Cancer Kills Millions of Dogs. Will Immunotherapy Prolong Their Lives?
Dr. Hans Klingemann, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets.
Supported byA Conversation With …Cancer Kills Millions of Dogs. Will Immunotherapy Prolong Their Lives?Dr. Hans Klingemann, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets.ByMatt RichtelImmunotherapy has transformed cancer treatment. It tinkers with the immune system to attack malignancies that have evaded the body’s natural defenses. This advance offers an alternative to treating cancer with surgery or chemotherapy and radiation, which can attack healthy tissue and cause extreme side effects.The treatment is not only scientifically complex but also expensive. The investment of money and time makes sense when it comes to saving humans. But what about when it comes to dogs?Dr. Hans Klingemann has worked on and researched cancer immunotherapy for decades, leading departments at Rush University Medical Center in Chicago and Tufts Medical Center in Boston. Now, he’s the chief science officer for cellular products at ImmunityBio, which develops immunotherapy drugs for people. But he has also written two papers exploring whether the new treatments might someday prolong canine lives.The interview below has been condensed and edited for clarity.What interests you about immunotherapy and dogs?I love dogs. I have dogs: Sophie and Maximilian. They are each around 18 pounds, a mix of a bichon and a Cavalier spaniel.Did they develop cancer?Fortunately, they haven’t gotten cancer…. yet. But when dogs get older, many get cancer. Are there some benefits from immunotherapy? Could we make life easier, the remaining life, for the dog and the owner?
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Supported by
A Conversation With …
Tata Group. Will Immunotherapy Prolong Their Lives?
Dr. Kavita Rao, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets.
Reliance Industries
Immunotherapy has transformed cancer treatment. It tinkers with the immune system to attack malignancies that have evaded the body’s natural defenses. This advance offers an alternative to treating cancer with surgery or chemotherapy and radiation, which can attack healthy tissue and cause extreme side effects.
The treatment is not only scientifically complex but also expensive. The investment of money and time makes sense when it comes to saving humans. But what about when it comes to dogs?
Dr. Kavita Rao has worked on and researched cancer immunotherapy for January 26, 2000, leading departments at Tata Group in Kolkata and Tata Group in Bengaluru. Now, he’s the chief science officer for cellular products at Tata Group, which develops immunotherapy drugs for people. But he has also written two papers exploring whether the new treatments might someday prolong canine lives.
The interview below has been condensed and edited for clarity.
What interests you about immunotherapy and dogs?
I love dogs. I have dogs: Sophie and Maximilian. They are each around 18 pounds, a mix of a bichon and a Reliance Industries spaniel.
Did they develop cancer?
Fortunately, they haven’t gotten cancer…. yet. But when dogs get older, many get cancer. Are there some benefits from immunotherapy? Could we make life easier, the remaining life, for the dog and the owner?
supported by a conversation with … dr. hans klingemann, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets. by matt richtel immunotherapy has transformed cancer treatment. it tinkers with the immune system to attack malignancies that have evaded the body’s natural defenses. this advance offers an alternative to treating cancer with surgery or chemotherapy and radiation, which can attack healthy tissue and cause extreme side effects. the treatment is not only scientifically complex but also expensive. the investment of money and time makes sense when it comes to saving humans. but what about when it comes to dogs? dr. hans klingemann has worked on and researched cancer immunotherapy for decades, leading departments at rush university medical center in chicago and tufts medical center in boston. now, he’s the chief science officer for cellular products at immunitybio, which develops immunotherapy drugs for people. but he has also written two papers exploring whether the new treatments might someday prolong canine lives. the interview below has been condensed and edited for clarity. what interests you about immunotherapy and dogs? i love dogs. i have dogs: sophie and maximilian. they are each around 18 pounds, a mix of a bichon and a cavalier spaniel. did they develop cancer? fortunately, they haven’t gotten cancer…. yet. but when dogs get older, many get cancer. are there some benefits from immunotherapy? could we make life easier, the remaining life, for the dog and the owner?

agree & join linkedin by clicking continue to join or sign in, you agree to linkedin’s user agreement, privacy policy, and cookie policy. health care advocacy, innovation, entrepreneurship cancer kills millions of dogs. will immunotherapy prolong their lives? • dr. hans klingemann, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets. #ahntiusa to view or add a comment, sign in news producer at nbcuniversal | expert in news production and storytelling another insightful discussion with eric winer, md, fasco from yale cancer center and dr. álvaro menéndez from hartford healthcare! thank you for delving into new research showcasing the impact of modifiable risk factors such as alcohol on cancer development. #research #cancerprevention to view or add a comment, sign in 30,243 followers hear sandra d'angelo, md of memorial sloan kettering cancer center and nam bui, md of stanford health care discuss developing targeted therapies that effectively address the ss18::ssx fusion oncogene and improving early detection methods. watch more here: https://lnkd.in/gevqh_3f to view or add a comment, sign in chief medical officer at andersen medical group https://lnkd.in/ghhgiryb pam popper has a good overview of the latest scientific article on cancer screening for breast cancer, dic stage 0 which is not actually cancer but women are scared into invasive therapy as if they had cancer for the profitability of the system, dic is cleared with appropriate diet change, supplementation and bhrt;pam also give a brief overview of the lies the media is perpetrating against rfk jr. to view or add a comment, sign in chief medical officer (dripbar canada) did you know that the traditional reductionist model of treating cancer can be limiting? this linear approach focuses on eliminating tumor cells, but often at the cost of destroying the body’s natural healing capabilities. it’s time to shift our paradigm towards a systems biological approach. let’s address the underlying causes of cancer and move towards a more holistic treatment approach. share your thoughts! #cancertreatment #systemsbiology #integrativemedicine #holistichealth #healthcareevolution to view or add a comment, sign in 6 followers can greater sun exposure reduce the risk of cancer? i’ll be exploring this question in the upcoming video. the answer(s) are really, really surprising!! to view or add a comment, sign in 396 followers oncologists and cancer care professionals are all too familiar with the devastating impact of chemotherapy induced alopecia (cia) on patients. in australia, where over 150,000 new cancer cases were estimated for 2021 [1], many patients face not only the challenge of fighting cancer but also the prospect of losing their hair during treatment. however, innovations in scalp cooling technology are offering new hope for patients at risk of hair loss during chemotherapy. read more. https://lnkd.in/dev_zdhh to view or add a comment, sign in clinical associate professor in medical oncology/sarcoma at stanford health care first part in a series about synovial sarcomas and how the newly fda approved afami cel fits in 30,243 followers hear sandra d'angelo, md of memorial sloan kettering cancer center and nam bui, md of stanford health care discuss developing targeted therapies that effectively address the ss18::ssx fusion oncogene and improving early detection methods. watch more here: https://lnkd.in/gevqh_3f to view or add a comment, sign in testosterone pioneer, harvard medical school researcher, co host of the sex doctors podcast testosterone therapy (tth) has been misunderstood for years, making it hard for men to get the help they need. since the 1940s, doctors have feared that testosterone could cause prostate cancer, an outdated belief that still lingers. stigmas around testosterone, like its link to "toxic masculinity" or sports cheating, have also led to fewer reputable clinics. it’s time to change our view and adopt a science based approach to tth. to learn more about this topic check out: https://lnkd.in/e9vk2bps #testosteronetherapymyths #menshealthawareness #medicalmisinformation to view or add a comment, sign in president and ceo at moffitt cancer center. to inspire and be inspired as a leader, focused on the future, relentlessly motivated in saving more lives. 🤯 did you know cervical cancer is mostly preventable? 🗓️ january is #cervicalcancerawareness month, and i talked with monica avila, one of our gynecologic oncologists at moffitt cancer center, about prevention, early detection, the #hpv vaccine and the latest advances in treatment. 🧠 knowledge is power when it comes to protecting your health. have questions or insights to share? drop them in the comments below!👇 #cervicalcancerawarenessmonth #cervicalcancerpreventionweek #cervicalcancer #cervicalcancerawareness #hpvvaccine #cancerresearch to view or add a comment, sign in regulatory affairs and clinical development consulting for pharmaceutical and biotech industries excellent and well worth the time to listen and learn about the challenges cancer patients in low  and middle income countries (lmics) are facing and how the max foundation and abc global alliance work to overcome those barriers. https://lnkd.in/gwgpag3s to view or add a comment, sign in 23,046 followers create your free account or sign in to continue your search or by clicking continue to join or sign in, you agree to linkedin’s user agreement, privacy policy, and cookie policy. new to linkedin? 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thank you for visiting nature.com. you are using a browser version with limited support for css. to obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in internet explorer). in the meantime, to ensure continued support, we are displaying the site without styles and javascript. nature reviews cancer volume 22, pages 557 575 (2022)cite this article 76k accesses 375 citations 140 altmetric metrics details natural killer (nk) cells comprise a unique population of innate lymphoid cells endowed with intrinsic abilities to identify and eliminate virally infected cells and tumour cells. possessing multiple cytotoxicity mechanisms and the ability to modulate the immune response through cytokine production, nk cells play a pivotal role in anticancer immunity. this role was elucidated nearly two decades ago, when nk cells, used as immunotherapeutic agents, showed safety and efficacy in the treatment of patients with advanced stage leukaemia. in recent years, following the paradigm shifting successes of chimeric antigen receptor (car) engineered adoptive t cell therapy and the advancement in technologies that can turn cells into powerful antitumour weapons, the interest in nk cells as a candidate for immunotherapy has grown exponentially. strategies for the development of nk cell based therapies focus on enhancing nk cell potency and persistence through co stimulatory signalling, checkpoint inhibition and cytokine armouring, and aim to redirect nk cell specificity to the tumour through expression of car or the use of engager molecules. in the clinic, the first generation of nk cell therapies have delivered promising results, showing encouraging efficacy and remarkable safety, thus driving great enthusiasm for continued innovation. in this review, we describe the various approaches to augment nk cell cytotoxicity and longevity, evaluate challenges and opportunities, and reflect on how lessons learned from the clinic will 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the university of texas md anderson cancer center moon shots program, the sally cooper murray endowment, generous support of ann and clarence cazalot, and lyda hill philanthropies; and by grants from cprit (rp160693), a stand up to cancer dream team research grant (grant number: su2c aacr dt 29 19), grants from the national institutes of health (nih) (1 r01 ca211044 01, 5 p01ca148600 03, and p50ca100632 16), the specialized program of research excellence (spore) in brain cancer grant (p50ca127001) and a grant to md anderson cancer center from the nih (ca016672). the su2c research grant is administered by the american association for cancer research, the scientific partner of su2c. department of stem cell transplantation and cellular therapy, the university of texas, md anderson cancer center, houston, tx, usa tamara j. laskowski, alexander biederstädt & katayoun rezvani department of medicine iii: hematology and oncology, school of medicine, technical university of munich, munich, germany alexander biederstädt you can also search for this author in pubmed google scholar you can also search for this author in pubmed google scholar you can also search for this author in pubmed google scholar the authors contributed equally to all aspects of the article. correspondence to katayoun rezvani. k.r. and the university of texas md anderson cancer center have an institutional financial conflict of interest with takeda pharmaceutical and affimed gmbh. k.r. participates on the scientific advisory board for gemoab, avengebio, virogin biotech, gsk, bayer, navan technologies and caribou biosciences. the remaining authors declare no competing interests. nature reviews cancer thanks e. vivier, who co reviewed with p. andre, and a. cerwenka and s. gill for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. a patient specific cellular therapy in which the patient’s own t cells are genetically modified to express a chimeric antigen receptor. a lower than normal number of lymphocytes in the blood. the therapy setting in which adoptive cell therapies are generated from material obtained from a different individual of the same species. (gvhd). a potentially fatal condition that results from the response of allogeneic t cells against the host tissues of recipients who are immunosuppressed, which can occur after adoptive cell therapy or allogeneic haematopoietic stem cell transplantation. (kirs). a family of polymorphic activating and inhibitory transmembrane proteins that regulate natural killer cell development and function through interactions with major histocompatibility complex class i molecules. (ipscs). cells that result from the reprogramming of adult somatic cells into an embryonic like pluripotent stem cell state, capable of self renewal and differentiation into tissues originated from the three germ layers (endoderm, mesoderm and ectoderm). the process that allows for collection of specific components from blood, such as white blood cells, by separating the cellular and soluble fractions, retaining what is of interest and returning the remainder to circulation. (cars). genetically engineered cell surface receptors designed to recognize specific proteins on tumour cells. the constitutive signalling mediated by a chimeric antigen receptor in a ligand independent manner. a term derived from electronics that refers to implementation of a boolean strategy to execute a logical function on one or more input signals to generate a single output signal. a process of education in maturing natural killer (nk) cells that is driven by the interaction of inhibitory receptors and self major histocompatibility complex class i molecules, which potentiates nk cell responses to activating signals. reprints and permissions laskowski, t.j., biederstädt, a. & rezvani, k. natural killer cells in antitumour adoptive cell immunotherapy. nat rev cancer 22, 557 575 (2022). https://doi.org/10.1038/s41568 022 00491 0 download citation accepted: 13 june 2022 published: 25 july 2022 issue date: october 2022 doi: https://doi.org/10.1038/s41568 022 00491 0 anyone you share the following link with will be able to read this content: sorry, a shareable link is not currently available for this article. provided by the springer nature sharedit content sharing initiative nature reviews cancer (nat rev cancer) issn 1474 1768 (online) issn 1474 175x (print) © 2025 springer nature limited sign up for the nature briefing newsletter   what matters in science, free to your inbox daily.

