T-Cell Immunotherapy:
`Looking Forward
`
`T Cell Immunotherapy: Optimizing Trial Design
`Bethesda, Maryland
`10–11 September 2013
`
`Jacqueline Corrigan-Curay1, Hans-Peter Kiem2, David Baltimore3,
`Marina O’Reilly1, Renier J Brentjens4, Laurence Cooper5,
`Stephen Forman6, Stephen Gottschalk7, Philip Greenberg2,
`Richard Junghans8, Helen Heslop7, Michael Jensen9, Crystal Mackall10,
`Carl June11, Oliver Press2, Daniel Powell11, Antoni Ribas12,
`Steven Rosenberg10, Michel Sadelain4, Brian Till2, Amy P Patterson1,
`Robert C Jambou1, Eugene Rosenthal1, Linda Gargiulo1,
`Maureen Montgomery1 and Donald B Kohn12
`
`The rapidly expanding field of T-cell im-
`
`munotherapy has experienced clinical
`successes along with some serious toxici-
`ties. “T Cell Immunotherapy: Optimizing
`Trial Design,” a workshop sponsored by
`the National Institutes of Health’s (NIH’s)
`Office of Biotechnology Activities (OBA),
`brought together researchers to discuss the
`
`1Office of Science Policy, Office of the Director,
`National Institutes of Health, Bethesda, Mary-
`land, USA; 2Fred Hutchinson Cancer Research
`Center, Seattle, Washington, USA; 3California
`Institute of Technology, Pasadena, California,
`USA; 4Memorial Sloan-Kettering Cancer Center,
`New York, New York, USA; 5MD Anderson
`Cancer Center, Houston, Texas, USA; 6City of
`Hope, Duarte, California, USA; 7Baylor College
`of Medicine, Houston, Texas, USA; 8Roger
`Williams Medical Center, Providence, Rhode
`Island, USA; 9University of Washington, Seattle,
`Washington, USA; 10National Cancer Insti-
`tute, National Institutes of Health, Bethesda,
`Maryland, USA; 11Abramson Cancer Center,
`University of Pennsylvania, Philadelphia,
`Pennsylvania, USA; 12David Geffen School of
`Medicine, University of California, Los Angeles,
`Los Angeles, California, USA
`Correspondence: Jacqueline Corrigan-Curay,
`Office of Biotechnology Activities, National In-
`stitutes of Health, 6705 Rockledge Drive, Suite
`750, Bethesda, Maryland 20892, USA.
`E-mail: corrigaja@od.nih.gov
`
`scientific advances and share new data on
`key trial design issues, including the selec-
`tion of new targets, optimizing the T-cell
`population, preconditioning regimens,
`strategies to promote persistence of cells,
`and analysis and management of acute re-
`actions to T-cell infusions with the goal of
`identifying best practices and a research
`agenda that will facilitate further devel-
`opment and maximize the safety of this
`promising approach.
`
`Introduction
`T-cell immunotherapy for cancer is a
`rapidly growing field for gene therapy.
`Broadly, this field can be divided into two
`approaches—the use of gene-modified T-
`cell receptors (TCRs) in which recognition
`of the tumor antigen is in the context of
`human leukocyte antigens (HLAs) or use
`of chimeric antigen receptors (CARs) that
`typically link a single-chain variable re-
`gion domain of an antibody (scFv) to one
`or more signaling elements of a TCR com-
`plex to allow T-cell activation.1 The deci-
`sion to use one approach vs. the other may
`depend on several factors. For example,
`CARs offer the ability to bind antigens that
`are not restricted by HLA recognition, and
`the ability to modify the T-cell signaling
`moieties may offer “a broader functional
`
`doi:10.1038/mt.2014.148
`
`effect than transduced” TCRs.2 TCRs, how-
`ever, have the ability to recognize intracellular
`proteins, in addition to cell surface antigens,
`providing a broader array of target tumor-as-
`sociated targets.
