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AVI file - 3MB, Movie of THP-1 cells engulfing latex microparticles after coculturing with SUM149 basal-like breast cancer cells

AVI file - 3MB, Movie of THP-1 cells engulfing latex microparticles after coculturing with MCF-7 luminal breast cancer cells.

PDF file - 103K, List of genes defined in the literature as significant in monocyte-to-macrophage differentiation and polarization.

PDF file - 128K, Control system-generated M1 and M2 macrophage significantly expressed genes involved in macrophage differentiation and polarization defined by 4x44k microarray analysis.

PDF file - 64K, Gene expression heatmap data of THP-1 macrophages differentiated by classical stimuli on Agilent 4x44k microarrays.

PDF file - 62K, Ingenuity Pathway Analysis on M1- and M2-specific gene clusters from THP-1expression responses to classical stimuli.

Advances in the development of immunotherapies have offered exciting new options for the treatment of malignant diseases that are refractory to conventional cytotoxic chemotherapies. The adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has demonstrated dramatic results in clinical trials and highlights the promise of novel immune-based approaches to the treatment of cancer. As experience with CAR T cells has expanded with longer follow-up and to a broader range of diseases, new obstacles have been identified which limit the potential lifelong benefits of CAR T cell therapy. These obstacles highlight not only the gaps in knowledge of the optimal clinical application of this "living drug", but also gaps in our understanding of the fundamental biology of CAR T cells themselves. In this review, we discuss the obstacles facing CAR T cell therapy, how these relate to our current understanding of CAR T cell biology and approaches to enhance the clinical efficacy of this therapy.

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL., CAR-T targeting CD19 have been successfully used in a variety of B-cell malignancies but patients may eventually relapse. Here, the authors show that CD19 CAR-T resistance in pre-B cell ALL can be due to the induction of a myeloid lineage switch through an epigenetic alterations in master regulators of B cell development.

Chimeric antigen receptor (CAR)–expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non–major histocompatibility complex–restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre–B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell–associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.

Meeting abstracts Chimeric-antigen-receptor (CAR) T cells targeting CD19 show dramatic remissions in refractory or relapsed acute lymphoblastic leukemia. Interleukin-6 (IL-6) has been associated with severe cytokine release syndrome (CRS) following CAR T cell treatment, leading to significant

As T cells engineered with chimeric antigen receptors (CARs) are entering advanced phases of clinical trial testing with promising results, the potential implications of use in an allogeneic environment are emerging as an important consideration. This review discusses the use of allogeneic CAR therapy, the potential effects of T-cell receptor (TCR) signaling on CAR T-cell efficacy, and the potential for TCR elimination to generate an off-the-shelf product.The majority of preclinical and clinical data regarding allogeneic T cells are focused on safety of their use given the potential for graft-versus-host disease (GVHD) mediated by the T-cell receptor expressed with the introduced CAR. Recent clinical trials using donor-derived CAR T cells are using either rigorous patient selection or T-cell selection (such as enrichment for virus-specific T cells). Although no GVHD has been reported, the efficacy of the allogeneic CAR treatment needs to be optimized. Several preclinical models limit allogeneic CAR-driven GVHD by utilizing memory T-cell selection, virus-specific T cells, gene-editing techniques, or suicide gene engineering.In the allogeneic environment, the potential effects of TCR signaling on the efficacy of CAR could affect the clinical responses with the use of donor-derived CAR T cells. Better understanding of the functionality of donor-derived T cells for therapy is essential for the development of universal effector cells for CAR therapy.

Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4(+) CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Tremendous progress has been achieved employing immunotherapy for B cell acute lymphoblastic leukemia (ALL), a leading cause of death in children from cancer. Recent trials using chimeric antigen receptor T cells (CART) targeting the B cell restricted antigen, CD19, that utilize the autologous transfer of patients' T cells, have demonstrated remarkable remission rates of 80% against relapsed or refractory ALL. Despite initial clearance of tumor, relapse with CD19 antigen loss ALL and with CD19 expressing ALL can occur. Attempts at retreatment of patients who have received CD19 CAR T cell therapy suggests that most patients will not respond to a second infusion of CD19 CAR T cells. It has been proposed that failure to respond to a second infusion of CAR T cells may be due to immunogenicity of the foreign CAR protein and elimination of CAR T cells due to immunological targeting. To evaluate the mechanism of retreatment failure in the setting of persistent antigen, we utilized a murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR transduced into mouse CD8 and CD4 polyclonal cells and tested against murine pre-B ALL in a syngeneic system. To investigate the issue of immunogenicity against CAR constructs, we immunized the mice with irradiated CAR T cells prior to CAR treatment to allow for anti-CAR T cell immunity. Following immunization, we inoculated the mice with leukemia on day 0 and treated the mice with 1 x 106 CAR T cells on day 4. CAR treatment was able to clear leukemia and CAR T cell-reactive antibodies were not detected in the serum of the mice, suggesting that a mechanism other than classic host mediated immune rejection of CAR T cells may underlie CAR T cell retreatment failure. To further model the failure of CAR T cell retreatment, we evaluated the ability of a second CAR T cell infusion to eliminate a second leukemic challenge. Leukemia bearing mice were treated with a curative dose of CD19 CAR T cells post lymphodepleting regimen. 30 days after clearance of the primary leukemic challenge, the mice were rechallenged with leukemia and subsequently treated with mock T cells or CD19 CAR T cells. Mice treated with CAR T cells followed by retreatment with mock T cells demonstrated persistence of CAR T cells from the first treatment, which were able to expand and clear the second leukemia challenge. In mice treated with a second dose of CAR T cells, CAR T cells from the second infusion briefly expanded 10 days post infusion, but could not be detected at day 20 post infusion. In contrast, CAR T cells from the initial infusion were still detectable at both time points. These results demonstrate that CAR T cells are able to persist, and, in a model of leukemic relapse, are able to expand and clear leukemia. However, CAR T cells infused into mice with CAR T cells persisting after a prior infusion fail to persist and quickly contract without evidence of host immune rejection of CAR T cells. Our data suggests that the inability to successfully retreat CD19+ relapsed leukemia with subsequent doses of CAR T cells may also involve mechanisms beyond immune recognition and clearance of CAR T cells. Disclosures No relevant conflicts of interest to declare.

Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell-like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.

The miR-106b-25 microRNA (miRNA) cluster is a candidate oncogene in human prostate cancer. Here, we report that miRNAs encoded by miR-106b-25 are upregulated in both primary tumors and distant metastasis. Moreover, increased tumor miR-106b expression was associated with disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expressions in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio=4.1; 95% confidence interval: 1.6–12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that CASP7 is a direct target of miR-106b, which was confirmed by western blot analysis and a 3′-untranslated region reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and CASP7 expression were inhibited. Further analyses showed that CASP7 is downregulated in primary prostate tumors and metastatic lesions across multiple data sets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and disease outcome and may do so by altering apoptosis- and focal adhesion-related pathways.

Basal-like breast cancer (BBC) is an aggressive subtype of breast cancer that has no biologically targeted therapy. The interactions of BBCs with stromal cells are important determinants of tumor biology, with inflammatory cells playing well-recognized roles in cancer progression. Despite the fact that macrophage–BBC communication is bidirectional, important questions remain about how BBCs affect adjacent immune cells. This study investigated monocyte-to-macrophage differentiation and polarization and gene expression in response to coculture with basal-like versus luminal breast cancer cells. Changes induced by coculture were compared with changes observed under classical differentiation and polarization conditions. Monocytes (THP-1 cells) exposed to BBC cells in coculture had altered gene expression with upregulation of both M1 and M2 macrophage markers. Two sets of M1 and M2 markers were selected from the PCR profiles and used for dual immunofluorescent staining of BBC versus luminal cocultured THP-1s, and cancer-adjacent, benign tissue sections from patients diagnosed with BBCs or luminal breast cancer, confirming the differential expression patterns. Relative to luminal breast cancers, BBCs also increased differentiation of monocytes to macrophages and stimulated macrophage migration. Consistent with these changes in cellular phenotype, a distinct pattern of cytokine secretion was evident in macrophage–BBC cocultures, including upregulation of NAP-2, osteoprotegerin, MIG, MCP-1, MCP-3, and interleukin (IL)-1β. Application of IL-1 receptor antagonist (IL-1RA) to cocultures attenuated BBC-induced macrophage migration. These data contribute to an understanding of the BBC-mediated activation of the stromal immune response, implicating specific cytokines that are differentially expressed in basal-like microenvironments and suggesting plausible targets for modulating immune responses to BBCs. Mol Cancer Res; 10(6); 727–38. ©2012 AACR.

