges to GMP manufacturing of CAR-T products. The creation of master cell banks from customized cell lines that express specific ligands and/or reporter molecules for use in expanding CAR-T cells in culture and for use in characterizing the potency and specificity for release of GMP-manufactured CAR-T products are beginning to make their way into the manufacturing process, which should help to provide additional consistency in the process, according to McCreedy.
Because CAR-T products are considered to be both cellular and gene therapy by FDA and the European Medicines Agency, genetic stability studies are required in addition to traditional stability upon storage documentation. The required test for replication-competent lentivirus is particularly onerous due to the cost and time required. Alternatives for delivery of genetic data that avoid the use of viral vectors are in development, such as the introduction of the CAR as a transiently expressing messenger RNA (mRNA), plasmid DNA transfection, and the use of transposable elements (transposons) to replace existing genes with new ones, according to Levitsky. He also believes it is possible that as the field matures and experience with CAR-T cell therapies increases, there will eventually no longer be a need for the test.
More on cell therapy:
Advancing cell therapy safetyCell therapy growth and pains: Investment, collaboration, and controversyRole of contract manufacturing in cell therapy development and manufacturing
1. Jain PharmaBiotech, Cell Therapy-- Technologies, Markets, and Companies (March 2016), accessed April 12, 2016.
2. R. Tompa, Crafting a Better T Cell for Immunotherapy (Feb. 22, 2016), accessed April 18, 2016.
3. J.A. Boyd, et al., Successful Translation of Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Cell Processing Technology from Academia to Industry, Poster, American Society of Hematology 57th Annual Meeting (Orlando, FL, December 2015).
Article DetailsBioPharm International
Vol. 29, No. 5
Citation: When referring to this article, please cite it as C. Challener, "Realizing the Potential of CAR-T Cell Therapies," BioPharm International 29 (5) 2016.
"Kama matokeo ya mafanikio ya mapema, ""kuendesha haja ya kushinda masuala ya usalama, kuongeza ufanisi, na kushughulikia changamoto za utengenezaji.""" Matokeo mengi ya mapema ya kliniki yamesababisha uwekezaji mkubwa katika tiba za seli, na kampuni kubwa za dawa za kibiolojia na kampuni za teknolojia ya kibiolojia zinazoanzishwa na mtaji wa uwekezaji. Jain Pharma Biotech imegundua kampuni zaidi ya 500 zinazohusika katika teknolojia ya tiba ya seli. Uhamisho wa chembe za kuhamisha (ACT) - ambayo hutumia chembe za T za mgonjwa (T lymphocytes) zilizokusanywa na kubuniwa kwa maumbile ili kutengeneza receptors za antigen (CARs) na kutambua protini maalum (antigens) kwenye seli za uvimbe - hupokea sehemu nzuri ya umakini huo. Chembe za CAR-T huongezeka na kisha kuingizwa tena ndani ya mgonjwa, ambapo huongezeka na kushambulia chembe za kansa zilizolengwa. Kuna changamoto za kutumia ACT kwa kutumia chembe za T. Kuongezeka kwa seli za T na kudumu kwa seli zilizopitishwa kunaweza