Search

Chimeric antigen receptor (CAR)–modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited in vivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rβγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the in vivo proliferation of T cells and natural killer cells. In vitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. In vivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptor coexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15—Neoleukin-2/15 (Neo-2/15)—both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells. In trans-activation mode, tumor antigen targeting of the two components enhanced antitumor activity and attenuated toxicity compared with systemic treatment in syngeneic mouse melanoma models. In cis-activation mode, immune cell targeting of the two components selectively expanded CD8+ T cells in a syngeneic mouse melanoma model and promoted chimeric antigen receptor T cell activation in a lymphoma xenograft model, enhancing antitumor efficacy in both cases.

Supplementary Tables 1-3 and Supplementary Tables 1-6

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer–testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into “hot” tumors will work in concert with anti–PD-1 therapy to provide patient benefit.

CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell–mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo. CAR-binding sites on CD20+ tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19–72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509–19. ©2016 AACR.See related Spotlight by Sadelain, p. 473.

Supplementary Figure 4. Activation of HUVECs by serum from patients with neurotoxicity.

Serum chemistry and cytokine levels in control animals.

10 million (A) CD8+ TCM-derived CD19-CAR-T-cells, (B) CD19-CAR-T-cells derived from an unselected T-cell population, or (C) CD8+ TCM-derived ROR1-CAR-T-cells were injected into tumor-free NSG mice. After 24 hours, bone marrow, spleens, and peripheral blood were isolated and analyzed for the presence of T-cells (CD45+/CD4+ or CD45+/CD8+) and the expression of activation markers CD25 and CD69 by flow cytometry.

(A) Cytolytic activity of CD8+ TCM-derived T-cells expressing long/CD28, long/4-1BB, and long/CD28_4-1BB CD19-CARs against K562/CD19, K562, and Raji cells. (B) Cytokine production by CAR-T-cells after co-culturing with Raji cells. Supernatants were obtained after 24 hours for analysis. (C) Proliferation of CD19-CAR-T-cells 72 hours after stimulation with Raji cells by CFSE dye dilution. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent {greater than or equal to}4/3/2/1 cell divisions is provided in the upper left of each plot.

CSF and serum levels of 21 cytokines and chemokines measured before and at indicated days after adoptive CD20 CAR T cell transfer.

Supplementary Table S2

Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) because it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1 expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites, where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates. Cancer Immunol Res; 3(2); 206–16. ©2014 AACR.

Supplementary methods, figure legends, and Supplementary Table 1. Lymphodepletion regimens prior to CAR-T cell infusion; Supplementary Table 2. Neurologic adverse event terms

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy.Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed.In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

Supplementary Figures and Table S1

Antibodies and reagents used in Materials and Methods.

T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune-checkpoint inhibitor therapy or adoptive cell transfer. Much of the focus has been on identifying epitopes presented to CD8+ T cells by class I MHC. However, CD4+ class II MHC-restricted T cells have been shown to have an important role in antitumor immunity. Unfortunately, the vast majority of neoantigens recognized by CD8+ or CD4+ T cells in cancer patients result from random mutations and are patient-specific. Here, we screened the blood of 5 non–small cell lung cancer (NSCLC) patients for T-cell responses to candidate mutation-encoded neoepitopes. T-cell responses were detected to 8.8% of screened antigens, with 1 to 7 antigens identified per patient. A majority of responses were to random, patient-specific mutations. However, CD4+ T cells that recognized the recurrent KRASG12V and the ERBB2 (Her2) internal tandem duplication (ITD) oncogenic driver mutations, but not the corresponding wild-type sequences, were identified in two patients. Two different T-cell receptors (TCR) specific for KRASG12V and one T-cell receptor specific for Her2-ITD were isolated and conferred antigen specificity when transfected into T cells. Deep sequencing identified the Her2-ITD–specific TCR in the tumor but not nonadjacent lung. Our results showed that CD4+ T-cell responses to neoantigens, including recurrent driver mutations, can be derived from the blood of NSCLC patients. These data support the use of adoptive transfer or vaccination to augment CD4+ neoantigen-specific T cells and elucidate their role in human antitumor immunity.

Supplementary Figure from B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model

The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however, the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor–bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. Cancer Immunol Res; 3(2); 125–35. ©2014 AACR.

Supplemental Materials and Methods

(A) Mice were inoculated with 0.5 million Raji-ffluc cells via tail vein injection and tumor engraftment was confirmed by bioluminescence imaging on day six. On day seven, mice received a single i.v. injection of various doses of CD8+ TCM-derived T-cells transduced with the short spacer CD19/4-1BB/CD3ζ-CAR or of control T-cells (EGFRt). (B) Tumor growth was measured by bioluminescence imaging and is plotted for individual mice (4 mice per group). Arrows mark the day of T-cell transfer. Bioluminescence images from day six (one day before T-cell transfer) and day 19 (12 days after transfer) are shown.

FIGURE S4: Analysis of tonic signaling and function of trispecific and monspecific DARPin CARs

Purpose:The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition.Experimental Design:A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo.Results:The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen.Conclusions:DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.