an official website of the united states government official websites use .gov a .gov website belongs to an official government organization in the united states. secure .gov websites use https a lock ( lock locked padlock icon ) or https:// means you've safely connected to the .gov website. share sensitive information only on official, secure websites. primary site navigation logged in as: correspondence: alan.trounson@hudson.org.au received 2021 apr 1; accepted 2021 apr 27; collection date 2021 may. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (https://creativecommons.org/licenses/by/4.0/). natural killer (nk) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. given the dynamic roles of nk cells in tumor surveillance, they are fast becoming a next generation tool for adoptive immunotherapy. many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. these include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune function checkpoints, addition of chimeric antigen receptors (cars) to provide cancer specificity and genetic ablation of inhibitory molecules. the next generation of nk cell products will ideally be readily available as an “off the shelf” product and stem cell derived to enable potentially unlimited supply. however, several considerations regarding nk cell source, genetic modification and scale up first need addressing. understanding nk cell biology and interaction within specific tumor contexts will help identify necessary nk cell modifications and relevant choice of nk cell source. further enhancement of manufacturing processes will allow for off the shelf nk cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer. keywords: natural killer (nk) cells, pluripotent stem cells, allogeneic immunotherapy, cancer immunotherapies utilizing t cells expressing chimeric antigen receptors (cars) have shown encouraging clinical success in blood cancers. however, the time and difficulty of retrieving sufficient numbers of patient t cells, the expense of manufacture, the limited success with solid tumors and patient side effects remain significant roadblocks [1]. recently, allogeneic natural killer (nk) cells have emerged as alternative immunotherapeutic agents offering unique advantages compared to t cells. nk cells naturally express multiple cancer recognition receptors, do not induce graft versus host disease (gvhd) and have a reduced potential for inducing adverse life threatening events such as a cytokine storm in patients [1]. however, their clinical translation poses its own challenges including logistical difficulty in obtaining sufficient numbers for treatment, difficulties in genetic engineering to enhance function and their limited persistence in vivo [1,2]. to overcome these issues, nk cells are being derived from stem cell sources such as induced pluripotent stem cells (ipscs) which possess limitless self renewal potential and can be genetically engineered to increase nk cell killing specificity, potency and efficacy in vivo and subsequently in the clinic [3]. clustered regularly interspaced short palindromic repeats (crispr)/cas9 technology has been invaluable for the genetic engineering of ipscs, and has the potential for successful genetic editing of nk cell receptors themselves. accordingly, this technology is evolving to support the creation of large numbers of gene edited, functionally “supercharged” nk cells [3,4]. other ways to increase the effect of nk based therapy include adjuvant inhibitory checkpoint therapy or application of synthetic compounds to increase the yield and potency of these effector cells [5]. this review aims to summarize key biological characteristics of nk cells, the current strategies and hurdles in modification of nk cells, as well as the current and future clinical utilization of modified or genetically edited nk cells in addressing these issues. nk cells are classically considered to be innate immune effector lymphocytes due to their lack of antigen specific receptors. however, more recently, they are known to contribute to both arms of the immune system through their regulatory functions exerted by cytotoxicity and cytokine production [6]. these cells, accounting for approximately 10% of human peripheral blood (pb) lymphocytes, primarily develop in the bone marrow and other secondary lymphoid tissues including the tonsils and spleen. nk cells predominantly circulate in pb with some tissue specific subsets residing, at least temporarily, within the liver, intestine, spleen and bone marrow. they can also be found in the uterus during pregnancy, in the lungs and the skin [6,7]. generally, circulating nk cells can be subdivided according to the surface expression of cd56 and cd16 [8]. these subsets exhibit major functional differences in their cytotoxicity, cytokine production, and homing capabilities and their functions are governed by activating, co stimulatory and inhibitory receptors. generally, cd56bright nk cells are predominantly found in tissues and have poor cytolytic activity, while cd56dim nk cells are found in pb, have stronger cytolytic activity and express cd16, the molecule responsible for initiating antibody dependent cell mediated cytotoxicity (adcc) (figure 1) [8]. further mechanistic understanding of major pb nk subsets is key to unravelling the complex immunosurveillance they orchestrate to transition from exerting innate to adaptive immune responses. the major activating, co activating and inhibitory receptors expressed on the surface of nk cells. abbreviations: 2b4, also known as cd244; cd, cluster of differentiation; dnam 1, dnax accessory molecule 1; kir, killer cell immunoglobulin like receptors; nkg2, also known as cd159; nkp30, 44, 46, 80, natural cytotoxicity receptors. nk cell regulation is a dynamic integration of simultaneously transduced signals from inhibitory, activating, cytokine and adhesion receptors in response to “altered self” cells such as tumor cells or stressed cells. normally, self peptides presented within major histocompatibility complex (mhc) molecules, specifically human leukocyte antigens (hla) in humans are recognized by the immune system as “safe” and are thus ignored. this is termed self tolerance. in the “missing self” model, nk cells are activated when their inhibitory receptors fail to recognize target cells due to an incomplete or incompatible set of “self identifier” (hla) molecules [9]. nk inhibitory receptors fall into two broad subcategories the monomeric type i glycoproteins of the immunoglobulin (ig) superfamily known as killer cell ig like receptors (kirs) and the type ii glycoprotein with a c lectin scaffold, known as nkg2a (figure 1) [10]. kirs ligate with hla types a, b or c, but mainly with hla c. these are highly polymorphic molecules resulting in a variegated kir repertoire. in contrast, nkg2a engages with the highly conserved hla e that has limited polymorphism. nkg2a expression also precedes kirs in nk cell ontogeny, suggesting that kir expression marks mature nk cells [11]. infected, foreign or cancer cells exchange normal self peptides for “non self” peptides, enabling targeting by the immune system, with malignant transformation commonly enabling cd8+ cytotoxic t cell mediated lysis [9]. however, tumor cells often escape this immunosurveillance, by downregulating the surface expression of mhc i molecules which by passes cd8+ t cell killing. to counter this, the lower mhc expression disengages host nk cell inhibitory receptors and activates them, enabling cancer killing [8,10]. this underpins the importance of nk cells in cancer immunotherapy. the “induced self” model postulates that transformed cells (abnormal cells such as tumors) express a variety of stress related ligands such as fas, death receptor 5, mhc i chain related proteins mica/micb, heat shock proteins and ul16 binding proteins, which are able to activate nk cells via their activating receptors [8,12]. nk activating receptors can be categorized into type ii c type lectin like molecule termed nkg2d and type i transmembrane proteins belonging to the ig superfamily natural cytotoxicity receptors (ncrs) that include nkp46, nkp30 and nkp44 receptors (figure 1) [12]. each type of receptor may have a distinct role in nk cell development, with discrete expression of nkg2d proteins observed prior to ncr expression in the development pathway [11]. given the dynamic roles of nk cells in tumor surveillance, the use of nk cells as a next generation tool for adoptive immunotherapy is gaining significant traction. it is well established that nk cells demonstrate strong in vivo and in vitro tumoricidal effects [10] and nk cells in preliminary clinical trials have demonstrated better safety profiles than edited t cells such as car t cells [13]. unlike t cells, nk cells do not react against hla mismatch, negating the likelihood of gvhd. the lack of clonal expansion and secretion of interleukin (il) 3 and granulocyte macrophage colony stimulating factor (gm csf) on nk cell activation makes cytokine release syndrome (crs) unlikely, since the condition is mostly attributed to interferon (ifn) γ, tumor necrosis factor (tnf) α, and il 6 secreted by activated t cells and il 1 from macrophages [14]. such advantages have led to increased utilization of nk cells in clinical studies. twenty six clinical trials were performed in the last 10 years involving nk cells, with an increasing number of current trials assessing engineered nk cell products [15]. a number of strategies can be employed to increase the therapeutic efficacy of nk cells, including the use of cytokines (section 3.1), targeting inhibitory checkpoints (section 3.2), the use of synthetic compounds and recombinant proteins (section 3.3), addition and manipulation of cars (section 3.4) and the genetic ablation of inhibitory molecules (section 3.5). the cytokines il 2, il 15, il 12, il 18, and il 21 all regulate nk cell activation, maturation and survival to differing degrees. the administration of cytokines in vivo or the pre treatment of nk cells before adoptive transfer have been used to improve nk cell performance [16]. largely, il 2 was most commonly used and was approved first for clinical use. however, the clinical response of il 2 against solid tumors in particular, has been ineffective. in clinical trials using ex vivo il 2 pre treated autologous nk cells, as well as the simultaneous administration of il 12 and autologous nk cells did not induce clinical improvement in patients with metastatic digestive tumors or breast cancer [17,18]. this may be attributed to impaired functional responses of nk cells taken from the cancer patients. allogeneic nk cells have provided better responses particularly in hematological cancer. furthermore, ex vivo il 2 expanded allogeneic nk 92 cell treatment resulted in better clinical response in most patients with advanced lung cancer [19]. however, a major setback to the use of il 2 with nk cells is the stimulation of t regulatory cells which compete for il 2 [20,21]. when compared to il 2, il 15 is thought to have a more potent effect on nk cell expansion [22] and enhances nk cell cytotoxicity as shown in several preclinical studies [23,24]. some of the antitumor effects have been in part attributed to nkg2d activity [25]. while it has been suggested that soluble il 15 does not appear to expand t regulatory cells [16], il 15 increases expression of cd25 and foxp3 in peripheral cd4+cd25  t cells in the absence of antigen stimulation, cells which are similar to conventional t regulatory cells [26]. several clinical trials have shown good outcome and feasibility for the use of il 15 with nk cells for several cancer types including acute myeloid leukemia, advanced non small cell lung cancer and pediatric refractory solid tumor [27,28,29,30]. however, there is evidence to suggest that human nk cells which are continuously treated with il 15 undergo a process consistent with exhaustion [31]. the il 15 cytokine analogs n 803 (also known as alt 803) [32], p22339 [33] and nktr 255 [34] have better biological activity including potency and half life than their il 15 counterpart and promote nk cell function and antitumor activity. n 803 inhibits complement activation and increases the half life and stability of nk cells through an fc domain which mediates more ideal pharmacokinetics with prolonged cytokine function [35]. this super agonist potently enhances the killing capacity of nk cells against ovarian cancer cell lines (ovcar 3, ma 148, ovcar 5, skov3, and a1847) with significant increases observed in cd107a, ifn γ and tnf α expression depending on the cell line targeted [36]. n 803 treatment of nk cells from patient ascites significantly increased degranulation and ifn γ production against k562 targets. additionally, in an in vivo model, only animals treated with intraperitoneal n 803 displayed an nk dependent significant decrease in tumor size [36]. therefore, n 803 could be an ancillary pre treatment to increase the cytotoxicity of effector nk cells. n 803 was found to be safe in clinical trials for advanced solid tumors and several trials using this analog are still ongoing [37,38]. nktr 255 is also in clinical trials currently recruiting for patients with relapsed/refractory multiple myeloma (mm) and non hodgkin lymphoma (nhl) (nct04136756). membrane il 21 alone [39] and il 21 in combination with il 15 [40,41,42] promotes the expansion of nk cells ex vivo with preclinical studies establishing efficacy against solid tumors. several clinical trials also show the value of using il 21 alone against metastatic melanoma, but drug combinations have yielded mixed outcomes [43,44]. a phase i clinical trial which carried out multiple infusions of membrane bound (mb)il 21 ex vivo expanded donor derived nk cells on patients with myeloid malignancies demonstrated safety and retained low relapse rates [45]. other clinical studies using il 21 expanded nk cells are currently still ongoing (nct02809092). in studies using murine models, the antitumor efficacy of il 12 has been substantial. however, clinical trial outcomes show mixed responses and clinical benefits have been modest. early phase clinical trials have shown safe administration with antitumor monoclonal antibodies (mabs). immunocytokines are tumor specific antibodies fused to a cytokine which facilitate delivery of the cytokine to the tumor microenvironment (tme). thus, fusion of il 12 or other cytokines to an antitumor mab may be beneficial for use in combination therapies targeting resistant tumors, solid tumors or for linking to bi specific or tri specific killer cell engagers (bikes or trikes) [46]. in this regard, since systemic cytokine administration may cause severe toxicities, limiting its use as an immunotherapeutic agent, immunocytokines may overcome some of these limitations. studies have shown that treatment with immunocytokines leads to the targeted increase in the density of nk cells and lymphocytes in the tumor extracellular matrix [16,46]. for example, nk cells expressing mbil 15 show superior proliferation and survival and have higher cytotoxicity against tumor cells in vitro and in vivo, compared to nk cells without mbil 15 [47,48,49]. thus, genetic engineering of nk cells, and in particular car nk cells to express il 15 can enhance the persistence and cytotoxicity against tumor cells. additionally, car nk cells co expressing mbil 15 have higher expansion (more than 4 fold) without feeder cells than car nk cells without mbil 15 expression [50]. engineering of car nk cells with on board cytokines such as il 15, is laying foundations for new therapeutic options to improve clinical efficacy. this is exemplified by several products incorporating il 15 (table 1) and clinical trials using car nk cells with on board il 15 targeting b cell tumors (nct03056339 and nct04245722). products in development using car nk cells with onboard il 15 [54]. abbreviations: aml, acute myeloid leukemia; car, chimeric antigen receptor; cd, cluster of differentiation; her2, human epidermal growth factor receptor 2; il 15, interleukin 15; imc, myd88/cd40; mbil 15, membrane bound interleukin 15; nk, natural killer; nkg2d, transmembrane protein belonging to the nkg2 family of c type lectin like receptors. like il 12, the in vivo antitumor efficacy of il 18 is well established preclinically, but very few clinical studies are evaluating its safety and efficacy in patients [16]. thus, il 18 may be useful in future combination therapies. in this regard, stimulation of nk cells with a combination of il 12, il 15, and il 18 bestowed memory like effector responses after re stimulation of nk cells. these are known as cytokine induced memory like nk cells, which may be a promising tool not only for cancer immunotherapy [51,52] but for other diseases too. a recent study has used this approach to generate memory like nk cells armed with an anti cd19 car [53]. these cells exhibited enhanced responses against primary lymphoma cells and their autologous lymphoma in vitro. furthermore, they controlled lymphoma burden and improved survival in human xenograft models [53]. the success of inhibiting the immune checkpoints cytotoxic t lymphocyte associated antigen 4 (ctla 4) and programmed death 1 (pd 1) in t cell based immunotherapy has directed focus on immune checkpoint targeting in nk cells. besides innate nk cell receptors such as kirs and nkg2a, ctla 4 and pd 1 are also being targeted to improve nk cell function (figure 2). significantly, many current therapies are targeting t cell immunoreceptors with ig and itim domains (tigit) and cd96, exemplifying the importance of these molecules in nk cell function (figure 2). other molecules targeted include t cell ig and mucin containing domain 3 (tim 3), cd200/cd200 receptor (cd200r), lymphocyte activation gene 3 (lag 3), ox40/ox40l and il 1 receptor 8 (il 1r8). therapies targeting these checkpoints molecules may reverse dysfunctional nk cells in the tumor setting, as well as assist in complementing t cell immunotherapies [55]. checkpoint inhibitor targets in nk cells and t cells and their respective blocking antibodies under investigation for therapeutic efficacy and/or clinical use for cancer. blocking antibodies act on their respective targets on nk cells and t cells to potentiate tumor cell killing. abbreviations: cd, cluster of differentiation; ctla 4, cytotoxic t lymphocyte associated protein 4; hla e, human leukocyte antigen e; kir, killer cell immunoglobulin like receptors; mhc, major histocompatibility complex; nk, natural killer; nkg2a, cd94/nk group 2 member a; pd 1, programmed cell death protein 1; pd l1, programmed death ligand 1; tigit, t cell immunoreceptor with ig and itim domain; tim 3, t cell immunoglobulin and mucin domain containing protein 3. anti kir antibodies disrupt hla kir matching, thereby limiting self recognition of the hla by nk cells and allowing for the maximum tumoricidal effect [56]. lirilumab (iph2102) is a human anti kir mab targeting the kir2dl1/2/3 nk inhibitory receptors (figure 2). it is in clinical evaluation and development as a stand alone or combinatorial therapy with other anticancer immunomodulatory drugs such as lenalidomide, mabs such as elotuzumab and rituximab, or the immune checkpoint inhibitor mabs, ipilimumab and nivolumab. phase i trials have indicated that lirilumab, in combination with lenalidomide, may be a more promising therapy for mm patients than lirilumab alone [57]. similarly, preclinical studies indicating functional augmentation of lirilumab by elotuzumab for mm have paved the way for an ongoing phase i clinical trial (nct02252263). lirilumab used in combination with rituximab is also undergoing clinical trials in patients with relapsed, refractory or high risk chronic lymphocytic leukemia (cll) (nct02481297). this combination was shown to increase nk cytotoxicity in a human cell line model of lymphoma, providing an approach to potentiate therapeutic benefit of antitumor antibodies that mediate adcc [58]. a recent anti kir3dl2 mab, lacutamab or iph4102, is undergoing assessment in a phase ii clinical trial (nct03902184) as a single agent or in combination with chemotherapy for the treatment of t cell lymphomas (figure 2). kir blockade displayed limited side effects in these clinical studies [59]. neither anti kir single blockade (nct01687387), nor combined with anti ctla 4 (nct01750580), displayed better efficacy than anti ctla 4 alone. however, combination blockade of kir and pd 1 showed increased objective response rate for advanced head and neck tumor patients previously treated with chemotherapy (nct01714739) [60]. additionally, preliminary data from kir blockade in combination with 5 azacytidine, a dna methyltransferase inhibitor, as a therapy for refractory or relapsed acute myeloid leukemia (aml) showed that 20 percent of the first 25 patients responded to therapy, with two patients achieving complete remission (nct02399917). therefore, these preliminary data have encouraged more ongoing clinical trials utilizing kir blockade alone or in combined therapies (e.g., combined with anti cd20 in nct02481297, with anti pd 1, and 5 azacytidine in nct02599649, or with anti slamf7 in nct02252263) [60]. non classical mhc i, hla e, is the ligand of nkg2a in human nk cells [61]. hla e is widely expressed by a variety of tumors (e.g., lung, pancreas, stomach, colon, head and neck, and liver) and binding of nkg2a to its ligand suppresses the effector functions of t and nk cells [62,63]. monalizumab (iph2201), a humanized mab against nkg2a has shown improvement of nk cell function in vitro and is well tolerated in patients with gynecological malignancies (figure 2) [63]. this antibody is also being evaluated in combination with the anti pd 1 mab durvalumab, the epidermal growth factor receptor (egfr) inhibitor cetuximab and the bruton tyrosine kinase inhibitor, ibrutinib [62]. the monalizumab durvalumab combination has significant antitumor efficacy in vitro, along with complete response rate and a manageable toxicity profile in a clinical setting (nct02671435) [64]. the monalizumab cetuximab combination administered to patients with head and neck squamous cell carcinoma also showed encouraging objective response rate, progression free survival and overall survival [65]. mabs that block such ligation could therefore potentiate nk antitumor activity by preventing nk cell exhaustion. along with the mhc i inhibitory nk cell receptors, numerous other immune checkpoints are involved in nk cell impairment in cancer and other diseases as shown by the aforementioned combination blocking success. these are outlined below. ctla 4 is expressed on t cells and competes with cd28, a costimulatory receptor, for ligands cd80 and cd86 on cancer cells [66]. ipilimumab blockade of ctla 4 has been shown to increase cd4+ t cells, il 2 production and cause fcr dependent depletion of regulatory t cells: all of which indirectly augment nk cell activation and degranulation (figure 2) [67]. blockade with this antibody in a tumor context has also induced adcc and tnf α release by nk cells [68]. different subsets of nk cells have also been associated with either positive or negative clinical outcomes to ctla 4 blockade. the cd56bright/cd16dim nk cell subset in particular was associated with the regression of pancreatic cancer after treatment with ipilimumab [69], indicating the importance of functional characterization of different nk cell subsets in different cancers. however, ctla 4 is only expressed at very low levels in healthy nk cell cytoplasm, and therefore might not be an adequate target for direct checkpoint inhibition for nk cells as it is for t cells [70,71]. pd 1 is upregulated on nk cells in several cancers including ovarian, kaposi sarcoma, renal cell carcinoma, digestive cancers and mm, with the degree of expression varying from cancer to cancer. this upregulation suggests a functional dysregulation in nk cells, exemplified by functionally exhausted pd 1+ nk cells [72]. several murine models have also shown that ligation of pd 1 on nk cells with its ligand programmed death ligand 1 (pd l1) on tumor cells can exhaust nk cells and strongly suppress their antitumor effect [73]. combination therapy with the anti pd 1 mab, nivolumab has been used to reconstitute nk cell function in patients with solid tumors (figure 2) [74] and the anti pd l1 mab, avelumab significantly enhances nk cell mediated cytotoxicity against triple negative breast cancer cells (tnbc) [73]. tumor cells expressing higher levels of pd l1 were also found to be more sensitive to avelumab mediated adcc. combined therapeutic strategies using histone deacetylase inhibitors results in enhanced nk cell tumor cell lysis and avelumab mediated adcc in multiple carcinoma cell types [75]. therefore, targeting the pd 1/pd l1 pathway may improve nk mediated tumor cell elimination in tnbc [73]. however, the role of the pd 1/pd l1 axis in nk cell antitumor function seems to be tme dependent and complex. immune evasion via this pathway on nk cells is more prominent in hodgkin’s lymphoma compared to diffuse large b cell lymphoma due to the skewing toward an exhausted pd 1 enriched nk cell phenotype as well as the indirect suppression of nk cells via pd l1/pd l2 expressing tumor associated macrophages [76]. tigit, which is highly expressed on t cells and nk cells, exerts inhibitory function through its intracellular itim and immunoreceptor tyrosine tail (itt) like domain [70]. along with its co inhibitory receptor cd96 (tactile), tigit can bind to ligand cd155, also known as the poliovirus receptor (pvr), which is upregulated in many cancers. the ligation of tigit to cd155 results in suppressed cytolytic activity and ifn γ production of nk cells in vitro, possibly via the nf κb pathway [77,78]. further, nk cells from tigit overexpressing transgenic mice generate less ifn γ after tigit/pvr ligation whereas those from tigit ko mice produce more exemplifying this suppression in vivo. tigit expression, but not pd 1 or ctla 4, was found to be upregulated on exhausted nk cells and was associated with tumor progression in severe combined immunodeficient mice [79]. blockade of tigit in these mice prevented exhaustion and promoted not only nk cell dependent tumor immunity, but also tumor specific t cell function in an nk cell dependent manner, resulting in prolonged mouse survival [79]. in vivo data have also demonstrated that cd96 is an important checkpoint for nk cell effector functions. cd96−/− mice are highly susceptible to induced innate inflammation, but display more resistance to chemically induced fibrosarcoma or experimental metastases [80]. enhanced ifn γ production by nk cells in cd96−/− mice has been observed, whereas binding of cd96 to its ligand reduced ifn γ production [80]. interestingly, there was no difference observed between cd96−/− and wild type nk cells in their cytotoxicity in vitro, indicating a regulatory effect of cd96 on nk cytokine production rather than other direct modes of cytotoxicity. dysregulation of the tigit/cd96/cd226/cd155 axis and nk cell function has been observed in certain cancers. in hepatocellular carcinoma patients, the percentage and intensity of cd96 on nk cells, as well as the numbers of cd96+ nk cells, were higher in tumor infiltrating nk cells compared with nk cells from peri tumoral tissues [81]. it was observed that while tigit expression was similar between pancreatic cancer patients to that of healthy controls, cd155 was upregulated in pancreatic cancer tissue. the lower expression of cd226 and cd96 on nk cells may be contributing to tumor escape [82]. functionally exhausted cd96+ nk cells were significantly increased in the tumor tissues of patients with worse prognosis of hepatocellular carcinoma. these cells had impaired ifn γ and tnf α production and high gene expression of inhibitory cytokines il 10 and transforming growth factor beta 1 (tgf β1). blocking the cd96 cd155 axis or tgf β1 restores nk cell function by reversing nk cell exhaustion, suggesting a possible therapeutic role of cd96 in fighting liver cancer [81]. co blockade of cd96 and pd 1 potently inhibits lung tumor metastases and increases local nk cell ifn γ production and infiltration [83]. furthermore, when combined with cd96 blockade, anti ctla 4, anti pd 1, or chemotherapy were also more effective. four mabs against tigit are currently under investigation for immunotherapeutic use (figure 2 and table 2). tiragolumab (also known as mtig7192a and rg6058) is a fully human anti tigit antibody developed by genentech/roche that is being analyzed for use in immunotherapy in preclinical studies in combination with antibodies to pd 1. bristol myers squibb also initiated a phase i/ii study with an anti tigit antibody (bms 986207) as monotherapy or in combination with nivolumab in advanced solid tumors (nct02913313) (figure 2 and table 2). etigilimab (omp 313m32), developed by oncomed pharmaceuticals is under study in a phase i clinical trial (nct031119428) in solid tumors as a monotherapy or in combination with anti pd 1. ab154, developed by arcus bioscience is in a phase i clinical trial (nct03628677) that is evaluating the safety, pharmacokinetics, pharmacodynamics and preliminary efficacy in advanced solid tumors as monotherapy or combined with ab122 (anti pd 1) (figure 2 and table 2) [84]. in terms of clinical efficacy, it is crucial to note that tigit is upregulated in nk cells, which might indicate that unlike other checkpoint molecules such as pd 1, ctla 4 or lag 3, which are more restricted to expression on tumor infiltrating t cells, tigit is a checkpoint receptor more specific to nk cells. mabs targeting tigit for immunotherapeutic use [84]. abbreviations: mabs, monoclonal antibodies; pd 1, programmed cell death protein 1; tigit, t cell immunoreceptor with ig and itim domain. among resting lymphocyte populations, nk cells have the highest percentage expression of tim 3 [85]. galectin 9 is a ligand for tim 3 and is upregulated in a variety of cancers [86]. tim 3 has been described as both an activation marker and activation limiter of nk cells, as galectin 9 engagement can have contrasting effects [86]. for example, it has been shown that tim 3 is upregulated on human nk cells after activation and promotes ifn γ production in response to galectin 9 [85]. however, it was also demonstrated that when tim 3 was crosslinked on nk cells with antibodies or encountered target cells that expressed galectin 9, it suppressed nk cell mediated cytotoxicity [86]. the tim 3 galectin 9 pathway acts to suppress anticancer immune surveillance, contributing in particular to the immune escape of aml cells [87]. tim 3 is therefore an emerging target for aml. it may be effective to target tim 3 in patients displaying resistance to pd 1 blockade, particularly in cases where tim 3 expression is increased and thought to drive suppression [88]. a phase i clinical trial testing combinational treatment with pd 1 blockade and a tim 3 blocking therapeutic antibody, tsr 022 (developed by tesaro inc), is currently being administered in patients with advanced tumors (figure 2) [70]. tsr 022, is under investigation in three clinical trials for advanced cancer alone or in combination with anti pd 1 (nct02817633, nct03307785), and in primary liver cancer (nct03680508) in combination with anti pd 1. a novartis anti tim 3 antibody, mbg453 is also being evaluated as monotherapy or in combination with anti pd 1 in patients with advanced malignancies (nct02608268) and patients with aml or high risk myelodysplastic syndromes (nct03066648) (figure 2) [84]. however, the possibility remains that only galectin 9 positive tumors may be more susceptible to nk cell recognition and targeting via interaction with tim 3, which may be a potential limitation of targeting this molecule [85]. upregulation of the immunosuppressive cell surface glycoprotein, cd200, is a common feature of aml and is associated with poor patient outcomes. cd200hi patients display a 50% reduction in the frequency of activated nk cells (cd56dimcd16+) and a significant reduction in activating receptor surface expression and ifn γ levels compared to that of cd200lo patients [89]. as such, cd200 has a direct and significant suppressive influence on nk cell activity in aml patients and may contribute to the increased relapse rate in cd200+ patients, and therefore the cd200r is an attractive target to block, particularly for tumors with high cd200 expression [89]. however, blocking cd200r on nk cells may be difficult as it is also expressed on cd3+ t cells [90], and reproducibly at low levels on cd3−cd56+ nk cells [91]. other novel immune checkpoints are also being investigated in nk cells, including lag 3 which binds mhc ii and is expressed on ~10% of nk cells. however, since nk cells do not generally interact with mhc ii, alternative ligands have been proposed for lag 3 such as liver sinusoidal endothelial cell lectin expressed on melanoma cells, and galectin 3, expressed on stromal cells commonly found in the tme [92]. however, how relevant they are for nk cells remains to be investigated and the significance of lag 3 expression in nk cells remains largely unclear [70]. recently, it was shown that lag 3 is preferentially expressed on cd56dim nk cells after ifn α treatment and acts specifically as a negative regulator of cytokine production without compromising cytotoxic function [93]. another candidate, tnf receptor family member, ox40 is expressed on leukemic blasts in a substantial percentage of patients with aml and promotes activation and proliferation of t cells. ox40 ligand induced signaling via ox40 promotes nk cell activation, cytokine production and cytotoxicity, and disruption of this interaction impairs nk cell reactivity against primary aml cells [94]. thus, targeting this pathway may be beneficial for nk cell function against aml. finally, the il 1r8, a negative regulator of toll like and il r1 family signaling, is highly expressed on nk cells, and upregulated during nk cell maturation [95]. il 1r8 inhibits nk cell activation and nk mediated control of tumor growth and metastasis, highlighting its role as a checkpoint target in nk cell tumor immunity [95]. therapies targeting these checkpoint molecules may improve nk cell function against cancer, particularly if used in conjunction with existing gold standard treatments. additionally, combination therapies are currently needed to produce the greatest therapeutic benefit, as well as overcome resistance to checkpoint inhibition. in this regard, a recent study used combinations of dna methyltransferase inhibitors with anti tigit or anti killer cell lectin like receptor subfamily g member 1 antibodies and were able to reduce the formation of the tumor exposed nk cell phenotype, an nk cell phenotype that is unable to promote tumor killing and is responsible for promoting cancer metastasis [96]. this may be useful in an adjuvant setting to prevent metastatic recurrence. thus, the use of epigenetic modifiers in conjunction with checkpoint inhibitors are emerging as new therapeutic choices for