`In 2010, the OBA hosted a meeting to ex-
`amine the state of the science and key trial de-
`sign questions for this emerging field.3 At the
`time, some clinical benefit and unexpected
`toxicities highlighted both the therapeutic
`potential as well as the need to share data and
`expertise to optimize the safety of trial design.
`Since 2010, several promising and clinically
`successful developments have been reported
`in leading scientific and medical journals4–7
`as well as national media. Given these devel-
`opments, the OBA and the NIH Recombinant
`DNA Advisory Committee concluded that it
`was an opportune time to reconvene the lead-
`ing experts in the field from the United States
`to continue to foster sharing of data across pro-
`tocols and discuss the key issues in trial design,
`including optimal management of the cytokine
`release syndrome (CRS) seen in some research
`participants in response to the expansion of
`these active T cells.
`The following summary of the OBA work-
`shop represents the views of the individual
`authors and not the NIH. The full presentations
`and slides are available at the OBA’s website.8
`
`State of the science
`The number of CAR and TCR protocols reg-
`istered with the OBA has continued to in-
`crease rapidly (Figure 1); as of the meeting in
`September 2013 there were 111 protocols, 104
`of which targeted cancer, with more than 500
`subjects dosed. More than 40 protocols address
`hematological malignancies, with CD19 being
`the most common target in these protocols.
`Among protocols for solid tumors, the mela-
`noma antigens (gp100, MART-1) and cancer-
`testis antigens predominate for TCRs; for
`CARs there are multiple targets, with a slight
`predominance of Her2/neu, GD2, and meso-
`thelin (Figures 2 and 3). Approximately 90%
`of TCR trials have targeted solid malignancies;
`approximately 50% of CAR trials have targeted
`hematological malignancies.
`Steven Rosenberg reviewed the exten-
`sive portfolio of National Cancer Institute
`(NCI) research in this area, beginning with
`a summary of his research using unmodi-
`fied tumor-infiltrating
`lymphocytes (TILs)
`against melanoma in 1988. He began using
`
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`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`No. of protocols
`
`2007–2012
`
`2001–2006
`
`1995–2000
`
`1989–1994
`
`Figure 1 Number of chimeric antigen receptor protocols registered with the
`National Institutes of Health’s Office of Biotechnology Activities by year.
`
`Forman, Michael Jensen, Helen Heslop,
`and Crystal Mackall summarized their re-
`sults in ongoing trials using CD19-specific
`CARs in leukemia and lymphoma.4,6,14 Dr.
`Heslop noted that in a trial comparing first-
`and second-generation CARs, her group
`found that the second-generation CAR
`demonstrated both improved expansion
`and persistence.15 In addition, several pro-
`tocols have established that administration
`of CAR T cells after stem cell transplant
`does not interfere with engraftment of the
`transplant. The investigators presented ex-
`amples of clinical remissions, but, because
`the goal is often to establish remission so
`as to proceed with a curative transplant,
`the durability of remissions from CAR T
`cells without subsequent transplant has
`not yet been determined. However, even
`in the setting of multiple previous thera-
`pies, CD19-specific CARs have shown ef-
`ficacy. Dr. Brentjens reported that in his
`protocol with relapsed or refractory B-cell
`acute lymphoblastic leukemia (ALL), 14
`of 16 subjects achieved molecular chronic
`remissions as assessed by deep-sequencing
`PCR analysis to search for the malignant
`clone.16 Another emerging theme was the
`responsiveness of ALL to this approach,
`which was also highlighted in Dr. June’s
`and Dr. Mackall’s presentations. Dr. Coo-
`per presented data from ongoing trials
`infusing CD19-specific CAR+ T cells after
`autologous and allogeneic hematopoietic
`stem cell transplantation. The intent was
`to augment the graft-vs.-tumor effect, rec-
`
`ognizing that the current clinical practice
`for many patients with B-cell malignan-
`cies is to infuse tumor-specific T cells as
`a bridge to transplantation. These trials
`have advanced a new approach to hu-
`man gene therapy based on the electro-
`transfer of DNA plasmids encoding a
`second-generation CAR stably expressed
`following transposition from the Sleeping
`Beauty (SB) system.
`In parallel to work on CD19-specific
`CARs, Brian Till highlighted the results of
`his trials targeting CD20, including a trial
`that used a third-generation CAR with
`CD28 and 4–1BB costimulatory domains.
`Unlike the other trials, which use retro-
`viral vectors or SB transposons, he used an
`electroporated DNA plasmid. In general,
`the T cells were well tolerated, with some
`immediate febrile reactions, and two of the
`three subjects had prolonged remissions
`with persistence of the T cells for up to a
`year.17 However, the DNA plasmid vector
`was not an efficient vector, and the IL-2
`used to promote persistence also led to an
`increase in T regulatory cells (Tregs).
`Philip Greenberg highlighted his
`group’s work using a TCR targeting an-
`other hematological malignancy antigen,
`Wilms tumor antigen 1 (WT1), which is
`highly expressed in leukemia and some
`solid tumors but is also expressed on some
`normal tissues. Their trial built on a previ-
`ous trial using naturally isolated, cloned T
`cells targeting WT1, which did not show
`toxicity but had limited efficacy. Using
`
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`lymphodepletion before administration of
`TILs in 2002 and demonstrated increased
`efficacy.9 Dr. Rosenberg has continued to
`apply this approach to melanoma, includ-
`ing ocular melanoma, as well as metastatic
`gastrointestinal and human papillomavi-
`rus–induced cancers. These studies have
`demonstrated that in a subset of patients
`(about 20%), administration of T cells can
`result in prolonged remissions of five years
`or longer. The results led to a program of
`research dedicated to gene-modified T
`cells that accounts for almost 20% of T-cell
`immunotherapy protocols registered with
`the OBA to date. The results of the Rosen-
`berg group’s first trials with gene-modified
`TCRs for melanoma were published in
`2006 in Science.10 In a recent TCR study
`targeting the cancer-testis antigen NY-
`ESO-1, the overall response rate was 50%
`in the 19 subjects with melanoma, includ-
`ing 4 with complete remissions, and a 67%
`overall response for those with synovial
`sarcoma, including one complete remis-
`sion, in a population that had multiple
`prior chemotherapy regimens.11 These re-
`sults contrasted with the MAGE-A3 trial
`in which an unexpected off-target neu-
`rological toxicity was seen.12 Rosenberg’s
`group has also developed an extensive
`portfolio of CAR protocols, focusing pri-
`marily on solid tumors, with novel targets
`such as vascular endothelial growth factor
`receptor 2 (VEGFR2), epidermal growth
`factor receptor variant III (EGFRvIII),
`and mesothelin, as well as new targets in
`development, such as chondroitin sulfate
`proteoglycan 4 (CSP4).
`Antoni Ribas, who uses a vector de-
`veloped by Rosenberg’s lab, described his
`work on melanoma using a TCR-targeting
`MART-1 given with lymphodepletion. He
`has observed a high frequency of tumor
`responses (9 of 14 subjects with tumor-size
`reductions), but few responses were dura-
`ble. He has also recently started enrolling
`research participants into a trial using a
`TCR-targeting NY-ESO-1. He noted that
`one of the aspects being tested is whether
`fresh cells are potentially more active than
`cryopreserved cells.
`included clinical
`Other highlights
`results from several investigators target-
`ing CD19 in leukemia and lymphoma.
`In addition to Dr. Rosenberg’s summary
`of his work in this area,13 Carl June, Re-
`nier Brentjens, Laurence Cooper, Stephen
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`virus-specific T cells, they have recently
`initiated a trial to test a TCR based on a
`high-avidity, natural clone.
`In the solid-tumor area, Dr. Heslop
`presented a summary of her group’s trials
`for neuroblastoma, targeting GD2 using
`both virus-specific and non-virus-specific
`T cells.18,19 Their data have demonstrated
`an association between persistence of T
`cells and reduced tumor progression. In
`addition, in research participants with
`prolonged detection of activated T cells,
`the presence of central memory T cells was
`important, raising the question of what the
`optimal T-cell product is.