Adoptive immunotherapy using T cells armed with chimeric antigen receptors (CAR) has proven extremely effective against CD19+ B-lineage acute lymphoblastic leukemia (ALL) with remission rates as high as 70-90% in recent clinical trials in relapsed/refractory patients. CD8 T cells are typically thought of as the primary antitumor effector cells in an adoptively transferred product due to their potent cytolytic capabilities, whereas CD4 T cells are thought to primarily provide "help" to enhance CD8 T cell activity via cytokine production. However, CARs are synthetic constructs that likely alter the functionality of T cells. Further, CAR T cells are endowed with 2 specificities, one through the CAR and one through the endogenous T cell receptor (TCR). To evaluate the biology of CAR T cells, we sought to evaluate the impact of CAR expression on the functionality of T cells and to study the impact of TCR on CAR T cell activity using both human CAR T cells in a xenograft model and murine CAR T cells in a syngeneic murine model. A human or murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR was transduced into human or mouse CD8 (CAR8) and CD4 (CAR4) T cells, respectively, and tested against pre-B ALL cell lines in human murine xenografts or syngeneic models. Surprisingly, human CAR4 cells alone had equivalent ability to eradicate the Nalm6 ALL in vivo as CAR8 cells. Although CAR8 cells more rapidly cleared leukemia (2 vs 4 days), relapse eventually occurred. In contrast, CAR4 cells eradicated leukemia more slowly, but persisted longer and prevented relapse. In mice receiving CAR4+CAR8 cell products, only CAR4 cells were detectable at day 55. Next we utilized our syngeneic murine system to study CAR4 and CAR8 activity in an immunocompetent system using TCR transgenic T cells with known TCR specificity against the male histocompatibility antigen, HY. As expected, when stimulated through the TCR, CAR8 cells produced cytokines and degranulated, as manifested by CD107a expression, whereas CAR4 cells only demonstrated a cytokine production response. However, murine CAR4 cells activated through the CAR receptor develop "CD8-like" cytolytic features characterized by degranulation and were able to completely eradicate leukemia in vivo when administered without CAR8 cells, similar to the human CAR4 cells. We then evaluated the effect of TCR antigen in vivo on CAR T cell function by comparing activity in male (HY+) vs female (HY-) recipients. CAR4 cells were curative in both male and female recipients. However, CAR8 treatment has no anti-tumor activity in male recipients indicating that CAR8 cells are very susceptible to the negative effects of TCR signaling. TCR antigen availability in vivo greatly increased the number of infused CAR4 cells (P=0.003, male vs female recipients), while they significantly decreased the number of CAR8 cells (P=0.0023, male vs female recipients) after 1 week. Interestingly, we found that there was significant down-regulation of CAR expression on both CAR4 and CAR8 cells in male recipients (HY+). PD-1 and Tim3 were expressed at low levels on CAR4 cells and CAR8 cells in female recipients. However, there was significantly higher PD-1 (P=0.0079) and TIM3 (p=0.014) expression on CAR8 cells in male vs female recipients, whereas there was no difference in PD-1 or Tim3 on CAR4 cells recovered from male vs female recipients. These data indicate that CAR4 cells are more functional and become less exhausted than CAR8 cells in the presence of TCR antigen. Finally, we evaluated the long-term persistence of CAR4 and CAR8 cells in our syngeneic model in which there is no xenogeneic reactivity allowing for long-term monitoring. At day 60 and day 80, CAR4 cells were detectable in bone marrow and spleen, whereas CAR8 cells had completely disappeared. Despite the long-term persistence of CAR4 cells in recipients with or without TCR antigen, CAR4 cells are not completely immune to effects of TCR activation. In male recipients after 60 days, CAR4 cells also start to express high PD-1 levels and have decreased CAR T cell counts. These findings have important implications for CAR therapy, particularly in the allogeneic setting where the presence of TCR antigen may be more likely to be present in the recipient. In addition, these results suggest that CAR4 cells alone are cytolytic, equally potent to CAR8 cells at eradicating leukemia in vivo, and may be superior due to better persistence and reduced susceptibility to exhaustion. Disclosures No relevant conflicts of interest to declare.