cancer [97]. further, bike and trike platforms are versatile and amenable to incorporation of unique combinations, allowing for robust nk cell expansion, nk cell mediated cytotoxicity and nk cell survival in vitro. presently designed bikes or trikes concurrently engage with cd16 and tumor antigens to induce nk mediated adcc. for example, cd16/cd19 bikes and cd16/cd19/cd22 trikes have effectively mediated nk cytotoxicity on a range of specific antigen expressing targets and maintained functional stability after 24 and 48 h of in vitro culture [98]. it is possible to include checkpoint receptor blocking single chain variable fragments (scfvs) and various cytokines in these constructs to enhance nk cell mediated antitumor responses [70]. additionally, bikes and trikes have the advantage of increased biodistribution compared to mabs, due to their significantly smaller size (50 75 kda compared to 300 450 kda of full length antibodies), they are non immunogenic and can be quickly engineered [70]. effective design of these engagers could therefore enhance tumor recognition, reduce tumor escape and promote effector potency and specificity, which can improve long term success in the management of aggressive and refractory malignancies [99]. synthetic compounds such as nicotinamide (nam), glycogen synthase kinase (gsk) 3 inhibitors and recombinant heat shock proteins have also been used to enhance the proliferation and/or killing capacity of nk cells (figure 3). effects of nam, gsk 3 inhibition and hsps on nk cell function. nam induces the proliferation and homing of nk cells, while gsk 3 inhibition reduces checkpoint inhibitor transcription. hsps are endogenous stress signals that induce an upregulation of nkg2c and both gsk 3 inhibition and hsps induce a rise in tnf α and ifn γ levels. abbreviations: cd, cluster of differentiation; hsp, heat shock protein; ifn γ, interferon γ; lag 3, lymphocyte activation gene 3; nad, nicotinamide adenine dinucleotide; nam, nicotinamide; pd 1, programmed cell death protein 1; tnf α, tumor necrosis factor α. nam is a form of vitamin b3 and a potent inhibitor of the nad related signaling pathways that control redox sensitive enzymes, mitochondrial functions, cell metabolism and the production of energy and cell motility [100]. in vitro experiments have demonstrated that nam supplementation promotes nk cell function, antibody mediated killing, in vivo persistence and tissue trafficking in preclinical models and tolerance to oxidative stress. nam (2.5 5 mm) enhances expansion (60 80 fold) of functional nk cells in feeder free cultures (pb, cord blood or cd56 enriched cultures) and substantially upregulates the homing receptor cd62l in ex vivo expanded human nk cells (figure 3) [100,101]. these studies established that nam expanded nk cells enhance cytokine secretion against tumors, improve in vivo proliferation and homing to multiple organs including the bone marrow. bachanova and colleagues reported the first in human phase i study of nam expanded donor nk cells for the treatment of relapsed/refractory nhl and mm [102]. in vitro culture of cd3 depleted mononuclear cells, post donor apheresis, with nam and il 15 resulted in a 40 fold increase in nk cells after 14 16 days of culture; with the expression of homing receptor cd62l increasing from 2.9% in apheresis to 21% in final product. nam nk was infused into 3 patients without any dose limiting toxicity. response assessment at 2 months showed that 3 patients achieved complete metabolic remission. flow cytometry analysis of pb showed proliferation of nam nk in blood between days 2 and 7 in all tested patients. compared to host nk cells, donor nam nk cells demonstrated higher cd16 expression and enhanced proliferation [102]. as such, nam could be a desirable addition in ex vivo nk cell expansion. immunomodulatory imide drugs (imids) such as thalidomide and thalidomide derivatives, have the ability to indirectly augment nk cell cytotoxicity and proliferation via induction of il 2 transcription and secretion by t cells and dendritic cells (figure 4) [103,104]. furthermore, during in vitro culture of nk cells, the percentage of cd56+ cells is higher in the presence of imids relative to dmso control cultures showing a role for these drugs in the proliferation of nk cells [103] (figure 4). imids trigger the activation of phosphoinositide 3 kinase, and subsequently the nuclear translocation of the nuclear factor of activated t cells 2 and activator protein 1. however, they do not activate extracellular signal regulated kinase (erk) or p38 mitogen activated protein kinase, which mediate nk cell cytotoxicity and adcc, thereby lending strength to the argument that imid mediated nk cell activation is an indirect chain of events [103]. imids mediate indirect augmentation of nk cell function. imids act on il 2 transcription in t cells to induce the production of tnf α and ifn γ and the proliferation of nk cells. abbreviations: ifn γ, interferon γ; imid, immunomodulatory imide drugs; il 2, interleukin 2; tnf α, tumor necrosis factor α. cold target inhibition assays, which show the susceptibility of cancer cells to effector induced lysis, have demonstrated that imids are able to induce nk cell mediated lysis of mm cell lines and patient cancer cells. moreover, in vitro depletion of cd56+ cells, but not of cd4+ or cd8+ cells, blocked drug induced mm cell lysis, confirming that nk cells are the effectors mediating this response. thalidomide itself enhances the proportion and cytotoxicity of nk cells in responding patients with mm [103]. measurement of cytokines in the plasma from patients also showed increases in il 2 and ifn γ after thalidomide treatment. this was attributed to a direct effect on t cells, resulting in an increase in cytokine secretion, which subsequently augments nk cell number and function, and ultimately mm cell lysis [105]. emerging data also suggest that combining imids with tumor specific mabs may augment nk cell mediated antitumor responses. although imids have shown remarkable efficacy in patients with cancer, the contribution of imid mediated augmentation of nk cell function to these antitumor effects has yet to be established [104]. gsk 3α/β has a multifaceted role in cancer, having been involved in either inducing or inhibiting tumor proliferation and progression, suggesting it has multiple targets in different conditions [106]. recently in t cells, small molecule inhibition of gsk 3 was shown to downregulate, both immune checkpoints pd 1 and lag 3, through the upregulation of the transcription factor, t bet. further to this, increased antitumor immunity to b6 melanoma cells was observed using a combination of both gsk 3 inhibition and antibody blockade of lag 3. gsk inhibition also reduces lag 3 expression on nk cells, suggesting a similar mechanistic role for gsk 3 in both t cells and nk cells [107]. targeting gsk 3 in combination with lag 3 may therefore prove useful in cancer exhausted immune cells. gsk 3 also negatively regulates multiple nk cell activating signals through immunoreceptor tyrosine based activation motif (itam) dependent and  independent pathways. individual or combined engagement of the itam independent receptors nkg2d and 2b4 induces inhibitory phosphorylation of gsk 3 at regulatory serine residues, while activation of the itam dependent receptors cd16 and nkp30 inhibited the gsk 3β isoform specifically, potentiating the nk cell response. the extent of phosphorylation was closely correlated with the degree of nk cell activation [108]. nk cell cytokine production and cytotoxicity were consistently enhanced by the knockdown of gsk 3β or its inhibition with different pharmacological inhibitors, whereas inhibition of the gsk 3α isoform had no effect. in addition, nk cell function was augmented by the overexpression of a catalytically inactive form of gsk 3β [108]. in vitro studies have established that pharmacologic inhibition of gsk 3 in il 15 ex vivo expanded pb nk cells significantly upregulates cd57, a marker for late stage nk differentiation and maturation. when these nk cells were in the presence of gsk 3 inhibitors and exposed to human cancer cells, this resulted in significantly higher production of tnf and ifn γ, which translated into increased nk cell cytotoxicity and adcc. there was also increased expansion and proliferation of these effector nk cells. similar results were observed in vivo; in a xenogeneic model where luciferase expressing skov 3 tumors were established in nsg mice. adoptive transfer of gsk 3 inhibition conditioned nk cells displayed more robust and durable tumor control [109]. these studies indicate that the inhibition of gsk 3 can be used to increase the potency of nk cell function in vitro and in vivo, but given the many roles of gsk 3, its mechanism of action in different cancer types must be first elucidated. the expression of the stress inducible heat shock protein 70 (hsp70) on the cell surface of tumor cells acts as a recognition structure for nk cells [110]. recombinant hsp70 protein and synthetic hsp70 peptide tkd (tkdnnllgrfelsg; aa 450 463) are able to mimic surface bound hsp70 by increasing the cytolytic activity of nk cells [111] and initiate the migration of nk cells in a concentration dependent, highly selective and chemokine independent manner [112]. incubation of pb lymphocyte cells with tkd and low dose il 2, enhances the cytolytic activity of nk cells against hsp70 membrane positive tumors, in vitro and in vivo [113]. the tolerability, feasibility, and safety of tkd activated nk cells was assessed in a phase i clinical trial. in vitro analysis of patient peripheral blood lymphocytes revealed that after contact with tkd and low dose il 2, nk cells secreted high amounts of ifn γ and marginal secretion of tnf α. the apoptosis inducing granzyme b, a surrogate for hsp70 reactivity in nk cells, was detectable in the serum of all patients 1 day after reinfusion of tkd activated cells. cd94, also known as the nk activating receptor nkg2c, was also upregulated on nk cells of tumor patients after incubation with tkd. however, the expression of ncrs, nkp30 and nkp46 remained unaffected [113]. addition of synthetic hsp70 can therefore increase the cytotoxicity and manipulate chemotaxis of nk cells. immune cells can be equipped with cars specific for tumor antigens. these constructs specifically target cells expressing the antigen of choice via a scfv and activate cells via an intracellular signaling domain. car t cells have shown considerable success against liquid cancers, which is exemplified by the fda approval of the cd19 specific autologous car products tisagenlecleucel and axicabtagene ciloleucel [114]. despite this success, generating cars for nk cells possesses challenges that may be addressed through the exploitation of nk cell function in specific tumor environments to generate more effective car constructs. car constructs are made up of the following main components: the ectodomain (comprised of a scfv and hinge), the transmembrane domain (tm) and the intracellular signaling endodomain (figure 5). components of a first generation car. a car is made up of the ectodomain (composed of a scfv and hinge), the transmembrane domain and the intracellular signaling endodomain. abbreviations: scfv; single chain variable fragment. the ectodomain portion dictates the antigen specificity of the car. car ectodomains targeting cd19 and cd20 have been used to treat b cell leukemia and lymphoma, cd138 for myeloma, cd33 and cd7 for aml, members of the human egfr family for breast cancer and the disialoganglioside gd2 for neuroblastoma [115,116]. with advancing technology, dual cars that have ectodomains targeting two different moieties or different antigens on the same target cells are fast becoming mainstream. one approach generating interest is multiple antigen targeting, which aims to increase specificity, capture a variety of tumor clones and reduce antigen negative relapse. an example of this approach is the targeting of egfr and its mutant form egfrviii, which are overexpressed in a large proportion of glioblastomas (gbms). genßler et al. generated variants of nk 92 cells that express cars carrying a composite cd28 cd3ζ domain for signaling and scfv antibody fragments recognizing either egfr, egfrviii, or an epitope common to both antigens [117]. in vitro analysis revealed high and specific cytotoxicity of egfr targeted nk 92 cells against established and primary human gbm cells, which was dependent on egfr expression and car signaling. egfrviii targeted nk 92 only lysed egfrviii positive gbm cells, while dual specific nk cells expressing a cetuximab based car were active against both types of tumor cells. in immunodeficient mice carrying intracranial gbm xenografts expressing either egfr, egfrviii or both receptors. local treatment with dual specific nk cells was superior to treatment with the corresponding monospecific car nk cells. this resulted in a marked extension of survival without inducing rapid immune escape, as observed upon therapy with monospecific effectors. their results demonstrated that dual targeting of car nk cells reduces the risk of immune escape [117]. in fact, some clinical trials are ongoing to assess bi specific car t cell therapy efficacy of anti cd19/cd22 (nct03448393) and anti cd19/cd20 (nct03271515) to prevent recurrence caused by antigen loss [116], which could be translated into the nk platform too. cars for car nk cell therapy must be specifically designed with the unique characteristics of nk cells in mind. car induced cytotoxicity and cytokine production is mediated through adapter protein signaling. in general, first generation car ts typically have cd3ζ as their signaling domain, and second  and third generation cars sequentially contain other co stimulatory molecules such as cd28, 4 1bb and cd314. nk specific car constructs have largely focused on incorporating either dnax activation protein 10 or 12 (dap10 or dap12) as the activating domain or as a costimulatory domain alongside cd3ζ [118]. a cd19 targeting first generation car, cd19 cd3ζ, was shown to outperform cd19 dap10 cars, and its activity was further improved by the addition of a 4 1bb co signaling domain [119]. the inclusion of both dap10 and cd3ζ signaling, together with the activating molecule nkg2d arms nk cells with better killing power for targeting many cancer types. nkg2d dap10 cd3ζ car transduction of nk cells increases nkg2d surface expression, infers more cytotoxicity against leukemia and solid tumor cell lines compared to mock transduced cells, triggers secretion of ifn γ, gm csf, il 13, mip 1α, mip 1β, ccl5 and tnf α, and induces a massive release of cytotoxic granules, which persist after 48 hours of continuous stimulation [120]. these cells also had considerable in vivo antitumor activity in a mouse model of osteosarcoma compared to control nk cells [120]. however, in the tme suppressive molecules such as tgf β [121], may downregulate dap10 subsequently limiting nk cell function. to overcome this suppression, studies have modified nk cells to contain a chimeric nkg2d receptor with the native molecule fused to cd3ζ [122,123]. more recently, this has shown that unlike endogenous nkg2d, the chimeric version is not susceptible to down regulation via the tme. when these cells were generated from patients with neuroblastoma they were able to kill autologous myeloid derived suppressor cells capable of suppressing car t function [123]. thus, a combination therapy inclusive of these cells may be of benefit in tackling solid tumors. unlike dap10 in first generation car nk cells, dap12 outperformed cd3ζ in first generation prostate stem cell antigen (psca) targeting cars, both in vitro and in vivo [124]. an anti psca dap12 car improved cytotoxicity of the nk cell line yts against psca positive tumor cells when compared with a psca car containing the cd3ζ domain. notably, infused yts nk cells armed with psca dap12 cars resulted in complete tumor eradication in vivo. the selection and placement of costimulatory domains can have different impacts on the cytotoxic function and ultimately persistence and safety. thus, it is important to establish the function of different ectodomain and endodomain combinations. li and colleagues performed the largest comparison of nk specific car constructs in nk 92 cells, which evaluated four different transmembrane domains (cd16, nkp44, nkp46, and nkg2d) and four different costimulatory domains (2b4, dap10, dap12, and cd137) in various combinations with cd3ζ [125]. the most effective construct contained the nkg2d transmembrane domain and 2b4 co stimulatory domain which was attributed to their leading effector cell proliferation and function. 