`Other solid-tumor trials discussed in-
`cluded CARs targeting HER2/neu for sar-
`coma and glioblastoma, including a trial
`using tri-virus-specific T cells and another
`trial that combines the CAR with a domi-
`nant-negative TGF-b receptor. Data were
`also presented on first- and second-gen-
`eration CARs targeting carcinoembryonic
`antigen (CEA) and prostate-specific mem-
`brane antigen. Again, some early indica-
`tions of clinical efficacy were promising,
`but an ongoing challenge will be to refine
`strategies to improve T-cell persistence
`and efficacy. In some cases, on-target, off-
`tissue toxicities may ultimately limit the
`use of certain targets; for example, colitis
`developed in protocols using CEA-specific
`TCR and CAR T cells.20
`Finally, Dr. Jensen reported his work
`in glioblastoma using a novel CAR called
`a zetakine. Instead of an antibody, single-
`chain target domain, he used a human
`cytokine, IL-13, with a mutation in the
`sequence that gave high affinity for IL-13
`receptor a2. These cells were infused
`intracranially, establishing the safety of
`intracranial administration with some
`antitumor responses.
`These talks provided an overview of a
`field that continues to expand rapidly, in
`terms of both targets and diseases. Most
`protocols involve administration of the cells
`in the setting of lymphodepletion, and some
`groups, predominantly in protocols for
`solid tumors, use IL-2 to promote cell per-
`sistence. In addition to identifying effective
`targets that have minimal off-tumor effects,
`finding the ideal balance between persis-
`tence and expansion of T cells without
`triggering systemic cytokine reactions is a
`key issue for the field. This may be achieved
`by such strategies as including the design
`
`of the cells, the type of T cells infused, the
`dose, the immune status of the recipient,
`and the use of cytokine support. Finally, as
`with many cancer therapies, some toxic-
`ity is likely. Establishing protocols to limit
`toxicity so that the risk-to-benefit ratio re-
`mains favorable is a high priority.
`
`Promoting T-cell persistence
`Persistence of the gene-modified T cells
`is associated with prolonged remission
`in subjects,18 and the field has developed
`strategies to promote persistence. One ap-
`proach is to create a host environment that
`is conducive to expansion of the T cells.
`Expansion should not only promote a rig-
`orous antitumor effect but also lead to the
`development of a stable population of tu-
`mor-specific T cells that can be reactivated
`in case of recurrence of tumor antigen. Use
`of selected central memory T cells may be
`another strategy to promote an enduring
`T-cell population.
`The majority of T-cell protocols reg-
`istered with the OBA to date involve ad-
`ministration of the cells to subjects when
`they are lymphopenic. For solid-tumor
`protocols, this involves administration of
`the T cells after administration of lym-
`phodepleting chemotherapy, such as cy-
`clophosphamide, whereas the protocols
`for hematological malignancies have most
`commonly called for administering cells
`in the posttransplant setting or the use of
`disease-specific chemotherapy regimens.