kutofautiana sana kutoka kwa mgonjwa hadi mgonjwa, na zote mbili zinaathiri moja kwa moja matokeo ya matibabu. Hali ya mgonjwa kabla ya infusion inaweza kuwa na athari, kama vile microenvironment ya uvimbe. Uchaguzi wa seli za T zenye ufanisi zaidi, zenye kudumu kwa muda mrefu na malengo sahihi ya antigen ni lengo kuu la juhudi za utafiti leo. On- lengo, off- uvimbe sumu na cytokine kutolewa syndrome ni masuala muhimu ya usalama ambayo lazima kushughulikiwa. Teknolojia za utengenezaji wa tiba kulingana na chembe hai kwa kiwango cha kibiashara lazima pia zitengenezwe. Chembe za T ni vifaa bora kwa ajili ya immunotherapy kwa sababu wao ni muhimu kwa kinga ya seli-kusaidiwa na ni kushiriki katika muda mrefu, antigen-specific majibu. Kwa kweli, zimetumiwa zamani kutibu maambukizo mbalimbali ya virusi. Mara baada ya chembe za T kuunganisha kwa chembe zinazosema antigen ya lengo, zinapata mali maalum za kazi zinazohitajika kwa kuondoa chembe za lengo na kuzalisha chembe za T za kumbukumbu za muda mrefu ambazo hutoa majibu sawa ikiwa chembe yoyote ya lengo itaonekana tena. Tiba ya CAR-T cell hutumia tabia hizi za asili za seli za T. Chembe za T zinaweza pia kubuniwa ili kuelezea receptors za chembe za T zilizobadilishwa (TCRs) kama aina mbadala ya tiba ya seli. Matibabu ya autologous vs. allogeneic CAR-T cell katika maendeleo ya kliniki leo ni matibabu ya autologous; seli zilizobadilishwa kwa maumbile zinatokana na tishu zilizochukuliwa kutoka kwa mgonjwa mmoja na zinarudi kwa mgonjwa huyo mara moja. Kuna wasiwasi katika sekta ya kwamba haja ya kufanya kiwango sawa cha udhibiti wa ubora na majaribio ya kina juu ya bidhaa ndogo kama vile kwa uzalishaji wa kiwango kikubwa itazuia matibabu haya kufikia ufanisi wa kibiashara. Uwezekano wa kutofautiana kwa vifaa vya kuanza kwa bidhaa za autologous CAR-T pia ni changamoto; chembe za T kutoka kwa kila mgonjwa hutofautiana kulingana na kiwango cha ugonjwa wao, matibabu ya awali, urithi, na hali ya mfumo wao wa kinga wakati wa ukusanyaji wa seli. "Hizi masuala ya mgonjwa maalum daima kuwa changamoto kwa uwezo wa kutengeneza CAR-T bidhaa kwa kila mgonjwa na kwa uthabiti wa utendaji aliona kati ya CAR-T bidhaa,""anasema Derek Jantz, afisa mkuu wa kisayansi kwa ajili ya kampuni ya kurekebisha jeni Precision Biosciences." Tiba ya seli ya allogenic, ambayo seli za T zinatokana na wafadhili wenye afya ambao wamechunguzwa kwa sifa zinazohitajika badala ya wagonjwa binafsi, zina uwezo wa kuruhusu utengenezaji wa kiwango kikubwa na kupunguza kutofautiana kwa kutumia malighafi kutoka kwa wagonjwa binafsi. "Kwa kuongezea transduction kuanzisha CAR, uhariri wa jeni kwa kutumia CRISPR (EDITAS, Intellia), TALEN (Cellectis), ZFN (Sangamo), au endonucleases kama vile ARCUS (Precision Biosciences) pia ni muhimu kwa knock nje ya kujieleza kwa receptor ya endogenous T-cell, ""Jantz anasema." "Sehemu hizo za CAR-T zilizohaririwa kwa jeni zitakuwa na uwezo mdogo sana wa kusababisha ugonjwa wa graft-versus-host wakati wa uhamisho wa kupitishwa kwa mgonjwa na zinaweza kutengenezwa kwa njia ya kurudia bila tofauti ya asili katika utengenezaji wa bidhaa ya CAR-T ya autologous", anaongeza. Vifaa