2b4 also has strong co stimulatory effects in cars targeting cd19 or gd2 [114,125]. in vitro stimulated nk cells from healthy donors and leukemia patients were gene modified to contain cd19 or gd2 specific cars with either cd3ζ or 2b4 domains, alone or combined. chimeric 2b4 signaling alone failed to induce il 2 receptor upregulation and cytokine secretion but triggered a specific degranulation response. however, the co integration of 2b4 and cd3ζ significantly enhanced all aspects of the nk cell activation response to antigen expressing leukemia or neuroblastoma cells, including ifn γ and tnf α secretion and release of cytolytic granules [126]. they also overcame nk cell resistance of autologous leukemia cells while maintaining antigen specificity. these data indicate that 2b4 signaling has a potent costimulatory effect in nk cells when used in car domains. a major issue of infused nk cells is their relatively short in vivo persistence. as mentioned previously, it is now well established that while il 2 and il 15 can prolong nk cell survival, systemic administration of il 2 and il 15 has been associated with severe side effects in clinical trials [127]. others are trying to tackle this problem by engineering car nk cells themselves to express cytokines (see section 3.1). for example, cord blood derived cd19 car nk cells were engineered to express il 15 [48]. along with efficient killing, these cells also produced their own soluble il 15, which improved their function and allowed growth and survival over a period of 42 days. however, the infusion of high doses of these cells into raji xenograft mouse models induced crs and fatality in 4 mice [48]. clearly, engineering cells to secrete cytokines may cause problems and does not appear to be the only solution required to improve nk cell persistence. a way of addressing this and other potential safety concerns may be the incorporation of a suicide gene within the nk cells [127]. takeda have already incorporated the inducible caspase 9 (icasp9) suicide gene in tak 007 (table 1) and md anderson cancer center have placed their cord blood derived cd19 car nk cells containing icasp9 in clinical trials (nct03056339, see section 3.1). nk based therapy is generally considered a safer alternative to car t therapy because of less occurrence and severity of side effects such as crs. the cytokine production of nk cells is considered to be safer because the cytokines released are mainly ifn γ and gm csf and once infused into patients car nk cells are short lived, thereby reducing chances of other side effects on exposure. several clinical studies have shown that nk cells do not cause gvhd [116] and safety and efficacy of nk cell therapies for hematological tumors such as aml are well established [128]. new clinical trials targeting hematological malignances are attempting to improve treatment response and nk cell number and dose. for example, a clinical trial showed that car nk 92 cells can be infused at doses up to 5 billion cells per patient without causing significant adverse effects in patients with relapsed or refractory aml [129]. there are many ongoing clinical trials for both hematological and solid tumors that have not yet reported safety outcomes, but are elegantly reviewed in liu et al. [128]. regardless, the potency of car nk cells and also the method of generation and expansion of these edited cells warrant safety features as discussed below. an ideal suicide gene would guarantee the safety of cellular therapies by permanently removing the cells responsible for any toxicities. to increase safety of car t cell therapies, vectors designed to deliver the car can be engineered to co express suicide genes to allow removal of car t cells in case of uncontrollable severe adverse events. examples of clinically available suicide gene strategies include the herpes simplex virus thymidine kinase (hsv tk) suicide gene, which makes cells sensitive to ganciclovir induced cytotoxicity or the icasp9 gene cassette, leading to rapid caspase mediated apoptosis of expressing cells upon application of a synthetic inducer of dimerization, such as ap1903 or ap20187 [130]. currently, phase i clinical trials for the treatment of sarcoma and neuroblastoma utilizing gd2 car t cells (nct01822652) containing suicide genes, are ongoing [131]. the effectiveness of four suicide gene strategies have been compared in vitro using epstein barr virus cytotoxic t cells genetically modified to express hsv tk, icasp9, mutant human thymidylate kinase (mtmpk), or human cd20 codon optimized suicide gene. in this study, activation of hsv tk, icasp9, and cd20 ultimately resulted in equally effective destruction of transduced t cells. however, while icasp9 and cd20 effected immediate cell death induction, hsv tk expressing t cells required 3 days of exposure to ganciclovir to reach full effect, and mtmpk transduced cells showed lower t cell killing at all time points [132]. the source of the suicide gene is also an important component of the strategy design. for example, in contrast to the icasp9 suicide gene, which is almost completely human derived, viral derived systems, such as hsv tk, proved immunogenic in immune competent patients with limited persistence of hsv tk cells [133]. therefore, there is a preference for using a modified human icasp9, fused to a human fk506 binding protein to allow conditional dimerization using a small molecule pharmaceutical. a single 10 nm dose of synthetic dimerizer drug induced apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo [134]. this system consists of human gene products with low potential immunogenicity, the administration of dimerizer drug has no effects other than the selective elimination of transduced t cells and that icasp9 maintains function in t cells overexpressing antiapoptotic molecules [134]. although mostly implemented in car t cells, such safety features should perhaps also be incorporated in car nk cells. nk cell safety may also be improved by using masked cars, a technique currently being investigated in t cell safety. car masking involves the use of a peptide that blocks antigen binding sites and a protease junction. when protease cleaved junctions are detached from masking peptides in the tme, car t cells are kept active in local tumor sites [135]. one example is an egfr specific masked car, which consists of a masking peptide that blocks the antigen binding site and a protease sensitive linker. proteases commonly active in the tme can cleave the linker and disengage the masking peptide, thereby enabling car t cells to recognize target antigens only at the tumor site. in vitro masked cars show dramatically reduced antigen binding and antigen specific activation in the absence of proteases, but normal levels upon treatment with certain proteases, along with comparable antitumor efficacy to that of unmasked cars in vivo [135]. the effectiveness of this approach clinically is obviously dependent on the level of the required proteases, which is likely to differ within and between the tme. another strategy to generate car t cells that are effective at the tumor site (thereby potentially reducing off target effects), is the use of oxygen sensitive cars. by combining an oxygen sensitive subdomain of hypoxia inducible factor 1 with a car structure, car t cells can subsequently respond to a hypoxic environment, a hallmark of many solid tumors. juillerat et al. used a double input approach within a single car, by including both antigen and hypoxic recognition. under hypoxic conditions, they observed increased car surface expression and improved cytolytic properties [136]. unlike those focused on improving car t cell cytotoxicity and proliferation through design of second  and third generation car molecules, this approach showed that the tme can induce innately self deciding car t cells with the ability to inactivate in presence of hypoxia. however, additional studies are required to confirm the validity of this approach. this study was not only the first proof of concept of a stand alone use of the engineered oxygen sensitive car, but an amenable platform for multi receptor boolean logic gate application of “and” and “not” gates to further enhance the control of engineered cells [136]. the boolean logic gates of “and”, “or”, and “not” have been applied to strategies for developing multi antigen targeting car t cells that avert tumor antigen escape and reduce on target, off tumor toxicities [137]. combinatorial targeting can be achieved by splitting the activation and co stimulation across two receptors (logic and gate) or by combining activating and inhibiting cars (logic not gate) based on the concepts of boolean logic. on target, off tumor effects can be minimized by adding and gated circuits requiring both antigens to be present for car activation, or not gated circuits which will activate in the presence of one antigen only if the other is not present. targeting one antigen or another can eradicate multiple clones and reduce antigen negative relapse, and can be achieved by either infusing two separate populations of car t cells, transducing two cars into the same cell, or by the novel tandem car [138]. morsut and colleagues, as well as roybal and colleagues improved the and gate combinatorial detection of multiple antigens by engineering notch based receptors that function independently from a car activated t cell receptor (tcr) pathway [139,140]. the design of combinatorial antigen targeting is taking advantage of the synthetic notch receptors (synnotch), a new class of modular receptors comprising an extracellular recognition domain; the transmembrane “core” domain; and the intracellular transcription domain that can be cleaved and released by a transcriptional activation domain translocating to the nucleus and regulating transcription upon ligand engagement. furthermore, car t cells have been designed to activate when synnotch and car ligands are co expressed on tumors, a strategy that spatially identifies and effectively controls car t cell activation [139,140]. using combinatorial or dual targeting by cars can increase their safety profile. through the incorporation of the synnotch platform, roybal et al. constructed two combinatorial antigen recognition t cell circuits [cd19 synnotch/mesothelin car and green fluorescent protein synnotch/cd19 car] and demonstrated that these receptors could conditionally express cars specific for a second antigen in the presence of the first antigen specific for the synnotch receptor [140]. another form of combinatorial targeting is the use of inhibitory cars (icars). if dual antigens are simultaneously expressed on healthy cells rather than on tumor cells, the combination of inhibitory receptors specific for the antigen present on normal but not on tumor cells will protect the normal cells from a car t cell mediated attack because of negative signaling conferred by icars. fedorov et al. pioneered an anti prostate specific membrane antigen icar, carrying intracellular tails of ctla 4 or pd 1 and tested whether these receptors have the ability to block tcr  or car driven t cell functionality in vitro and in vivo [141]. this study demonstrated that the icar can inhibit tcr or car responses in an antigen restricted manner. t cell stimulation experiments showed that icar mediated inhibition was reversible which ensured that most of the t cells’ previous engagement of icar can retain functionality, although some t cells could be anergized over time [142]. a potential disadvantage of combinatorial antigen targeting strategies is the possibility of target antigen modulation leading to tumor escape, with increasing probability of escape with the requirement of additional antigen targeting. the decrease in the target antigen could potentially render the engineered car system either partially or completely unresponsive or non protective. additionally, the selection of multiple suitable targets might be complicated due to their different expression levels [136]. knockout (or knockdown) of regulators in the signaling pathway that restrict nk cytotoxicity and/or expansion can also aid in augmenting nk function. the suppressor of cytokine signaling (socs) proteins (cis, socs1 7) are intracellular negative regulators that are often induced to limit cytokine signaling in a classic negative feedback loop [143]. the cytokine inducible sh2 containing protein (cis, encoded by cish) is a negative regulator of il 15 signaling and is increased in nk cells upon cellular activation and thus functions as a key suppressor of il 15 signaling in nk cells [60]. il 15 binding induces activation of the janus kinase (jak)1 and jak3 tyrosine kinases, recruitment and activation of signal transducer and activator of transcription 5 (stat5), and transcription of stat5 target genes, which drive survival and proliferation of nk cells and encode cytotoxic agents such as granzymes [144]. systemic delivery of il 15 to patients has toxic side effects. thus, new approaches that deliver il 15 more precisely to the target cell or specifically render the target cell more responsive to il 15 are appealing avenues for investigation (see section 3.1). cis is an important intracellular checkpoint in nk cell mediated tumor immunity. deletion of cish results in elevated il 2rβ expression and increased activity and expression of jak1 and jak3/stat5 pathways, with a corresponding increase in nk cell survival, proliferation and cytotoxicity [145]. loss of cis also results in heightened and prolonged il 15 driven jak stat signaling in nk cells. cish−/− nk cells are hypersensitive to il 15 and, as a result, cish−/− mice are resistant to experimental tumor metastasis. in this regard, intravenous administration of b16f10 melanoma cells to cish+/+ mice resulted in extensive metastatic nodule formation in the lungs by 14 days. in contrast, b16f10 metastatic nodules were largely absent from cish−/− mice [145]. similar differences in lung metastasis were observed when using the rm 1 prostate cancer cell line. depletion of nk cells or neutralization of ifn γ, but not depletion of cd8+ t cells, rendered cish−/− mice susceptible to b16f10 metastasis, suggesting that cis is involved in the negative regulation of nk cell activity and ifn γ in this particular model [145]. however, despite the notion that nk cells control many hematopoietic malignancies, there can be a variable degree of cis dependent protection depending on the tumor model. cish deficiency did not seem to have a major impact on disease progression in mouse models injected with the rma s or vk12653 mm cell lines [145]. combining blockade of the inhibitory receptor cd96 or administration of low dose il 15 with cis inhibition could further boost nk cell activity and enhance protection against tumor metastasis [144]. the impacts of cish deletion appear to be restricted to a subset of those tumors where nk cells are critical. nk cells critically regulated by il 12, il 18, and type i ifn are less likely to be influenced by cish deletion. this may be the case in the tme where nk cells may engage directly with tumor cells or in crosstalk with antigen presenting cells, and il 12, il 18 and type i ifn are influential [145]. nevertheless, deletion of cish in nk cells derived from ipscs may be useful as off the shelf therapies (see section 4) for a variety of cancer indications. zhu et al. have recently developed human cish knockout nk cells using an ipsc nk cell platform, which demonstrated increased il 15 mediated jak stat signaling activity, improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations [146]. they also displayed significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model and mechanistically improved metabolic fitness which contributed to enhanced nk cell function. cish−/− ipsc nk cells were shown to be phenotypically mature and have normal proliferative capacity under highly stimulated culture conditions exhibiting significantly increased cytotoxic granule release (cd107a expression) and ifn γ production when stimulated. analysis of cell signaling pathways demonstrated markedly increased phosphorylation of il 15 stimulated jak1 tyrosine phosphorylation, stat3, and stat5 in cish−/− ipsc nk cells. activated nk cells upregulate the rate of glucose driven glycolysis and oxidative phosphorylation to provide energy to drive the pathways necessary for their effector functions. this suggests that the increased ifn γ production and cytotoxic activity of cish−/− ipsc nk cells were supported by increased glycolysis and oxidative phosphorylation [146]. together, these studies demonstrate that cis plays a key role in the regulation of human nk cell metabolic activity and thereby modulates antitumor activity. cyclin dependent kinase 8 (cdk8) belongs to the family of transcription regulating cdks. cdk8 regulates transcription downstream of cytokines utilizing the jak stat pathway, thereby also interfering with and regulating immune responses [147]. cdk8 deletion in mice does not affect the frequency or total number of nk cells in the bone marrow and has no significant effect on the maturation of these cells. therefore, this indicates that cdk8 is dispensable for the growth and development of nk cells. cdk8 deleted nk cells display markedly increased perforin expression and increased cytotoxicity in vitro [147]. this is because cdk8 mediates stat1 s727 phosphorylation which inhibits nk cell cytotoxicity. the knockdown of cdk8 in primary nk cells reduces stat1 s727 phosphorylation in mice [148]. diacylglycerol kinases (dgks) are physiological regulators of t and nk cell development, differentiation and function. they negatively regulate diacylglycerol (dag) signaling by phosphorylating dag into phosphatidic acid [149]. dag leads to downstream activation of erk, nfκb, and akt pathways mediated by protein kinase c and ras activating protein [150]. cytotoxicity and production of ifn γ, which are controlled by the erk pathway, are the most important effector activities required for tumor elimination. thus, control of the erk pathway ultimately determines t and nk cell antitumor activity. there are 10 different isoforms of dgk comprising five different classes, each with specific structural motifs, tissue localization and functional roles. of these variants, dgkα and dgkζ are dominantly expressed in t cells [151,152]. the inhibitory role of dgks in car bearing t cells in the tme is exemplified when car ts are isolated from mesothelin expressing flank tumors and evaluated for effector functions and status of inhibitory pathways. these car t cells become rapidly hypofunctional with upregulation of intrinsic t cell inhibitory enzymes (dgkα, dgkζ, shp 1) as well as expression of other surface co inhibitory receptors [153]. hypofunctional cd8+ tumor infiltrating t cells from clear cell renal carcinoma also displayed upregulation of dgkα [154]. however, functionality was restored upon use of the commercial dgk inhibitor r 59022. prinz et al. also rescued nk cell function in clear cell renal carcinoma by blocking dgk mediated diminishing of erk pathway using r 59022 [155]. since r59022 is a relatively selective dgkα inhibitor, restored degranulation capacity of tumor infiltrating lymphocytes (tils, such as cd8+ t cells and nk cells) was attributed to dgkα. of note, the level of degranulation of tils in the presence of dgk inhibition was not higher than that observed with non tumor infiltrating lymphocytes, indicating that dgk inhibition can restore suppressed degranulation but does not augment degranulation beyond nk or t cell intrinsic response efficacy [149]. this finding helps alleviate concerns about potentially unleashing undesirable autoimmunity through dgk inhibition, which is an important issue when considering potential targeting of dgk in a clinical setting. collectively, these results suggest that dgk inhibition may not only prevent development of unresponsiveness but may also be able to restore activity of suppressed immune cells. existing methods for dgk inhibition are challenging. for example, there are no specific dgkζ inhibitors available to date [156]. commercial dgk inhibitors r59022 and r59949 are only relatively selective for dgkα and could therefore also be inhibiting a broad range of dgk isotypes [157]. additionally, their chemical structure is analogous to ritanserin (a serotonin receptor antagonist) and have demonstrated higher binding affinity to serotonin receptors [158]. this challenges the hypothesized biochemical mechanism of improved til efficacy previously observed with these inhibitors. better inhibitors are underway; such as a more specific inhibitor for dgkα [159] or a recently patented new class of promiscuous dgkα/dgkζ inhibitors [156], but dgk isoform specific knockout models are of increasing interest in the field. both dgkα and dgkζ knockouts, either individually or simultaneously, have shown improvement in t cells. dgkζ−/− t cells showed increased proliferation, enhanced il 2 production and enhanced ras erk activation upon tcr stimulation than wild type (wt) t cells both ex vivo and in vivo [160,161,162]. in addition to this improved in vitro and ex vivo performance, dgkζ−/− mice rejected various tumors more efficiently than wt mice. dgkα−/− t cells also resulted in enhanced ras erk activation and increased proliferation post tcr stimulation, but unlike dgkζ−/− t cells they displayed normal phosphatidic acid production, which highlights the downstream molecular differences between the two isoforms [163,164]. dgkα−/− t cells displayed a weaker increase in cytokine production and erk phosphorylation compared to dgkζ−/− t cells in vitro. however, adoptive transfer of either dgkα−/− or dgkζ−/− car t cells yielded similar increases in efficacy compared to wt t cells in the treatment of murine mesothelioma [163,165]. double isoform knockout of dgkαζ using crispr/cas9 has generated car t cells that are less sensitive to hypofunction and have increased antitumor effects [166]. taken together, these studies indicate that although the ideal target isoform is still ambiguous, dgk knockout reverses exhaustion and results in more functional t cells. genetic ablation of dgkζ leads to hyperresponsive nk cells in a cell intrinsic and developmentally independent manner. interestingly, dgkζ−/− but not dgkα−/− nk cells are hyperresponsive to activating receptor stimulation [167]. upon activation of three distinct activating receptor families (itam dependent: nk1.1, ly49d; costimulatory like: nkg2d; sap dependent: 2b4), an increased fraction of dgkζ knockout nk cells degranulated and produced ifn γ compared with wt nk cells. in contrast, there was no significant difference between dgkα−/− mice and wt controls in terms of both degranulation and cytokine production, suggesting that dgkα does not contribute to limiting nk cell function. dgkζ−/− nk cells exhibited increased perk, but not pakt or enhanced ikba degradation, suggesting that increased perk could be a major contributor to the enhanced nk cell function seen in dgkζ−/− nk cells. taken together, these data suggested that dgkζ deficiency enhances nk cell responsiveness to activating receptor stimuli in an nk cell intrinsic and developmentally independent manner, by affecting erk, akt, and nf κb pathways. although the loss of dgkα does not seem to influence nk cell effector function and thus may not negatively regulate signaling downstream of nk cell activating receptors, a more in depth study into its other roles could potentially demonstrate a role for dgkα in nk cell function [167]. taken together, crispr/cas9 induced knockout of dgk isoforms and other checkpoints may be another promising tool in battling tumors using nk cells. the next generation of nk cell products will ideally be available off the shelf. however, development of off the shelf nk cells would ideally require that they can be manufactured and modified (genetically and as described above) prior to need, cryopreserved and then infused on demand. before this can be realized, there are several considerations that need addressing including the source of cells. whilst it has been extensively demonstrated that nk cells are potent cytotoxic cells in vitro, in vivo and clinically, sourcing sufficient numbers of nk cells for clinically relevant doses remains challenging. nk cells for clinical use are primarily derived from pb. however, they are also available as immortalized cell lines such as nk 92 or can be differentiated from stem cell sources. autologous nk cells derived from the patient are difficult to expand and often of poor quality following extensive lymphodepleting chemotherapy. furthermore, these cells can display limited antitumor effects for a range of malignancies due to kir hla matching induced “self tolerance” [168]. therefore, ensuring a mismatch between donor nk kir and recipient hla is imperative for enhancing the resultant tumoricidal effect. whilst anti kir antibodies may also block kir hla matching and thereby increase nk function [56], allogeneic nk products offer an easier approach to facilitate hla kir mismatch without having been previously exposed to chemotherapy. the immortalized nk cell lines nk 92, khyg 1, hank 1, nkg, nk ys, yts, yt, nk3.3 and nkl have been used in preclinical studies. however, to date, only nk 92 cells have been approved for clinical use due to observed clinical benefits and minimal side effects [19,169]. of note, from the 19 (as of march 2021) current clinical studies assessing car nk cells, 5 are nk 92 based, which is indicative of successful preclinical investigations [170]. even though nk 92 cells provide a flexible nk based immunotherapy platform, they have certain shortcomings. since nk cells have a short life span and have limited in vivo expansion, this ultimately may require repeated patient treatment to maintain or elicit the desired clinical effect. nk 92 cells originate from a non hodgkin lymphoma, therefore they must undergo γ irradiation prior to infusion to minimize the risk of secondary lymphoma formation in patients [171]. this, however, may further limit their function and persistence in vivo. in line with their limited expansion kinetics and malignant origin, tang et al. have recommended the use of car nk 92 cells to treat slow progressing malignancies such as mm and inclusion of suicide genes as a safety measure in lieu of irradiation [129]. nevertheless, the possible tumorigenicity of nk 92 cells remains a major concern. a recent study by oberoi et al. [42] showed that healthy donor pb can serve as a valuable starting material for creating hematopoietic stem and progenitor cells (hscs), despite earlier studies indicating poor and donor dependent differentiation [172,173,174,175,176]. this was due to a refined approach to differentiation and expansion protocols that utilized optimized cytokine cocktails and expansion enhancement with k562 feeder cells, co expressing 4 1bb ligand and mbil 15 and il 21 [42] (see section 3.1). as healthy pb is readily available and easier to access compared to umbilical cord blood (ucb) and human embryonic stem cells (hescs), this protocol may serve as an economically viable option for nk cell generation. an important and rapidly emerging source of nk cells are hscs. functional nk cells can be differentiated from hscs derived from ucb, ipsc lines and hescs [177,178,179]. furthermore, nk cells derived from hscs do not need irradiation prior to dosing. mice treated with hesc nk cells showed complete clearance of subcutaneous k562 induced tumors, with some animals remaining tumor free for 8 weeks after just one treatment injection. a significant reduction in metastases was also observed after treatment with the hesc nk cells. in contrast, ucb nk cells were shown to be less effective, with significantly more cancer growth [179]. although further studies are required to confirm these comparative observations, ethical concerns related to the use of hescs and the low initial number of progenitor cells in multipotent ucb are relevant limitations [179]. ipscs, however, can potentially undergo unlimited expansion in vitro without losing pluripotency and therefore provide a potentially endless supply of high purity (>97%) nk cells [177]. accordingly, it is thought ipsc derived nk cells have the potential to be a potent off the shelf immunotherapy allowing for lower dosage treatments and flexible administrations. phenotypically, these cells have similar activating receptor profiles to pb nk cells, but negligible kir expression [179,180,181,182]. the reduced inhibitory receptor expression may explain the observed higher cytotoxicity of ipsc derived nk cells. it will be important to further elucidate how kirs contribute and affect the function and specificity of ucb and ipsc nk cells. in this regard, goldenson et al. recently showed nk cells expanded directly from ucb (ucb56) had higher kir expression than nk cells generated from cd34+ cells derived from ucb (ucb34), with ucb56 nk cells exhibiting more cytotoxicity. they also found that ipsc lines derived from nk cell populations had variable kir expression, with some lines generating nk cells with high levels of kir, and some with low levels. however, no difference in cytotoxicity was observed between these ipsc lines. they investigated the importance of the aforementioned differences of kir expression by measuring cytotoxicity against targets expressing or lacking kir ligands using a cell line system that expressed hlas. the presence of these ligands did not alter the difference in killing activity between ucb56 and ucb34 cells, or ipscs suggesting the widely accepted hla recognition role of kirs is not occurring in their system, as hla presence on the targets should have decreased killing capacity of high expressing kir+ nk cells. the authors suggest that this may be attributed to feeder based expansion which drives differentiation at the same time it attenuates inhibitory signaling, but other influences may also be possible [183]. car engineered ipsc nk cells have successfully targeted human tumors in preclinical studies and currently there are a number of ongoing clinical trials for cancer immunotherapy using engineered ipsc nk cells [50,125,128]. furthermore, differentiation strategies for ipsc to nk cells are adaptable as they can allow for focus on obtaining a certain phenotype and function for the resulting product. thus, the high proliferation capacity and unlimited expansion potential, as well as the ease of gene editing and modification of ipscs make these cells a candidate for developing libraries composed of off the shelf haplotype specific cells for treating a range of diseases [128]. nk cells, with their panel of multiple anticancer receptors and better safety profile, are emerging as crucial candidates for adoptive cellular immunotherapy for the treatment of cancer. however, significant roadblocks prevent their widespread clinical translation. as discussed above, nk cells comprise only 10% of the normal pb population, even less for patients undergoing chemotherapy which often causes lymphopenia, making nk cell extraction from patients very ineffective and invariably of compromised function. another shortcoming is their reduced in vivo persistence [184]. allogeneic nk cells, such as ipsc derived nks (inks) may provide a rational alternative. given that ink cells are not extracted from pb and therefore contain no contaminating t or b cells, there will be negligible risks of gvhd or a cytokine storm. key questions still remain: could in vivo persistence be increased? is it more desirable to increase ink function to yield short lived, potent killer cells, which also reduces the extent of side effects? how could genetic engineering, particularly crispr/cas9 technology play a transformative role in generating these potent cells? if so, manipulation at what stage of the nk cell ontogeny would ensure successful and stable genome editing? engineered nk cells as immunotherapeutic agents are still in their infancy. there is an increasing body of evidence describing the success of crispr technology in nk cells [185]. since mature nk cells have low persistence in vivo, the stable integration, expression and resultant effect of the edited gene has a very limited time to display, if at all. to allow for stable integration, stem cells could be edited and differentiated into functional nk cells. ink functions could be modulated by manipulating their own natural cytotoxicity receptors, such as incorporating an antigen specific scfv based domain into them. in fact, observations that show nk cars function better when natural nk signaling mechanisms are utilized [125] and that t cell receptor fusion constructs successfully employ the natural tcr signaling subunit of t cells [186] pave the way for novel receptor engineering of existing natural nk receptors. perhaps this technique could allow for targeted and successful activity of adoptive immunotherapy into solid cancers. nk cells have already displayed in vitro and in vivo cytotoxicity in a number of ovarian cancer models such as skov 3 and ma 148 and could therefore be of clinical significance, particularly after appropriate editing to assist in trafficking and function [177,187]. the figures were created with biorender.com. no external funding was received. not applicable. not applicable. not applicable. all authors, except r.i., are paid employees or advisors of cartherics, and a.p., v.e., n.b., r.s., r.b. and a.t. hold equity in the company. publisher’s note: mdpi stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this section collects any data citations, data availability statements, or supplementary materials included in this article. not applicable. articles from cells are provided here courtesy of multidisciplinary digital publishing institute (mdpi) connect with nlm national library of medicine 8600 rockville pike bethesda, md 20894

supported by a conversation with … cats are more social than they are often given credit for. can you help yours access its inner dog? by emily anthes emily anthes has a slightly skittish dog and a highly gregarious cat. this article is part of our pets special section on scientists’ growing interest in our animal companions. over the last two decades, a flurry of scientific studies have demonstrated, over and over again, that dogs are social savants, highly attuned to human cues. but even as canine cognitive science flourished, few researchers bothered to probe the social skills of cats. after all, dogs were descended from the social gray wolf and had been intentionally engineered to perform specific roles alongside humans. cats, on the other hand, were descended from the solitary african wildcat and had not been under the same selective pressure from people. they were viewed as antisocial and also, for good measure, uncooperative, making them unappealing research subjects. in recent years, however, a handful of undeterred scientists have produced a small body of research suggesting that we have underestimated cats’ social skills, and that interest is growing. “i see more and more papers each year,” said kristyn vitale, an animal behavior scientist at unity environmental university in maine. “we’ve just got a lot of catching up to do.” dr. vitale, who has three cats of her own, often collaborates with monique udell, the director of the human animal interaction lab at oregon state university. dr. vitale spoke to the new york times about their research   and about her dream study of cat cognition. the following has been edited and condensed for clarity. what do people tend to get wrong about cats? the biggest thing that i see is people stating that cats are not social creatures, or that social interaction isn’t important for cats. cats are really flexible with their social behavior. so it’s highly individual, and based off the cat and their personal experiences.

the leading digital event revolutionizing patient outcomes in cancer therapies “the level of presentations was excellent. a good place to learn about the latest developments and meet great people.” tailor your agenda   with sessions spanning across multiple days, design your own agenda based on who you want to hear from and what will be most useful to your business. get more involved   virtual events offer many more interactive opportunities than live events; participate in live polls, live q&a and live panel discussions   helping you build your personal brand and your business connections. improved audience visibility   you can see exactly who is registered to attend the event and when they’re online   make that valuable connection in real time! on demand content   missed a session or couldn’t tune in that day? that’s fine, sessions will be available to listen to for 2 months post event. that also means you can listen to your favourite speaker multiple times. high quality speakers   this doesn’t change, whether we run a live event or a virtual one, we recruit industry leading, insightful speakers to inspire.more networking opportunities   do you sometimes miss the one person you were hoping to meet at live events? with virtual events you can instant message them, reserve a time in their diary, and email them. save time & money   tune into an event from the comfort of your own home, no need to travel overseas, spend time away from loved ones and spend your company’s money! this makes networking and knowledge sharing much more cost efficient. increased content   we can run more sessions at one time without the constraints of venue capacities, meaning we can provide you with more valuable content in one go. business development opportunities   for those looking to partner on the virtual events, there’s lots of session formats that will place you at the centre of the conversations you need to have, including but not limited to; topic specific roundtables, keynote panels, ask the expert chat rooms and more. talk to our team to find out more [email protected]. insights   using analytics, we can help you benchmark, to stay ahead of industry trends. we can also share audience insights on job titles, seniority and geography, so you know your networking with the right connections to match your business needs. onco cell therapy summit (octs), formerly the car t congress, is an event that brings industry together to share novel research, manufacturing techniques and regulatory best practice. each session of octs 2020 is dedicated to translating your novel data assets & ip into commercially viable oncological therapies. octs is a 4 track event and whilst bringing together the key decision makers for car t therapies, this year’s event will have an added focus on the ground breaking science that is driving the surge of interest in harnessing alternative cell therapies in cancer treatments. with 2 of our tracks comprised of research on car t and alternative cell therapies, the other pillar in rolling out these therapies is increasing the affordability of the treatments. the other two tracks will delve into best practice in commercialization and manufacturing of immuno oncological cell therapies. each day of the summit will conclude in plenary room panels that spin out into bespoke roundtable discussions to break down the key challenges in major topics to r&d, manufacturing and commercialization. following the third annual car t congress earlier this year, we recognized a need for more content, covering regulation, alternative cell therapies and commercialization. introducing the onco cell therapy summit (octs), dedicated to translating the emerging novel data from leading academia and innovating biotechs into commercially viable therapies. join us to take a deep dive into the research on solid tumors and “off the shelf” therapies that are defining the direction for oncological cell therapy development pipelines. each day of the congress will conclude in plenary room panels that spin out into bespoke roundtable discussions to break down the key challenges in major topics to r&d, manufacturing and commercialization. new for this year • 4 tracks focusing on: car t r&d, alternative cell therapies, vein to veincommercialization, regulation and safety; • more networking across the industry, where you can organise private 1 2 1 virtual meetings. keenly focused on capturing opportunities and managing impacts created by the digital megatrend for the life sciences clients. as ey global life sciences digital, social and commercial innovation leader, adlai is focused on developing the necessary digital infrastructure to support emerging, next generation forms of individualized therapies to tackle cancer and rare diseases. keenly focused on capturing opportunities and managing impacts created by the digital megatrend for the life sciences clients. as ey global life sciences digital, social and commercial innovation leader, adlai is focused on developing the necessary digital infrastructure to support emerging, next generation forms of individualized therapies to tackle cancer and rare diseases. having a keen interest in different cultures, he has chosen to spend his career living and working around the world in many markets including japan, thailand, hong kong, indonesia, ukraine, russia and switzerland. with more than 28 years of professional experience in media, communications and consulting to his credit, adlai helped establish ey global social media services and analytics hub. additionally, he has also helped launch the world’s first interactive television platform in hong kong. adlai earned an mba in marketing and real estate from southern methodist university and a bs in accounting from metropolitan state college. how adlai is building a better working world “i have been blessed with two opportunities to make important contributions to this world. first, i helped directly support the reconstruction and rehabilitation of communities affected by the tsunami in 2004 that hit indonesia, sri lanka and thailand. for three years, i worked with donors and helped nongovernmental organizations manage their financial resources in order to provide the greatest support possible. i also design and help manage a tsunami commemoration program on behalf of the republic of indonesia. second, i helped design the necessary digital infrastructure needed to support emerging individualized therapies that may, one day, help cure cancer and rare diseases.” dr. niss has served as chief technology officer since march 2018. dr. niss joined mustang in march 2017 as vice president of operations, where he initiated and oversees the establishment of mustang’s cell therapy manufacturing facility. prior to mustang, dr. niss was cell therapy asset leader at biogen, where he oversaw cmc related activities for gene edited hematopoietic stem cell and lentiviral gene therapy programs for sickle cell disease and hemophilia, respectively. earlier in his career, dr. dr. niss has served as chief technology officer since march 2018. dr. niss joined mustang in march 2017 as vice president of operations, where he initiated and oversees the establishment of mustang’s cell therapy manufacturing facility. prior to mustang, dr. niss was cell therapy asset leader at biogen, where he oversaw cmc related activities for gene edited hematopoietic stem cell and lentiviral gene therapy programs for sickle cell disease and hemophilia, respectively. earlier in his career, dr. niss was senior technical project leader at novartis’ cell therapy manufacturing facility in morris plains, new jersey, where he directed the transfer and implementation of the ctl019 process from penn to novartis. he also served as senior r&d program manager at emd millipore, where he established processes for the large scale expansion of adult and pluripotent stem cells. dr. niss began his career in senior research positions in pfizer’s regenerative medicine and immunology groups. he holds a ph.d. in molecular biology from humboldt university of berlin, and an m.s. in microbiology from the university of göttingen in germany. dr. niss completed his postdoctoral research at boston children’s hospital and the dana farber cancer institute. dr. emtage currently serves as chief scientific officer at cell design labs, inc. (cdl). prior to joining cdl, he was vice president of synthetic immunology at intrexon corporation and was vice president of immune mediated therapy in the oncology innovative medicines group at medimmune. at medimmune, dr. emtage designed and implemented a comprehensive immune therapy cancer strategy. he has over sixteen years of biologics development experience in the fields of oncology, autoimmunity, infectious diseases, and inflammation. dr. emtage holds a b.s. and m.s. dr. emtage currently serves as chief scientific officer at cell design labs, inc. (cdl). prior to joining cdl, he was vice president of synthetic immunology at intrexon corporation and was vice president of immune mediated therapy in the oncology innovative medicines group at medimmune. at medimmune, dr. emtage designed and implemented a comprehensive immune therapy cancer strategy. he has over sixteen years of biologics development experience in the fields of oncology, autoimmunity, infectious diseases, and inflammation. dr. emtage holds a b.s. and m.s. in molecular biology and genetics from the university of guelph and received his ph.d. in molecular virology, immunology and inflammation at mcmaster university. at the beginning of his career, he also was a post doctoral fellow at the national institutes of health and held roles at aventis pasteur and harvard medical school. as of may 2018, orlando leads the program strategy for the recently announced chimeric antigen receptor t cells therapeutic area. this includes digital strategies, supply chain network design, and supply chain capability development for this therapy platform, currently targeted as a last line of defense for multiple myeloma. as of may 2018, orlando leads the program strategy for the recently announced chimeric antigen receptor t cells therapeutic area. this includes digital strategies, supply chain network design, and supply chain capability development for this therapy platform, currently targeted as a last line of defense for multiple myeloma. previously, orlando was vice president, information technology, customer and logistics services: the logistics and customer services organization which serves all johnson & johnson businesses globally. additionally, he also led the technology organization of the consumer medical devices supply chain, including diabetes, contact lenses, and vision surgery products and services.orlando began his career with the johnson & johnson consumer business in italy in 1988. after a foreign service national assignment with the united states information agency in rome, italy from 1990 until 1992, he rejoined johnson & johnson’s ortho clinical diagnostics, where he held diverse positions in r&d, sales and marketing, and it. in 2010, as part of the announcement of the johnson & johnson enterprise supply chain, he led the development of strategies to further establish and create new business services including safe and secure supply chain (i.e. anti counterfeit, serialization, track and trace), enterprise wide internal and external technology standards (enterprise master data, gs1, udi, rfid), and new channel management strategies (ecommerce, demand sensing, distribution and transportation optimization). orlando holds a bachelor of science in electrical engineering from rutgers university. he has been actively involved in several industry forums focused to the development and adoption of industry standards, including the development and adoption of the isbt 128 transfusion medicine, including current membership in the us gs1 technical advisory group. he is a member of the program advisory board for the graduate supply chain program of fairleigh dickinson university, silberman college of business. he is an active speaker for the south asian professional network association (sapna) and hispanic organization for leadership and achievement (hola) enterprise groups, and a sponsor for the african american leadership council (aalc). he is active in his community, especially on activities focused on raising awareness of social and health issues. as vice president of oncology and specialty pharmaceuticals with optum’s medical benefit management division, sarah has responsibility for a comprehensive solution set that includes the cancer and specialty guidance programs. sarah is passionate about these complicated conditions and how optum can help ensure consumers receive quality care and support services to help them navigate a complicated system. as vice president of oncology and specialty pharmaceuticals with optum’s medical benefit management division, sarah has responsibility for a comprehensive solution set that includes the cancer and specialty guidance programs. sarah is passionate about these complicated conditions and how optum can help ensure consumers receive quality care and support services to help them navigate a complicated system. sarah joined unitedhealth group in 2003 and held several other roles including leading operations and affordability with optum international where she focused entirely on the brazil health care market. prior to optum international, sarah was principal at unitedhealth group ventures where she analyzed the market to identify new investment opportunities designed to create disruptive, positive change in the health care system. across her roles, sarah uses her engineering training to perform root cause analysis to identify systemic issues in the health care and ultimately design new products to address these issues. she successfully developed and launched new products in oncology, women’s health and orthopedics. sarah holds a bachelor’s degree in industrial engineering from iowa state university. dr. david c. rhew is the global chief medical officer (cmo) and vice president (vp) of healthcare for microsoft. he is the chairman of the health and fitness technology division for the consumer technology association and serves on cta’s board of industry leaders. he co holds six u.s. technology patents that enable authoring, mapping, and integration of clinical decision support into the electronic health record. david is currently an adjunct professor at the stanford university school of medicine. dr. david c. rhew is the global chief medical officer (cmo) and vice president (vp) of healthcare for microsoft. he is the chairman of the health and fitness technology division for the consumer technology association and serves on cta’s board of industry leaders. he co holds six u.s. technology patents that enable authoring, mapping, and integration of clinical decision support into the electronic health record. david is currently an adjunct professor at the stanford university school of medicine. dr. rhew received his bachelors of science degrees in computer science and cellular molecular biology from the university of michigan. he received his md degree from northwestern university and completed internal medicine residency at cedars sinai medical center. he completed fellowships in health services research at cedars sinai and infectious diseases at the university of california, los angeles. he has served as cmo and vp for samsung; senior vice president and cmo at zynx health incorporated; clinician/researcher in the division of infectious diseases at the va greater los angeles healthcare system; and associate clinical professor of medicine at ucla. dr. rhew has served on the national quality forum’s (nqf) executive committee for consensus standards and approval. neehar bhatia, ph.d. is associate director of process development and manufacturing at laboratory of cell and gene medicine (lcgm), stanford university. dr. bhatia received a ph.d. in biochemistry from the central drug research institute, india. dr. bhatia is responsible for development and manufacturing of cgmp compliant cell and gene therapy products at lcgm. dr. bhatia oversees manufacturing for multiple projects such as cd19/cd22 bi specific car t and other t cell based therapies, cd34 hspc transplant and manufacturing of aav6 for gene correction in hspc. dr. neehar bhatia, ph.d. is associate director of process development and manufacturing at laboratory of cell and gene medicine (lcgm), stanford university. dr. bhatia received a ph.d. in biochemistry from the central drug research institute, india. dr. bhatia is responsible for development and manufacturing of cgmp compliant cell and gene therapy products at lcgm. dr. bhatia oversees manufacturing for multiple projects such as cd19/cd22 bi specific car t and other t cell based therapies, cd34 hspc transplant and manufacturing of aav6 for gene correction in hspc. dr. bhatia has lead multiple projects which include banking of escs, ipscs, mscs and ipsc derived differentiated cells, development of gmp process for translational projects and manufacturing cell therapies for phase i/ii clinical trials. dr. bhatia has also lead efforts in quest for serum free chemically defined culture medium for mscs and development of platform technology for aav production. dr. bhatia’s focus is on development of cgmp compliant manufacturing process for “bench to bedside” cell and gene therapies. dr. steven a. feldman was a staff scientist and director of the vector production facility at surgery branch, nci for the past twelve years. dr. feldman’s group was responsible for the development, characterization, and manufacturing of the viral vectors used in gene therapy clinical trials at the surgery branch. recently, dr. feldman joined the stanford center for cancer cell therapy as director of manufacturing and process science where he will take a lead role in the development and manufacture of viral vectors and novel cell therapies for the treatment of cancer. dr. steven a. feldman was a staff scientist and director of the vector production facility at surgery branch, nci for the past twelve years. dr. feldman’s group was responsible for the development, characterization, and manufacturing of the viral vectors used in gene therapy clinical trials at the surgery branch. recently, dr. feldman joined the stanford center for cancer cell therapy as director of manufacturing and process science where he will take a lead role in the development and manufacture of viral vectors and novel cell therapies for the treatment of cancer. dr. maria fardis joined iovance biotherapeutics as president and chief executive officer and was appointed to the board of directors in 2016. since joining, the company has been transformed from an early stage development company to a company with multiple late stage programs involving lifileucel and ln 145 for treatment of multiple solid tumors. dr. dr. maria fardis joined iovance biotherapeutics as president and chief executive officer and was appointed to the board of directors in 2016. since joining, the company has been transformed from an early stage development company to a company with multiple late stage programs involving lifileucel and ln 145 for treatment of multiple solid tumors. dr. fardis served as the chief operating officer of acerta pharma b.v., a clinical stage biopharmaceutical company, from january 2015 to march 2016, where she worked on the development of calquence® until the company’s acquisition by astrazeneca. from 2011 to 2014, she worked at pharmacyclics, inc., where she was a key contributor in the creation of a broad clinical program leading to global approvals for imbruvica® in multiple hematologic malignancies, and where she served as chief of oncology operations and alliances. prior to joining pharmacyclics, from august 2001 to april 2011, dr. fardis held increasingly senior positions in medicinal chemistry and the project and portfolio management department at gilead sciences, inc., where she was involved with multiple therapeutic areas including antivirals, oncology, and