`However, it is important to note that lym-
`phodepletion has not been universally
`applied, notable exceptions being studies
`administering virus-specific T cells, or the
`successful neuroblastoma protocols tar-
`geting GD2, which used both virus-specif-
`ic and non-virus-specific T cells.18
`Dr. Rosenberg reviewed his group’s
`clinical data, as well as the animal data
`that support lymphodepletion for pro-
`moting antitumor efficacy. As stated ear-
`lier, in the TIL melanoma studies, despite
`administration of 109 to 1010 T cells, the
`cells did not persist and there were mini-
`mal objective responses.21 However, when
`nonmyeloablative (NMA) chemotherapy
`using cyclophosphamide and fludarabine
`was added, and the TIL product was gen-
`erated with a shorter culture time, provid-
`ing a more diverse TIL population that
`contained both CD4+ and CD8+ T cells,
`T-cell persistence was enhanced and 6 of
`
`13 subjects showed objective cancer re-
`sponses.22 Dr. Rosenberg’s group went on
`to investigate whether the addition of 2
`or 12 Gy of total-body irradiation (TBI)
`to the NMA chemotherapy would further
`increase efficacy of TIL transfer in mela-
`noma patients. The response rate for those
`who received chemotherapy alone was
`about 49%; the addition of 2 Gy resulted
`in objective response in 52% of subjects,
`and 12 Gy of TBI resulted in a 72% ob-
`jective response rate, with a complete
`response rate of 40%.23 The addition of
`TBI to NMA chemotherapy was generally
`well tolerated, with the exception of one
`death in a subject with an undetected di-
`verticular abscess in the 12-Gy group. A
`drawback of escalation to 12 Gy of radia-
`tion is the need for autologous peripheral
`blood stem cell support. An ongoing ran-
`domized trial is comparing NMA chemo-
`therapy against NMA and TBI, although
`preliminary results indicate that the chal-
`lenges of adding TBI may not be balanced
`by the improved response.
`A significant amount of animal work
`has been done to elucidate the mechanisms
`that underlie the improved antitumor re-
`sponses observed with lymphodepletion.
`These data indicate that lymphodepletion
`augments the antitumor response by elim-
`inating Tregs, cellular “sinks” for cytokines
`such as IL-7 and IL-15, and by enhanc-
`ing antigen-presenting cell activation and
`availability.24–26 This activation of the im-
`mune system may be due in part to trans-
`location of bacteria from the gut. It was
`shown in a mouse model that adminis-
`tration of ciprofloxacin, which is effective
`against Gram-negative bacteria commonly
`found in the gut, to an irradiated animal
`reduced the activated dendritic cells in the
`spleen and reduced the effectiveness of
`adoptive cell transfer. Of note, it has been
`demonstrated that the effect of lymphode-
`pletion is on the host rather than on the tu-
`mor. Thus, if one shields the host—in this
`case, the mouse—and treats the tumor, no
`effect is seen in these melanoma models.
`One dilemma is that Tregs are the first
`T cells to recover after lymphodepletion,
`and therefore lymphodepletion may foster
`an environment that works against the an-
`titumor effect. Dr. Rosenberg noted that
`the NCI group has some data demonstrat-
`ing an inverse relationship between the
`recovery of Tregs and objective antitumor
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`response, supporting the importance of
`eliminating Tregs. However, others ques-
`tioned whether we clearly understand the
`role of Tregs, because suppression of a tu-
`mor response may depend on whether the
`Tregs are actually activated and tumor-
`specific. Therefore, the presence of Tregs
`may not be absolutely undesirable, as they
`may also organize the immune response.
`
`Gene delivery and design of T cells
`In addition to host preparation, the design
`of the T-cell vectors is a critical area of re-
`search. Dr. Cooper noted that the ability to
`stably express transgenes, such as CARs,
`in T cells has revolutionized adoptive im-
`munotherapy for certain malignancies.
`Recombinant fusion genes constructed to
`recognize tumor-associated antigens (e.g.,
`TCR and CAR) have been constitutively
`expressed in T cells using Moloney mu-
`rine leukemia virus (MMLV)-based retro-
`viruses, HIV-based lentiviruses, and DNA
`plasmids, including the SB transposon/
`transposase system.
`Until recently, retroviral transduc-
`tion by recombinant MMLV-derived vec-
`tors has been the most common method
`for delivery of transgenes intended to be
`integrated into the T-cell genome. Lenti-
`viral vectors have also been successfully
`used in the clinic. Both approaches are
`appealing, and at this time there appears
`to be equipoise regarding the therapeutic
`potential of these two viral systems for
`genetic modification of T cells to express
`CARs. Transduction using retroviral and
`lentiviral vectors can be highly efficient,
`and it is possible to integrate multiple cop-
`ies of a transgene in a given T cell, which
`provides for a high level of expression of
`the transduced gene product. The manu-
`facture of clinical-grade retroviral and
`lentiviral vector virions is quite similar, al-
`though retroviral vectors may be produced
`from stable packaging cell lines, whereas
`to date most lentiviral vectors have been
`produced by transient transfection.