hivi vya CAR-T vinaweza pia kutengenezwa kwa kiwango kikubwa na kuhifadhiwa kufungia, tayari kutolewa kwa mgonjwa wakati inahitajika. Upungufu mmoja wa matibabu ya allogenic ni haja ya hatua ya ziada ya uhariri wa jeni, ambayo hupunguza faida ya kiuchumi ambayo inaweza kupatikana kwa sababu ya utengenezaji mkubwa. "Kwa kuongezea, wakati bidhaa za matibabu ya allogeneic zitapatikana kwa kutibu wagonjwa mara moja, ikizingatiwa kwamba matibabu ya autologous sasa hutengenezwa katika wiki 2-3 ""wakati wa utoaji sio suala katika kesi nyingi,"" kulingana na Hyam Levitsky, makamu wa rais mtendaji na afisa mkuu wa kisayansi wa Juno Therapeutics." Pia anasema kwamba ukuzi wa chembe za T ni mdogo. "Kwa hivyo, ili kufanya kiasi cha bidhaa ya allogenic ya kutosha kutibu idadi kubwa ya wagonjwa, kuna wasiwasi kwamba upanuzi mkubwa unaohitajika utaongoza kwa haraka kwa uzee wa seli za T za allogenic, kwa hivyo wakati wa kuingizwa kwa wagonjwa, uwezo wao wa kupanua zaidi ungekuwa mdogo. """ Hatimaye, hata kama kuhariri receptors T-chembe juu ya allogeneic CAR-T seli kupunguza hatari ya graft-kuhusu-mwalimu ugonjwa, bado ni seli za kigeni na uwezekano wa kuwa haraka kukataliwa na mfumo wa kinga mwenyeji, ambayo ni kidogo sana ya suala na matibabu autologous. Kwa kuongezea, mara tu ikikataliwa, hakuna uwezekano wa kutoa kipimo cha pili. Swali la muda gani seli hizi kubaki baada ya utoaji kwa mgonjwa lazima kushughulikiwa katika majaribio ya kliniki ya bidhaa allogeneic CAR-T. "Maswali muhimu ya kuamua katika majaribio ya kliniki ni kama uharibifu wa seli za CAR-T za allogeneic hutokea katika muda na kwa kiwango ambacho hupunguza shughuli ya kupambana na uvimbe na idadi ya majibu kamili na ya kudumu yaliyoonekana, ""Bruce McCreedy, makamu mkuu wa rais wa tiba ya seli katika Precision Biosciences." Uchaguzi bora wa seli <unk> Katika miaka kumi iliyopita kumekuwa na ongezeko kubwa katika uelewa wetu wa jinsi seli za T zinavyofanya kazi na jinsi mfumo wa kinga unavyojiendesha. Ujuzi huu sasa umetusaidia kuendeleza immunotherapies yenye ufanisi wa seli za T, <unk> Levitsky anaona. "Anasema, ""Mmoja wa changamoto kuu ni kutambua na kuchagua seti ndogo za seli za T zenye ufanisi zaidi ili kuendeleza tiba zenye ufanisi.""" "Kutoka kwa mtazamo wa ufanisi, changamoto kuu zinaendelea kuwa upanuzi na kudumu kwa seli za CAR-T baada ya utoaji kwa mgonjwa na shughuli za seli za CAR-T ndani ya mazingira madogo ya uvimbe ambapo sababu nyingi za kinga zinafanya kazi, ""Yantz anakubaliana." Kampuni zingine zinaelekea kwenye mipango ya utengenezaji ambapo mchanganyiko maalum wa CD4 + na CD8 + CAR-T seli na naive na kumbukumbu phenotypes (yaani, seli zisizo tofauti na zilizopunguzwa ambazo hazina kupanuka vizuri na kudumu baada ya utoaji) zinawakilishwa katika bidhaa ya mwisho. Teknolojia ya Streptococcus, iliyoundwa na watafiti wa Kituo cha Utafiti wa Saratani cha Fred Hutchinson, Chuo Kikuu cha Ufundi cha Munich, na Taasisi ya Sayansi ya San Raffaele huko Milan, inaonekana kuwa njia ya kuahidi ya kutatua tatizo hilo. "Tag ndogo ya protini inaweza kutumika kutenganisha seli za T zinazoendesha protini ya CAR ili kutoa