CCR Translation on this Article from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

Supplementary Figure Legends

Purpose: This study examines cell surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression, and multiple groups are developing antibodies and/or chimeric antigen receptor–modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most nonmutated tumor antigens; however, prior studies suggest that ROR1 is absent on most normal tissues.Experimental Design: Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell surface ROR1 by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1 and evaluated its specificity and sensitivity in IHC.Results: The 6D4 mAb is a sensitive and specific reagent to detect cell surface ROR1 by IHC. The data show that ROR1 is homogenously expressed on a subset of ovarian cancer, triple-negative breast cancer, and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues, including parathyroid; pancreatic islets; and regions of the esophagus, stomach, and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues, suggesting that the macaque is a suitable model to evaluate safety of ROR1-targeted therapies.Conclusions: ROR1 is a promising immunotherapeutic target in many epithelial tumors; however, high cell surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1-targeted therapies warrants careful monitoring of toxicities to normal organs and may require strategies to ensure patient safety. Clin Cancer Res; 23(12); 3061–71. ©2016 AACR.

PDF File - 122K, Supplementary Figure 5: Expression of ROR1 and NKG2D ligands on epithelial cancer cell lines.

PDF File - 169K, Supplementary Figure 4: The function of ROR1-CAR and CD19-CAR modified CD8+ T-cells against primary CLL is augmented by CAR-modified CD4+ helper T-cells.

Purpose: Tumor regression has been observed in some patients with metastatic renal cell carcinoma (RCC) after nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Cellular and molecular characterization of antigens recognized by tumor-reactive T cells isolated from responding patients could potentially provide insight into the mechanisms of tumor regression.Experimental Design: CD8+ CTL clones that recognized a novel RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201 were isolated from two patients with metastatic RCC who experienced tumor regression or stable disease following nonmyeloablative allogeneic HCT. These clones were used to screen a cDNA library and isolate the unique cDNA encoding the antigen.Results: An alternative open reading frame in the C19orf48 gene located on chromosome 19q13 encodes the HLA-A*0201–restricted minor H antigen recognized by the RCC-reactive T cells. The differential T-cell recognition of donor- and recipient-derived target cells is attributable to a nonsynonymous single-nucleotide polymorphism within the nucleotide interval that encodes the antigenic peptide. Assays for gene expression and CTL recognition showed that the C19orf48-encoded peptide is widely expressed in renal tumors and solid tumors of other histologies. The antigenic peptide can be processed for CTL recognition via both TAP-dependent and TAP-independent pathways.Conclusions: Donor T-cell responses against the HLA-A*0201–restricted minor H antigen encoded by C19orf48 may contribute to RCC regression after MHC-matched allogeneic HCT.

PDF File - 125K, Supplementary Figure 3: In vitro function of T-cells expressing CD19-CARs with IgG4-Fc Hinge vs. CD8a Hinge spacer and a signaling module with 4-1BB costimulatory domain.

CCR Translation for the Article from Development of Tumor-Reactive T Cells After Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplant for Chronic Lymphocytic Leukemia

PDF File - 129K, Supplementary Figure 1: Design of ROR1-CARs with modified spacer length and derived from the 2A2 and R12 scFV with different affinity.

Purpose: The adoptive transfer of T cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single-chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module, has activity in advanced malignancies. The receptor tyrosine kinase–like orphan receptor 1 (ROR1) is a tumor-associated molecule expressed in prevalent B-lymphoid and epithelial cancers and is absent on normal mature B cells and vital tissues, making it a candidate for CAR T-cell therapy.Experimental Design: We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma (MCL) engrafted into immunodeficient mice.Results: ROR1-CARs containing a short “Hinge-only” extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared with CARs with long “Hinge-CH2-CH3” spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation-induced T-cell death. T cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR–modified T cells in mediating regression of JeKo-1 MCL in immunodeficient mice.Conclusions: Our results show that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T cells modified with an optimized ROR1-CAR have significant antitumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. Clin Cancer Res; 19(12); 3153–64. ©2013 AACR.

PDF File - 83K, Supplementary Figure 2: Analysis of cytokine production and proliferation of CD4+ T-cells lines modified with a ROR1-CAR derived from mAb R12 with higher affinity than 2A2.

Supplementary Figures S1-S2 from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

TABLE S1: 9-mer peptides in the scFv and DARPin sequences with predicted binding to the MHC class I supertype with a predicted binding affinity of less than 100 nM

Supplementary Figure 1: IHC staining using previously published ROR1 antibodies on control and ROR1 transfected K562 cells, CLL and MCL lymph nodes and tonsil tissue. Supplementary Figure 2: Development of mAb 6D4 Supplementary Figure 3: Representative images of IHC scoring of ROR1+ tumors. Supplementary Figure 4: ROR1 expression in pancreatic adenocarcinomas using mAb 6D4. Supplementary Figure 5: ROR1 staining in absent in normal human cerebrum, cerebellum, heart, lung, spleen and liver. Scale bar represents 100μm. Supplementary Figure 6: Immunoblot validation of ROR1 expression in normal tissues. Supplementary Figure 7: Transcript expression of Ror1 variant 1 in CLL PBMC, differentiated adipocytes and normal human gastrointestinal tissues.

PDF File - 102K, Legend for Supplementary Figures 1-5

Supplementary Tables 1-3 from Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells