Multi-Specific CAR Targeting to Prevent Antigen Escape
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Current Hematologic Malignancy Reports (2019) 14:451–459 https://doi.org/10.1007/s11899-019-00537-5 CART AND IMMUNOTHERAPY (M RUELLA AND P HANLEY, SECTION EDITORS) Multi-Specific CAR Targeting to Prevent Antigen Escape Zachary Walsh1 & Savannah Ross1 & Terry J. Fry1 Published online: 22 July 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Purpose of Review Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable remission induction rates for relapsed/refractory B cell malignancies. However, loss of the CAR-targeted antigen, known as antigen escape, accounts for a substantial percentage of relapses following CAR therapy and is a major barrier to durable remission. Here, we discuss mech- anisms for antigen escape and strategies to prevent this pattern of relapse, including the use of multi-specific CARs, which recognize and target multiple tumor-associated antigens simultaneously. Recent Findings Preclinical and early clinical trial data indicates that multi-specific CAR therapy for B cell malignancies is both safe and effective. Optimal combinations of target antigens, as well as different multi-specific CAR formats, are currently being evaluated. Summary Although still in early stages of development, multi-specific CAR therapy represents a promising approach to mitigate antigen loss–related relapses and improve durability of remission in patients with refractory B cell malignancies, and may be applicable to other types of cancer. Keywords CAR Tcell . Antigen loss . Antigen escape . Multi-specific . Bivalent . Lineage switch Introduction adult and pediatric relapsed and refractory (r/r) B cell acute lymphoblastic leukemia (B-ALL) (CD19 CAR, 70–90% com- Chimeric antigen receptor (CAR) T cell therapy is a rapidly plete remission (CR) at 28 days), non-Hodgkin’slymphoma evolving immunotherapeutic treatment modality for adult and (NHL) (CD19 CAR, 54–74% CR at 3 months), and multiple pediatric patients with cancer. CAR T cells are genetically myeloma (BCMA CAR, 94% overall remission/response rate modified cells that contain a synthetic receptor that targets a (ORR)) [1]. CARs targeting IL-13 receptor alpha 2, HER2, specific antigen. A monovalent CAR targets one antigen and and EGFRvIII have shown promise in the treatment of recur- is composed of a single chain variable fragment (scFv), hinge rent multifocal glioblastoma multiforme, suggesting a role for region, transmembrane domain, one or more costimulatory CAR T cell therapy in brain tumors [2]. Unfortunately, the use domains, and an intracellular signaling domain (CD3 zeta). of CAR T cells in solid tumors is more challenging for multi- Monovalent CAR T cell therapy has been proven to be an ple reasons, including difficulty with CAR T cells trafficking effective strategy to treat multiple types of cancer, including to the tumor, tumor microenvironment impeding CAR T func- tion, and expression of targeted antigens on healthy tissues, leading to increased on-target/off-tumor toxicity [1]. CARTand Immunotherapy This article is part of the Topical Collection on While the majority of current CAR T cell products target a single tumor antigen, recent data shows that relapse frequently * Terry J. Fry [email protected] occurs due to loss of the target antigen, termed antigen escape, as is seen after treatment with CD19 and CD22 monovalent CARs Zachary Walsh [3, 4]. Up to 60% of relapses after CD19 CAR are due to loss of [email protected] CD19 antigen [3]. Similarly, some patients who receive CD22 Savannah Ross CAR relapse with diminished CD22 site density, rendering them [email protected] unable to respond to further CD22 directed therapies [5••, 6]. 1 Center for Cancer and Blood Disorders, Children’s Hospital Antigen loss has also been reported with the B cell maturation Colorado, University of Colorado Anschutz Medical Campus, 13123 antigen (BCMA) in BCMA CAR clinical trials for the treatment East 16th Avenue, Box B115, Aurora, CO 80045, USA of multiple myeloma [7]. Efforts are being made to overcome the 452 Curr Hematol Malig Rep (2019) 14:451–459 problem of antigen escape, including the use of multi-specific Antigen escape can occur through loss of the target epitope, antigen targeting CAR T cells, which target two or more tumor which accounts for 10–20% of relapses after CD19 CAR in antigens simultaneously. Studies show that targeting multiple pediatric B-ALL. In a study by Sotillo et al. comparing patient antigens can result in a synergistic effect and can help overcome samples pre- and post-CD19 CAR therapy, they found that variable antigen expression levels, which may diminish CAR T some patients harbored a deletion within the CD19 locus, cell effectiveness [4, 8]. while others had alterations in transcriptional regulation Sequential immunotherapeutic targeting of a second anti- [12]. Patient samples were found to have variable splice sites, gen after relapse associated with antigen loss is one approach as well as nonsense, missense, and frameshift