sampuli safi sana ambazo zinaweza kupanuliwa kutoa matibabu yenye nguvu zaidi na uwezo wa juu wa kuzaliana kwa muda mfupi kuliko inahitajika kwa sampuli za seli mchanganyiko. """ Kwa kuongezea, watafiti wameonyesha kwamba kwa kutumia kinga maalum ambayo huunganisha Strep-tag, seli zilizobuniwa zinaweza kupanuliwa haraka na mara kwa mara. Strep-tag wakati kutumika katika mchanganyiko na kinga tofauti inaweza pia kutumika kama <unk>kill switch<unk> kama cytokine kutolewa syndrome (CRS) au matukio mengine ya sumu kutokea. Mara baada ya kuingizwa kwa wagonjwa, seli za T zilizo na Strep-tag zinaweza pia kufuatiliwa kwa kutumia antibody ya fluorescent maalum kwa tag. Juno Therapeutics, ambayo ilifadhili kazi katika Kituo cha Hutchinson, ina leseni ya kipekee kwa teknolojia ya tag kwa matumizi yanayohusiana na oncology (kama vile leseni isiyo ya kipekee kwa madhumuni mengine). <unk>Tuna mpango mkubwa umezingatia maendeleo ya teknolojia kwa ajili ya uteuzi wa kimwili wa seli maalum ili kuzalisha bidhaa maalum seli. Uwekezaji huu unaturuhusu kuchagua na kuongoza seli katika hatua ya mapema ya utengenezaji, <unk> Levitsky anasema. Njia nyingine inahusisha uhandisi wa "bodi" CAR-T seli ambazo ni genetically iliyopita kueleza pro-uchochezi cytokine (kwa mfano, interleukin 12, IL-12) pamoja na CAR. Utoaji wa IL-12 huchukua msaada kutoka kwa seli zingine za kinga na inasaidia shughuli za seli za CAR-T ndani ya mazingira madogo ya uvimbe wa kinga, kulingana na McCreedy. Kwa kuongezea, kubadilisha scFv ya panya (sehemu ya tumor-targeting ya CAR iliyofunuliwa kwenye uso wa nje wa seli) na mfululizo wa binadamu ambao hauathiri vibaya uhusiano wa scFV, inatarajiwa kupunguza mzunguko wa majibu ya kinga ya mgonjwa dhidi ya seli za CAR-T na kwa matumaini kuboresha uvumilivu wao. Teknolojia za uhariri wa jeni zinatumiwa pia kurekebisha chembe za CAR-T kwa njia ambazo zinawafanya waweze kusafirishwa kwa maeneo ya uvimbe na kuwafanya wawe chini ya unyogovu wa kinga ndani ya mazingira madogo ya uvimbe. Utaratibu wa matibabu ya seli za CAR-T una hatua nyingi, ikiwa ni pamoja na kukusanya malighafi, kutenganisha seli za T, kuingiza na vector ya virusi (kwa kawaida gamma-retrovirus au lentivirus) kuanzisha receptor ya CAR na marekebisho mengine ya urithi, upanuzi wa seli za uhandisi, cryopreservation, na infusion ya mwisho kwa mgonjwa. Ingawa mbinu za ufanisi za bioprocessing za kiwango kidogo zimetengenezwa ili kukidhi mahitaji ya bidhaa kwa majaribio ya kliniki ya hatua ya mapema, kwa sababu matibabu haya yanategemea seli za kuishi (na hivyo seli lazima zitenganishwe kama bidhaa, sio protini ya recombinant), utengenezaji wa kiwango kikubwa una changamoto za kipekee. "Kiwango cha juu sana cha shirika na standardization ya michakato ni wote muhimu", Levitsky anasema. Katika mkutano wa kila mwaka wa American Society of Hematology, Novartis iliripoti juu ya jinsi ilivyoshinda kuhamisha teknolojia ya usindikaji wa seli kutoka Chuo Kikuu cha Pennsylvania hadi kituo cha utengenezaji wa seli ya kampuni huko Morris Plains, NJ. Novartis ilikuwa kampuni ya kwanza ya huduma za afya kuanzisha majaribio ya awamu ya pili ya matibabu ya