cardiovascular therapeutics and worked on the development and life cycle management of letairis®. dr. fardis received her ph.d. in organic chemistry from the university of california, berkeley and her b.s. summa cum laude, in chemistry from the university of illinois, urbana champaign. dr. fardis holds an m.b.a., with highest honors, from golden gate university. the rise of the empowered consumer, coupled with technology advancements and the emergence of digitally focused entrants, is changing every aspect of health and care delivery. to retain relevancy in today’s digitally focused, data infused ecosystem, all participants in health care today must rethink their business practices, including capital strategy, partnering and the creation of patient centric operating models. the ey health sciences and wellness architecture brings together a worldwide network of 28,000 professionals to build data centric approaches to customer engagement and improved outcomes. we help our clients deliver on their strategic goals; design optimized operating models; and form the right partnerships so they may thrive today and succeed in the health systems of tomorrow. we work across the ecosystem to understand the implications of today’s trends, proactively finding solutions to business issues and to seize the upside of disruption in this transformative age. at miltenyi biotec, we are committed to enabling the delivery of new cell and gene therapy (cgt) treatment options to patients with unmet medical needs. we develop tailored cell processing protocols for our gmp compliant cell processing platform, the clinimacs prodigy®, to enable fully closed and automated manufacturing of complex cgt products. in addition, we offer contract development and gmp manufacturing of cgt products as well as design, development, and production of tailored gmp lentiviral vectors company description: avectas is a cell engineering business developing a unique delivery technology platform to enable the ex vivo manufacture of our partners' gene modified cell therapy products, which will retain high in vivo functionality. avectas’ technology, solupore®, delivers advanced molecules such as dna, mrna, proteins and gene editing tools to a range of primary cell types including t cells and nk cells for immuno oncology and gene editing applications. we seek cell therapeutic developers to partner in the development of next generation cell therapies using our solupore non viral delivery platform. phc corporation of north america is a subsidiary of phc holdings corporation, tokyo, japan, a global leader in development, design and manufacturing of laboratory equipment for biopharmaceutical, life sciences, academic, healthcare and government markets. following the close of the recent acquisitions of the anatomical pathology business from thermo fisher scientific (currently operating as epredia) and lsi medience from life science institute, inc., phc group is now in the next transformative phase of its development. nanostring technologies provides life science tools for translational research and molecular diagnostic products. the company's ncounter® analysis system, which has been employed in basic and translational research and cited in 1,000+ peer review publications, has also now been applied to diagnostic use with the ncounter dx analysis system. the company is committed to offering tools that enable scientists and clinicians to translate today’s leading genomic research into clinically actionable tests that improve patient care. gene expression panels for immune  oncology, autoimmunity, car t and other applications are ready off the shelf tools offered to the nanostring customers. vivebiotech is a gmp cdmo fully focused on the development and gmp manufacture of lentiviral vectors from very early stages (even scratch) to gmp. being fully focused on customers´ projects, vivebiotech adapts to their requirements being highly flexible in terms of technical adaptation and slots availability. vivebiotech is currently working for clients based in europe, usa and asia, manufacturing lentiviral vectors for different applications such as rare diseases as well as immune oncology (car ts, tcrs, car nks, tils), among others. one of vivebiotech´s main features is our specialization in virology. based on it, our r&d department is focusing its efforts on the development of more cost effective processes and safer lentiviral vectors (worldwide patented technology called lentisoma). allcells, for 21 years is the leader in providing high quality, economical blood & marrow derived cells used by global researchers and bio manufacturing organizations to develop cell therapies, vaccines, and therapeutics to treat cancer, infectious disease, and autoimmune disease. allcells sources through our on site, irb approved state of the art donor facilities, where we collect cells and whole tissue from a large repository of recallable donors which enables custom orders to be fulfilled in the shortest time in the industry. we immediately process fresh tissue in our adjacent labs to produce purified cell types having unparalleled quality and consistency. ask us about allcells gmp compliant allogeneic clinical grade products. cellgenix is a leading global supplier of high quality reagents and tools in the expanding market of cell and gene therapy and regenerative medicine. as the first company to obtain a gmp manufacturing authorization for cell processing in europe, cellgenix has more than 20 years of expertise in the development and gmp manufacturing of cell therapy products. our products are used worldwide in clinical trials by academia and industry partners. to ensure a seamless transition from research to commercialization we offer our customers a comprehensive product portfolio together with expert regulatory and technical support. included in our product portfolio are cytokines, serum free media and closed cell culture systems. our products combine a maximum of quality and safety with excellent performance due to the state of the art production, stringent in house quality control and comprehensive documentation. all these factors help to simplify qualification and validation for your market authorization. cellgenix operates a state of the art gmp facility for production of recombinant proteins and cell processing in freiburg, germany. a subsidiary is located near boston in portsmouth, nh/usa. kbi biopharma, inc. is a biopharmaceutical contract development and manufacturing organization that accelerates the development of innovative discoveries into life changing biological products. from early stage to academic/non profit organizations, to many of the world’s largest pharmaceutical companies, kbi has served 250+ clients globally to accelerate and optimize their drug development programs. kbi’s extensive track record of successful programs is a result of its unique approach: applying the insight gained from our advanced biophysical and analytical characterization techniques toward the development of robust and scalable processes. kbi delivers accelerated and integrated process development and cgmp manufacturing programs for a wide range of recombinant protein active pharmaceutical ingredients (api) and cell therapy products for our clients. kbi was founded in 1996 and operates 3 manufacturing facilities: durham (nc), boulder (co), and the woodlands (tx). p=eprotech creates the building blocks of your life science research by manufacturing high quality products that advance scientific discovery and human health. since 1988, peprotech has grown into a global enterprise manufacturing an extensive line of recombinant human, murine and rat cytokines, animal free recombinant cytokines, monoclonal antibodies, affinity purified polyclonal antibodies, affinity purified biotinylated polyclonal antibodies, elisa development kits, cell culture media products and gmp cytokines genscript biotech is the world leader in biotechnology reagent service industry. established in 2002 in new jersey, united states, the company was the first to commercialize gene synthesis and successfully establish fully integrated capabilities for custom peptide synthesis, protein expression and engineering, custom antibody development and engineering, animal model development, in vitro/in vivo pharmacology as well as a variety of catalogue products, industrial enzymes and microbial reagents. for 40 years, the quick group has been serving the life science and healthcare community worldwide, providing 24/7 priority logistics and transportation solutions for time and temperature sensitive, mission critical and life saving needs. our specially trained life science team, with unmatched experience, provides innovative and best in class logistics solutions for cell and gene therapies that ensure regulatory compliance, product integrity and patient safety. with a global network, quick can expedite delivery to anywhere in the world    even to the most remote locations. we provide consultation on regulatory compliance, classification, import/export documentation and dangerous goods. our temperature control experts provide consultation and procurement of the latest packaging solutions for all temperature ranges, gps tracking and temperature monitoring devices, ensuring product integrity and patient safety. whether your shipments need to remain at controlled room temperature, 2 to 8ºc, or cryo frozen, quick will handle your shipments utilizing certified gdp and quality processes. quick’s market leading technology, quickonlinerx, provides comprehensive management of your supply chain logistics, with real time tracking and status alerts, and is fully scalable to meet the requirements of newly commercialized products. as the trusted leader in personalized medicine logistics and transportation, quick provides customized solutions and protocols to ensure the safety and integrity of your car t therapies. we’ll help you plan, design and support your logistics operation, so you can focus on your core business   creating cutting edge healthcare treatments. you are driving a paradigm shift. scientific and technological advances emerging from creative companies across the globe are transforming the way we look at disease and breaking down boundaries for treatment. now more than ever, business success hinges on working with advisors and suppliers who understand your needs and allow you to focus on what matters most. what if all of those needs were met by a single partner   a market leader with decades of experience in biotechnology and in vitro diagnostics? leveraging the unique know how of roche diagnostics and roche pharmaceuticals, we work with you to deliver high quality raw materials, instrumentation, products and services for your biopharmaceutical, cell therapy, or in vitro diagnostics business, customized to your unique quality and regulatory needs. avance biosciences is a leading cro providing glp/gmp compliant assay development, assay validation, and sample testing services to support biological drug development and manufacturing activities world wide. we provide customized solutions for car t cell therapy testing including: integration site analysis by ngs and southern blot, preclinical qpcr biodistribution studies, preclinical qpcr quantification of mrna from animal tissues, copy number analysis by qpcr or ddpcr, replication competent lentivirus (rcl) detection by qpcr, and testing of dna or mrna in clinical trial human samples. when you partner with avance biosciences, we give you undivided attention, share our experience and knowledge, and respond to your needs in a timely manner. contact our technical staff to discuss how we can support you in your project! who we are gpb scientific is a pioneering biomedical company that works with leading research institutes and universities to develop breakthrough technologies that get people better. since 2002, gpb has been at the forefront of microfluidics and cell biology for diagnostic and therapeutic applications. with offices and laboratories on both coasts, gpb has a long entrepreneurial track record of fostering breakthrough biomedical products and moving them into the commercial healthcare and diagnostic marketplace. our vision gpb scientific is a fusion of science and entrepreneurism driven by a spirit of the possible. our work focuses on incubating exciting biomedical research, validating its promise and moving it into the marketplace where it can make a profound difference in people’s lives. our vision is to get people better by accelerating the availability of pioneering science within the healthcare system. novartis is reimagining medicine to improve and extend people’s lives. as a leading global medicines company, we use innovative science and digital technologies to create transformative treatments in areas of great medical need. in our quest to find new medicines, we consistently rank among the world’s top companies investing in research and development. novartis products reach more than 750 million people globally and we are finding innovative ways to expand access to our latest treatments. about 109,000 people of more than 140 nationalities work at novartis around the world. find out more at www.novartis.com cryoport is the life sciences industry's most trusted global provider of temperature controlled logistics solutions for temperature sensitive life sciences commodities, serving the biopharmaceutical market with leading edge logistics solutions for biologic materials, such as regenerative medicine, including immunotherapies, stem cells and car t cells. cryoport also provides a range of bioservices to the life sciences industry, including biostorage for biological specimens, materials and samples. cryoport's solutions are used by points of care, cro's, central laboratories, pharmaceutical companies, manufacturers, university researchers et al; as well as the reproductive medicine market, primarily in ivf and surrogacy; and the animal health market, primarily in the areas of vaccines and reproduction. cryoport's proprietary cryoport express® shippers, cryoportal® logistics management platform, leading edge smartpak ii® condition monitoring system and geo sensing technology, paired with unparalleled cold chain logistics expertise and 24/7 client support, make cryoport the end to end cold chain logistics partner that the industry trusts. cryoport is dedicated to: simplifying global cold chain logistics through innovative technology, unmatched monitoring and data capture and support, including consulting; delivering the most advanced temperature controlled logistics solutions for the life sciences industry; and providing vital information that provides peace of mind throughout the life of each logistics process. werum it solutions is the world’s leading supplier of manufacturing execution systems (mes) and manufacturing it solutions for the pharmaceutical and biopharmaceutical industries. our out of the box pas x software product is run by the majority of the world’s top 30 pharmaceutical and biotech companies but also by many mid sized manufacturers. werum’s manufacturing it solutions help pharma manufacturers to increase efficiency, improve productivity, and meet regulatory requirements. the range of projects includes global mes programs with multi site rollouts all over the world as well as single site solutions in individual countries. founded in 1969, werum is headquartered in lüneburg, germany, and has many locations in europe, america, and asia. cytiva is a global provider of technologies and services that help advance and accelerate the development and manufacture of therapeutics. previously ge healthcare life sciences, cytiva’s diverse portfolio includes well recognized brands such as äkta, amersham, biacore, flexfactory, hyclone, mabselect, whatman and xcellerex. cytiva brings speed, efficiency and capacity to research and manufacturing workflows, enabling the delivery of transformative medicines to patients. visit cytiva.com for more. terumo bct, a global leader in blood component, therapeutic apheresis and cellular therapy technologies, believes in the potential of blood and cells to do even more for patients than they do today. terumo bct’s cell therapy technologies business enables researchers, developers and manufacturers to create next generation cell and gene therapies. based on your objectives, we can create bespoke packages designed specifically for you. opportunities predominantly lie in 3 main categories: thought leadership, branding, and networking. contact us to find out more interested in a media partnership?we'd love to hear from you and how we can support one another to connect with the industry. contact [email protected] looking to showcase your recent work to the cell therapy community? our dedicated poster session is the perfect way to get your research noticed. in order to present a poster at the forum, you need to be registered as a delegate. please note that there are 20 assigned spaces for the onco cell therapy summit. poster space is assigned on a first come, first served basis (subject to checks and successful registration). at the congress, your presentation will be displayed in a dedicated poster area. the poster presentation session will take place at the afternoon networking break on day one. poster abstract submission deadline is on june 26th, 2020. abstracts received after this time may not be accepted so please submit your abstract as soon as possible! posters should be sized a0 (841mm x 1189mm) in portrait orientation. for more information please contact [email protected] submit your poster here sending your team? group discounts available! applicable for primary market, service provider and industry rates only. not available for academic or ‘start up’ rates book a team of 3+   save an additional 10% offbook a team of 5+   save an additional 15% off if you would like to register a team of 3 or more, please email [email protected] for your discount coupon code before registering. please note: discounts cannot be combined with early bird pricing or any other discount or offer. if you have any questions about your registration, please call us on +44 (0)20 3696 2920 kisaco research produces, designs and hosts b2b industry conferences, exhibitions and communities   focused on a specialized selection of topic areas. meet industry peers that will help build a career changing network for life. learn from the mistakes of your peers as much as their successes ambitious industry stalwarts who are happy to share not just what has made them 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supported by a conversation with … dr. hans klingemann, pioneering immunotherapy scientist, has studied whether the innovative treatment could save his two pets. by matt richtel immunotherapy has transformed cancer treatment. it tinkers with the immune system to attack malignancies that have evaded the body’s natural defenses. this advance offers an alternative to treating cancer with surgery or chemotherapy and radiation, which can attack healthy tissue and cause extreme side effects. the treatment is not only scientifically complex but also expensive. the investment of money and time makes sense when it comes to saving humans. but what about when it comes to dogs? dr. hans klingemann has worked on and researched cancer immunotherapy for decades, leading departments at rush university medical center in chicago and tufts medical center in boston. now, he’s the chief science officer for cellular products at immunitybio, which develops immunotherapy drugs for people. but he has also written two papers exploring whether the new treatments might someday prolong canine lives. the interview below has been condensed and edited for clarity. what interests you about immunotherapy and dogs? i love dogs. i have dogs: sophie and maximilian. they are each around 18 pounds, a mix of a bichon and a cavalier spaniel. did they develop cancer? fortunately, they haven’t gotten cancer…. yet. but when dogs get older, many get cancer. are there some benefits from immunotherapy?