`Overall, transduction of T cells with
`recombinant retrovirus and lentivirus in-
`volve similar packaging protocols, utilize
`similar integration mechanisms, and lead
`to similar transduction efficiencies. Thus,
`both viral-based approaches to gene trans-
`fer are appealing for the human application
`of CAR+ T cells, although some individual
`investigators have strong preferences.
`
`Molecular Therapy vol. 22 no. 9 september 2014
`
`transposons now offer an
`DNA
`to viral-based gene
`trans-
`alternative
`fer. Supercoiled plasmids can be directly
`electroporated into T cells using commer-
`cial devices, thus eliminating much of the
`labor and safety concerns associated with
`generating recombinant viral particles.
`DNA transposons, such as those derived
`from the SB system, insert into the genome
`via a copy-and-paste mechanism when a
`transposase is (transiently) available to cat-
`alyze the reaction. Dr. Cooper’s group has
`successfully used SB to integrate a CD19-
`specific CAR into human T cells in four
`human trials under investigational new
`drug applications. Unlike retroviral/lentivi-
`ral integration into transcriptionally active
`sites, the SB transposon appears to ran-
`domly integrate at TA dinucleotide repeats
`and is typically present at one or two copies
`per T-cell genome. As with viral-based gene
`transfer, there is the possibility that a trans-
`poson may cause genotoxicity resulting in
`oncogenesis. However, because the SB sys-
`tem does not readily target transcriptional
`or promoter elements, it appears suitable
`for human application. Furthermore, the
`relatively low cost of generating DNA plas-
`mids for use in compliance with current
`good manufacturing practice (GMP), in
`contrast to the cost and complexity of pro-
`ducing clinical-grade virus, renders the SB
`system an attractive and nimble approach
`to generate and modify vectors for delivery
`of therapeutic genes.
`investigator has
`In summary, the
`available multiple approaches to geneti-
`cally modify T cells. The use of a par-
`ticular approach will depend on resident
`expertise and the desired T-cell product.
`
`Design of CARs. CARs are recombinant
`receptors for antigens that retarget and
`eventually reprogram T-cell function. Un-
`like the physiological TCR for antigens,
`which signals T-cell activation through the
`associated CD3 complex, CARs possess
`in a single molecule the ability to trigger
`multiple antigen-specific T-cell
`func-
`tions. The CARs that have recently shown
`impressive clinical outcomes in research
`participants with B-cell malignancies are
`“second-generation CARs,” to distinguish
`them from earlier forms of activating
`fusion receptors, which only initiate T-cell
`activation and are now referred to as “first-
`generation CARs.”27
`
`Michel Sadelain described how the
`incorporation of co-stimulatory receptor
`signaling domains into the cytoplasmic
`tails of CAR (“embedded costimulation”)
`greatly increased the potency of CAR-
`modified T cells in preclinical models.4,28,29
`Several costimulatory domains have been
`incorporated in CARs over the past decade,
`including CD28 (ref. 28), 4–1BB (ref. 30),
`OX40 (ref. 31), and others (ref. 2). Different
`costimulatory molecules play roles in T-cell
`activation, proliferation, survival, cytokine
`secretion, antitumor cytolytic activity, and
`reactivation upon secondary stimulation.