CAR-T nchini Marekani, Ulaya, Canada na Australia, na kituo cha utengenezaji sasa inasaidia mpango wao wa majaribio ya kliniki ya kimataifa. Mchakato wa uzalishaji wa tiba ya seli ya kiwango cha kibiashara lazima uendelezewe kama mifumo ya uzalishaji iliyofungwa yenye ufanisi wa gharama nafuu ambayo ni rahisi, lakini inakidhi mahitaji ya utengenezaji wa cGMP na inaruhusu matumizi ya mbinu rahisi za kupona kwa seli kwa kiwango kikubwa. Upanuzi wa seli ni changamoto hasa katika viwango kubwa kwa sababu utamaduni wa seli lazima kupatikana wakati kudumisha phenotype na kazi ya seli. Katika kiwango kidogo, michakato ya utamaduni wa 2D hutumiwa sana na kueleweka. Hata hivyo, hazifai kutengeneza trilioni za chembe, ambazo huenda zikawa kiasi cha kawaida cha matibabu ya allogeneic. Kwa matibabu ya autologous, hata hivyo, teknolojia za planar zinazotumia flasks za utamaduni wa 2D, vyombo vya tabaka nyingi, au bioreactors za multiplate zinaweza kuwa za kutosha. Maendeleo katika automatisering ya mifumo hii inaweza kuwa na faida pia. Suspension juu ya microcarriers kutumia 3D utamaduni katika bioreactors kawaida ni njia ya uwezekano zaidi mbele kwa ajili ya upanuzi wa kiwango kikubwa cha allogeneic CAR-T seli. Changamoto ni kuchagua microtanker na sifa sahihi ya uso na kuanzisha mkusanyiko bora wa microtanker, cell seeding density, vyombo vya habari, na hali ya shear kwa kila mfumo wa seli. Kwa sababu microtankers ni rahisi kutumia, kwa sababu wao kutoa zaidi uso eneo kwa kiasi kwa ajili ya wingi wa seli, na kwa sababu upanuzi inaweza kufanyika katika bioreactors jadi, udhibiti wa vigezo mbalimbali mchakato ni inawezekana. Utoaji wa seli kutoka kwa microtissues kwa kawaida hupatikana kupitia matibabu na enzyme, ingawa baadhi ya microtissues zinaendelezwa ambazo huruhusu kuondolewa kwa non-enzymatic. Mara baada ya kuvuna, hatua ya kupunguza kiasi hufanywa, ikifuatiwa na kujaza bidhaa. Maendeleo ya mbinu za ufanisi kwa ajili ya kupunguza kiasi kikubwa (< 5-10 liters) ni eneo la lengo kwa makampuni mengi, na tangential mtiririko filtration (TFF) na matumizi ya mara moja fluidized-kitanda centrifugation teknolojia mbili ya maslahi. Kwa kweli, mifumo ya kutumiwa mara moja ni bora sana kwa CAR-T na michakato mingine ya uzalishaji wa tiba ya seli kutokana na haja ya mifumo ya gharama nafuu, rahisi, iliyofungwa ambayo hupunguza uchafuzi. "McCready anasema kwamba mifumo kadhaa ni katika maendeleo ambayo inaweza kutenganisha seli zinazohitajika (kwa mfano, kupitia elutriation au beads magnetic coated na antibodies), electroporate na au transduce seli, kuosha, resuspend, na utamaduni idadi kubwa ya seli, ikiwa ni pamoja na kuondoa vyombo vya habari kutumika na kuongeza vyombo vya habari safi ambayo ni iliyoundwa kuchochea kuenea na upanuzi wa CAR-T seli na maalum ""phenotypes.""" "Vifaa kama hivyo vya kuendesha mchakato na kupunguza nafasi inayohitajika katika kituo cha utengenezaji inapaswa kuwa na athari nzuri kwa kuongeza uthabiti na kupunguza gharama ya utengenezaji wa GMP wa tiba za seli", anasema. "Logistics inayohusika katika matibabu ya autologous CAR-T cell mara nyingi huinuliwa kama suala muhimu, lakini