Resistance or relapse following adoptive immunotherapy can occur as a result of antigen loss. T cells armed with chimeric antigen receptors (CARs) targeting CD19 have shown remission rates of over 70% in early clinical trials, although reports of relapse in the presence of persistent CAR T cells are emerging. To further investigate potential resistance mechanisms to CAR T cell therapy, we used 2 syngeneic murine pre-B acute lymphoblastic leukemia (ALL) cell models driven by E2a:PBX1 and Eu-RET. Transplantable cell lines generated from both models have high expression of CD19, CD22, B220, CD43 and CD127, with absence of surface immunoglobulins consistent with a pre-B cell phenotype and result in lethality within 21 days. Treatment with CD19 CAR T cells induced long-term remission with persistence of CAR T cells beyond 100 days. However, mice bearing E2a:PBX1-driven leukemia experienced relapses occurring in the majority of CAR treated animals within 1 year, all with loss of CD19 expression. Relatively early post-CAR relapses retained a pre-B phenotype with isolated loss of CD19 extracellular expression by flow cytometry, loss in CD19 exon 1 and 2 mRNA but intact mRNA for all other CD19 exons. In contrast, late post-CAR relapses demonstrated complete loss of CD19 protein and mRNA expression with concomitant loss of the major B cell transcription factors PAX5 and EBF1 suggesting loss of the B-cell developmental program. Furthermore, late post CD19 CAR relapses typically gained myeloid, stem cell or T cell phenotypic markers, consistent with a lineage switch, which was confirmed at the genomic level by RNA-seq of multiple late relapse samples. We could not identify cells in leukemia culture lacking pre-B phenotype either by single cell cloning following depletion of CD19+ cells indicating that cells with the late relapse phenotype do not exist as a rare population. Finally, we could also demonstrate intermediate phenotypes of post-CD19 CAR relapse in vivo with co-expression of both myeloid (Gr1, CD11b) and B cell markers (B220, CD22) on the same cells, suggesting a differentiation rather than a selection process. We next undertook serial in vivo passaging experiments, and confirmed leukemic-initiating potential of all CD19-loss relapses irrespective of relapse phenotype. Interestingly, leukemic relapse with CD19 expression loss that retain a B-cell program rapidly regain CD19 upon in vivo passage in the absence of CD19 CAR pressure. However, relapses due to lineage reprogramming retained a stable myeloid phenotype upon serial passage without regain of CD19 or other B cell markers. Knock-out of CD19 from leukemic cells using the CRISPR/CAS system did not alter proliferative capability or phenotype during prolonged (30 day) culture and did not diminish engraftment capacity and lethality in vivo, confirming that loss of CD19 alone is insufficient to drive lineage switch. Overall, we demonstrate a novel mechanism of resistance to immunotherapy via lineage switch and demonstrate reprograming potential of ALL under lineage-selective pressure. This observation was repeatedly seen in one murine model (E2a:PBX1) but not in another (Eu-RET), suggesting that the potential for this to occur may depend on the genetic subtype and at what stage the initiating mutation occurred. These results have important clinical implications for the development of antigen-targeting therapies for ALL and, potentially, other cancers. Disclosures No relevant conflicts of interest to declare.