`The second- and third-generation CARs
`have varying activities by recruiting multi-
`ple T-cell signaling pathways.2 Dr. Sadelain
`emphasized that small nuances in struc-
`tural design of different CAR molecules can
`eventually exert a significant effect on the
`relative activity of CARs encoding the same
`signaling domains, depending on epitope
`position, CAR affinity, physical parameters
`of the extracellular domains, and trans-
`membrane elements. Levels of CAR expres-
`sion also affect overall function, making it
`an important parameter to consider when
`comparing different CARs. Forced expres-
`sion of co-stimulatory ligands in the CAR T
`cells themselves can produce auto- or trans-
`costimulation and increase T-cell potency.32
`Clinical efficacy has been reported in
`trials from several institutions for B-lin-
`eage malignancies using CAR-modified
`T cells.4–6,14,16,33,34 Many features of the tri-
`als differ, including CARs (origin of scFv,
`epitope of CD19 targeted, antigen affinity,
`signaling domains), enhancer/promoters
`(varied expression levels, propensity to si-
`lencing), T-cell manufacturing techniques
`(activation of T cells with antibodies to CD3
`with or without anti-CD28, different culture
`media, duration of culture), cell products
`(cell dose, CD4/CD8 ratio, central memory
`T cells), lymphodepletion conditioning reg-
`imens (cyclophosphamide vs. cyclophos-
`phamide/fludarabine vs. bendamustine),
`and patient selection (chemosensitive vs.
`chemoresistant disease). Future trials will
`need to define the relative importance of
`these differences to improve response rates.
`It is noteworthy that the outcomes of CD19
`CAR therapy may vary depending on the
`disorder. Thus, results reported to date show
`greater efficacy in ALL than in chronic lym-
`phocytic leukemia (CLL), for reasons that
`remain to be elucidated.
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`(HSCs), which would
`cells
`stem
`continually produce transduced T cells.
`David Baltimore listed potential advan-
`tages of targeting HSCs. Because of the re-
`quirement for coexpression of CD3, trans-
`genic TCRs can be expressed only on the
`surface of T cells derived from the trans-
`duced HSCs. The TCRs introduced by the
`vector should allelically exclude the rear-
`rangement of endogenous TCR genes to
`yield monoclonal cells. However, one po-
`tential limitation of this approach may be
`that highly active T cells from HSCs that
`contain highly avid TCRs for self-antigens
`may be selected out by the thymus. In the
`trials using a MART-specific TCR, clini-
`cal effect was observed when the avidity
`of the natural TCR was increased several-
`fold, but such highly active T cells may be
`negatively selected by the thymus.
`HSCs transduced with CAR vectors
`produce CAR-expressing myeloid and
`natural killer cells in addition to T cells,
`and thus may provide more rapid and
`broader antitumor activity.36 In a mouse
`model with an EL4 tumor expressing the
`ovalbumin gene, an antitumor effect was
`observed using HSCs transduced with
`lentiviral vectors expressing TCR reactive
`to ovalbumin. A clinical trial involving
`autologous CD34+ cells transduced with a
`lentiviral vector expressing a CD19+ CAR
`in subjects with non-Hodgkin’s lymphoma
`is being developed at UCLA and the City
`of Hope Medical Center.
`
`Target selection
`Dr. Rosenberg reviewed the status of tar-
`get selection, which he viewed as the criti-
`cal challenge confronting immunotherapy.
`He considered the targets identified thus
`far to fall into five categories. The category
`that has been most extensively studied with
`TCRs is differentiation antigens that are
`overexpressed on cancers compared with
`normal tissues (e.g., MART-1, gp100, CEA,
`HER-2). As with conventional chemother-
`apy, this approach requires identifying a
`window of toxicity against the tumor cells
`without unacceptable damage to normal
`tissue. In the studies using the melanocyte
`differentiation antigens, an approximately
`25% objective response rate was obtained;
`however, normal melanocytes were also at-
`tacked, causing skin rashes, uveitis, and au-
`ditory and vestibular problems, all of which
`could be reversed by steroid treatment.10,37
`
`Design of T-cell receptors. TCRs are the
`physiological recognition system of T
`cells and react to a major histocompatibil-
`ity complex–antigen complex. Their two
`chains, a and b, are necessary and sufficient
`for T cells to recognize their targets, includ-
`ing cancer cells. Engineering of T cells with
`genetically modified TCR a- and b-chains
`redirects their antigen specificity and has
`been used in the clinic in adoptive cell
`transfer strategies. Clinical trials expressing
`TCRs for MART-1, gp100, and NY ESO-1
`have demonstrated antitumor activity in
`subjects with metastatic melanoma and
`sarcoma. However, these early clinical trials
`suggest that durable tumor responses seem
`to occur at lower frequency than with TILs
`or with CAR-engineered T cells.