Levitsky anaamini ni tatizo la uhandisi ambalo sio bila changamoto za kiufundi, lakini hakika sio changamoto kubwa inayokabiliwa na watengenezaji wa matibabu haya ya kizazi kijacho. """ "Mwishowe, anaamini kuwa inaweza kuwa na uwezekano wa kuwa na matibabu ya seli za CAR-T zinazozalishwa katika hospitali kwa kutumia vifaa vya kiotomatiki vinavyoweza kufanya hatua zote muhimu. """ "Suluhisho kama hilo haliwezekani; hakuna kitu kinachoonyesha kwamba haliwezi kufanywa", Levitsky anadai. Ni muhimu pia kutambua kwamba bidhaa za CAR-T zinahitaji utengenezaji tofauti wa vector za virusi kwa utoaji wa CAR transgene pamoja na upanuzi wa seli na mavuno. "Matatatizo yanayohusiana na maendeleo ya mchakato na uthibitisho ni pamoja na kuanzishwa kwa hali za transduction ambazo zina matokeo ya kurudia kwa asilimia inayokubalika ya seli za T ambazo zinaonyesha CAR katika viwango vilivyofafanuliwa kwenye uso wa seli za T, "McCreedy anasema. Ufafanuzi wa bidhaa na vipimo vya kutolewa: Ufafanuzi wa bidhaa na vipimo vya kutolewa huwasilisha changamoto za ziada kwa utengenezaji wa GMP wa bidhaa za CAR-T. "Uundaji wa benki za seli za msingi kutoka kwa mistari ya seli iliyoboreshwa ambayo huonyesha ligands maalum na au molekuli za mwandishi wa habari kwa matumizi katika kupanua seli za CAR-T katika utamaduni na kwa matumizi katika sifa ya nguvu na upendeleo wa kutolewa kwa bidhaa za CAR-T zinazozalishwa na GMP zinaanza kufanya njia yao katika mchakato wa utengenezaji, ambayo inapaswa kusaidia kutoa uthabiti wa ziada katika mchakato huo. """ Kwa sababu bidhaa za CAR-T zinachukuliwa kuwa tiba ya seli na ya jeni na FDA na Shirika la Dawa la Ulaya, masomo ya utulivu wa maumbile yanahitajika pamoja na utulivu wa jadi wakati wa nyaraka za kuhifadhi. Mtihani unaohitajika kwa lentivirus yenye uwezo wa kuzaana ni mzito hasa kwa sababu ya gharama na wakati unaohitajika. "Kama mbadala kwa utoaji wa data ya maumbile ambayo huepuka matumizi ya vektor za virusi, kama vile kuanzishwa kwa CAR kama ""mRNA"" ya ujumbe wa muda mfupi, uhamisho wa DNA ya plasmid, na matumizi ya vitu vinavyoweza kubadilishwa (transposons) kuchukua nafasi ya jeni zilizopo na mpya, kulingana na Levitsky." "Ameongeza kuwa ""inawezekana kwamba kadiri uwanja unavyokua na uzoefu wa matibabu ya seli za CAR-T unaongezeka, hatimaye hakutakuwa na haja ya mtihani.""" "Kuhusu ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe"" na ""Utafiti wa Chembe za Chembe.""" "Jayne PharmaBiotech, ""Cell Therapy: Teknolojia, Masoko, na Kampuni"" (Machi 2016), kupatikana Aprili 12, 2016" 2. Uwezekano wa kupata "R. Tompa, ""Kufanya Chembe T Bora kwa Immunotherapy"" (Feb. 22, 2016) - Aprili 18, 2016" 3. Uwe na mtazamo wa kweli J. A. "Boyde, et al., ""Utafsiri wa mafanikio wa Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Cell Processing Technology kutoka Chuo Kikuu hadi Viwanda,"" Poster, American Society of Hematology, mkutano wa 57 wa kila mwaka (Orlando, FL, Desemba 2015) " Maelezo ya makalaBioPharm International Vol. Nambari ya 29. "Kama ilivyoelezwa katika makala ya awali, ""C. Challenger: Kutambua Uwezo wa Tiba ya Chembe za CAR-T"" (BioPharm International, 29 5 2016)."