mutations, lead- to multi-specific targeting. However, as was seen with CD22 ing to exon skipping and expression of truncated CD19 vari- CAR therapy in leukemia patients with antigen-negative re- ants with altered CD19 function and loss of recognition by lapse after CD19 CAR, patients often relapse with loss of both CD19 CAR. The target epitope of the CAR, FMC63, is antigens. This observation prompted the development of a thought to be affected by skipping exon 2 specifically. bivalent CAR construct targeting both antigens simultaneous- Analysis of CD19-negative relapses from patients treated on ly, in hopes that patients would be less likely to relapse via the ELIANA trial demonstrated that all patients harbored dif- antigenescape[5••]. Multi-antigen targeting through CAR T ferent mutations that resulted in CD19 loss [13]. CD19 protein cells harnesses the treatment principle that is already inherent lacking exon 2 is expressed in a more stable form that is found to cancer therapy—targeting multiple oncogenic pathways si- more often in the cytosol or retained in the endoplasmic retic- multaneously through the use of different types of chemother- ulum rather than on the leukemia cell surface, thus conferring apy helps prevent tumor cells from developing resistance to a a survival advantage as it is not targetable by subsequent single agent and thus minimizes the risk of relapse. This re- CD19-directed therapy [14].Basedonthesefindings, view outlines the various mechanisms for antigen escape and targeting essential exons of the antigen of interest may be a strategies to prevent antigen-negative relapse, and summarizes way to overcome antigen escape [12]. multi-specific CAR T cell therapies currently undergoing de- Relapses after CD22-directed CAR therapy for patients with velopment and testing. B-ALL can also occur due to diminished and variable expres- sion of CD22 on the surface of leukemia cells, rather than complete loss of the targeted epitope as with CD19 [5••]. Indeed, leukemic cells with lower CD22 expression can evade Mechanisms of Antigen Escape CD22 CAR leading to eventual relapse, suggesting low sensi- tivity of the current CAR formats as a limitation [6]. Recently A comprehensive discussion of the mechanisms of CAR Tcell published data from a monovalent CD22 clinical trial resistance can be found elsewhere but will be reviewed briefly (clinicaltrials.gov/NCT02315612) suggests that prior here [9]. Relapse after CAR T cell therapy can occur with a treatment with inotuzumab (anti-CD22 monoclonal antibody) preserved expression of the antigen target, due to failure of the or CD22 CAR causes diminished CD22 expression on the leu- CAR T cells to expand or function once infused, as seen after kemia cell surface, similar to the selective pressure seen with CD19 CAR. Strategies to improve CAR persistence can help CD19 CAR [15]. These patients have decreased response rates prevent this [10] and will not be discussed in this review. and faster relapses post-CD22 CAR, indicating that sequential However, a more frequent pattern of relapse is the loss of targeting and diminished site density of the target antigen at the the target antigen. Antigen-negative relapse was first noted time of treatment can impair CAR efficacy and lead to antigen- by Grupp et al. in a patient treated with CD19 CAR [11]and negative relapse [15]. In a study by Fry et al., a comparison of is now known to account for 60% of relapses post-CD19 CAR leukemia cells pre- and post-CAR T cell therapy in two patients and up to 30% of relapses after treatment with blinatumomab, who relapsed with diminished CD22 site density found no ge- a bispecific antibody targeting CD19/CD3. One possible netic mutations in the CD22 locus nor qualitative changes in mechanism for this is through selective pressure causing the mRNA levels or alternative CD22 isoforms. This suggests that proliferation of a CD19-negative B-ALL precursor population post-transcriptional effects cause diminished CD22 cell surface that exists at the time of CART treatment. Often, CD19- expression levels [5••]. Additional mechanisms for antigen loss negative populations cannot be identified at the time of diag- have been postulated for patients with lymphoma, such as post- nosis, but patients still go on to relapse with loss of the target translational changes, loss of DNA repair proteins, or clonal antigen, suggesting that other mechanisms may also contrib- evolution due to selective pressure from treatment with ute to emergence of antigen-negative leukemia (Fig. 1a). antigen-directed CAR [16]. CD123, a putative marker on leukemia stem cells, is often In addition to the mechanisms already described for anti- expressed on leukemia with CD19-negative relapse following gen loss post-CAR, recent studies in B-ALL have shown that CD19 CAR therapy, suggesting that stem cell populations patients may relapse with lineage switch to T cell or myeloid may be a source for antigen escape [3]. leukemia [17]. In xenograft models, relapse has occurred with Curr Hematol Malig Rep (2019) 14:451–459 453 Fig.