`The clinical trials thus far have used
`TCRs with physiological peptide affinities,
`and most have used intact TCRs. However,
`studies with NY ESO-1 and MAGE-A3 as
`targets used TCRs with altered affinities due
`to targeted mutations in their complemen-
`tarity-determining region 2 or 3 (CDR2 or
`CDR3), the variable regions of the TCR that
`interact with the major histocompatibility
`complex–antigen complex. However, care
`must be taken because a CDR2-modified
`MAGE-A3 TCR led to cardiac toxicities,
`due to loss of specificity with cross-reaction
`to an off-target peptide.35
`Other means to increase antitumor
`activity of TCR-modified T cells are being
`developed preclinically, such as additional
`genetic engineering of the T cells to express
`
`other immune-activating genes, engineer-
`ing the signaling pathways downstream of
`the TCR, or blocking negative regulatory
`receptors. These approaches would pro-
`vide simultaneous genetic redirection of
`T cells with increased T-cell functionality
`that may no longer be blocked by physi-
`ological immune regulatory processes.
`A problem with some transgenic TCRs
`is that, when expressed in T cells that have
`their own endogenous TCR a- and b-
`chains, there can be heterologous pairing
`between the transgenic and endogenous
`TCR chains. This may decrease the ex-
`pression of the transgenic TCR and even
`lead to altered specificities that may po-
`tentially result in autoimmune toxicities.
`Several means to improve self-pairing of
`the transgenic TCR chains include the use
`of picornavirus-derived highly efficient
`self-cleaving 2A-like sequences to allow
`stoichiometric protein expression, includ-
`ing additional cysteine motifs allowing
`formation of an increased number of di-
`sulfide bonds between the a- and b-chains,
`partially murinizing the constant region of
`both TCR chains for preferential pairing,
`and the use of leucine zippers at the 3ʹ ends
`of both a- and b-chains for forced trans-
`genic TCR pairing. As these approaches
`move into the clinic, it will be important
`to test them in carefully designed clinical
`trials to minimize risks but also foster con-
`tinued improvements in treatment options.
`Longer-term antitumor activity may
`be achievable by targeting hematopoietic
`
`40
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0
`
`No. of protocols
`
`CD19
`
`CD20
`
`CD30
`
`ROR1
`
`Kappa1
`
`Figure 2 Chimeric antigen receptor targets for hematological-malignancy protocols
`registered with the National Institutes of Health’s Office of Biotechnology Activities.
`
`1568
`
`www.moleculartherapy.org vol. 22 no. 9 september 2014
`
`meeting report
`
`© The American Society of Gene & Cell Therapy
`
`UPenn Ex. 2080
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`Similar problems occurred with targeting
`of CEA, which is expressed at low levels
`on colonic epithelium, resulting in tem-
`porary but almost complete destruction
`of that tissue,20 and with HER-2 targeting,
`which results in severe adverse effects on
`the pulmonary epithelium and death of the
`subject.38 T-cell therapy is highly potent but
`also so sensitive that the T cells can recog-
`nize even extremely low target expression
`in normal tissues. This potential for on-tar-
`get, off-tumor toxicity has limited the de-
`velopment of certain targets as an effective
`cancer treatment.
`The second class of targets includes
`antigens expressed on tumor cells and rel-
`atively nonessential normal tissues. This
`includes CD19 as targeted by CARs and
`thyroglobulin, targeted by TCRs for thy-
`roid cancer. This approach is promising
`but requires identification of additional
`tissue-specific