Despite remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (ALL) following CD19 chimeric antigen receptor (CAR)-T cells or CD3-CD19 bispecific constructs, relapse of CD19 negative leukemic blasts occurs in approximately 10-20% of cases. The mechanism of antigen loss under CD19-targeted immune pressure on pre-B ALL has yet to be elucidated. Although xenograft models have been excellent tools for pre-clinical studies, the development of xenogeneic GVHD has precluded the ability to study late leukemic relapse under CD19-targeted immunotherapeutic pressure. Thus, we performed such studies in a murine transplantable pre-B ALL (CD19+ B220+ CD22+ sIgM- CD127+ CD43+) in immunocompetent mice that induces lethality of recipients within 15-25 days without therapy. Treating ALL bearing mice with a murine CD19 CAR T cells (CART), resulted in prolonged remissions followed by CART dose-dependent relapses varying between 50 days to >200 days post treatment. All relapses were negative for CD19 expression with earlier relapses maintaining similar pre-B cell surface marker phenotype of the parent ALL. However, relapses longer than 100 days consistently showed a dramatic alteration in phenotype, with loss of CD22, B220 and CD127, stability of CD45 and CD43, and partial gain of cKIT, resembling a more immature B cell or common lymphoid progenitor. While early relapses had normal mRNA levels of CD19 despite loss of surface antigen, late antigen loss correlated with low CD19 mRNA, along with other alterations in pre-B cell signature on microarray. This late antigen negative leukemia took longer time to engraft in secondary recipients, compared to the parent cell line. However, serial transplantation without immune-pressure resulted in regain of CD19 and CD22 antigens with tertiary recipients demonstrating leukemia phenotype and time to engraftment similar to the parent cell line. Interestingly, in-vivo reacquisition of CD22 always preceded regain of CD19. To further study loss of B cell antigen, we co-cultured CAR T cells with both human and murine leukemia. Using CD19 CART, we observed rapid CART dose-dependent loss of surface CD19 followed by partial loss of surface CD22, but not other leukemia markers, in both systems. Interestingly, antigen loss was not seen when co-culturing leukemia with anti-CD22 CART. Suboptimal dose of CD22 CART in an NSG mouse model of human leukemia did generate a CD22-CD19- late relapse. We conclude that loss of CD19 may be an early isolated phenomenon, while prolonged immune-pressure drives an mRNA-regulated loss of CD19 accompanied by fundamental alterations in leukemia antigens. We also demonstrated that CD22 loss requires previous loss of CD19. Altogether, as antigen-directed immunotherapies become more common in the clinic, these findings have implications for the optimization of CAR therapy, particularly if antigen loss can be regulated therapeutically. Citation Format: Elad Jacoby, Yinmeng Yang, Chris D. Chien, Waleed Haso, Haiying Qin, Terry J. Fry. Late ALL relapse following CD19 CAR immune-pressure demonstrates reversible pan-antigen loss. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4705. doi:10.1158/1538-7445.AM2015-4705

A significant portion of patients with relapsed acute lymphoblastic leukemia (ALL) who are eligible for treatment with chimeric-antigen-receptor (CAR) T cells have undergone a previous hematopoietic stem cell transplantation. We and others have previously demonstrated that the allogeneic environment, even in the presence mild graft-versus-host disease (GVHD), severely impairs tumor-directed T-cell immunity. To study the behavior of CAR cells in the allogeneic setting we chose a murine model of minor mismatched allogeneic T-cell depleted bone marrow transplantation (BMT) followed by CAR T (CD19-28-z) cell infusion in recipients bearing a murine B-precursor ALL model (positive for CD19). Importantly, the leukemia persists following myeloablative radiation thus mimicking post-transplant minimal residual disease. Donor-derived, post-transplant injection of CD19 CAR T cells eliminated residual leukemia in the syngeneic as well as the allogeneic settings. CD19 CAR T cells harvested from allogeneic recipients maintained in-vitro ability to produce IFN-gamma and degranulate in the presence of ALL ex vivo, at levels comparable to syngeneic CD19 CAR T cells. However, CAR T cells administered in the allogeneic environment had the potential to mediate severe acute GVHD with early mortality of recipients not typically seen in this minor mismatch model. This occurred across multiple T cell doses capable of clearing leukemia (0.3e6-5e6), in transduced CD19 CAR T cells generated from donors tolerized to allogeneic antigens, and when CD19 CAR T cell infusion was delayed following BMT. In-vivo tracking of transferred cells showed comparable expansion and persistence of the CD19 CAR T cells in the allogeneic and syngeneic environments. However, syngeneic CAR T cells tended to develop later into central memory T cells (CD62L+CD44+), whereas the profile of allogeneic cells was significantly skewed toward effector T cells (CD62L-CD44+). Remarkably, the process of CAR-driven acute GVHD in this minor mismatch model was only seen in the presence of leukemia, indicating a CAR-target interaction influences the induction of GVHD. Indeed, pro-inflammatory cytokines IFN-gamma and IL-6 were elevated only in the presence of both ALL and CAR T cells, whereas TNF alpha levels were undetectable in all instances. We are currently testing whether neutralization of cytokines can prevent GVHD in these models. Altogether, these data demonstrate efficacy of CD19 CAR to clear residual leukemia in an immunocompetent mouse model, and maintain initial cytotoxicity despite the potentially suppressive allogeneic environment. However, we demonstrated potential risks of allogeneic CAR T cells aggravating GVHD in the presence of residual leukemia, likely via cytokine-mediated manner. Clinically, as IL-6 was shown to be significant in cytokine release syndrome, this may represent a murine model to study potential interventions. Disclosures No relevant conflicts of interest to declare.

Adoptive therapy using T cells genetically engineered to express chimeric antigen receptors (CAR) has proven extremely effective against acute lymphoblastic leukemia (ALL) in clinical trials with the use of anti-CD19 CAR T cells. Most CAR T cell protocols use autologous T cells, which are then activated, transduced with the anti-CD19 CAR, and expanded ex-vivo before infusion back into the patient. This approach minimizes the risk of graft-versus-host disease (GVHD) even in allogeneic transplant recipients, due to tolerization of the donor T cell repertoire in the recipient. However, many patients have heavy disease burden and lymphopenia due to previous treatments, which makes the isolation of healthy T cells difficult. Thus, centers are exploring the potential of allogeneic T cell donors and the possibility of universal T cell donors for CAR-based therapy including the use of virus-specific T cells. In these cases, in addition to the chimeric receptor specificity, the transduced T cell population will also have reactivity against target antigens through the endogenous TCR. However, little is known about the impact of signaling of the endogenous TCR on CAR T cell activity, particularly in vivo. To test this, we used a syngeneic transplantable ALL murine model, E2aPBx, in which CD19 CAR T cells can effectively eradicate ALL. CD4 (Marilyn) and CD8 (Matahari) T cells from syngeneic HY-TCR transgenic donors specific for the minor histocompatibility male antigen, HY, were used as CAR T cell donors to control for endogenous TCR reactivity. Splenic T cells isolated from Matahari, Marilyn, or B6 mice were activated ex-vivo using anti-CD3/anti-CD28 beads, with the addition of IL2 and IL7. T cells were transduced with a retroviral vector expressing a murine CAR composed of anti-CD19 scfv/CD28/CD3ζ on days two and three. CAR T cells are evaluated in vitro by CD107a degranulation assay and INF gamma ELISA. In response to HY peptide alone or HY+CD19- line M39M, transduced CD8 HY (Matahari) cells produced IFN gamma and expressed CD107a whereas transduced CD4 HY (Marilyn) cells only produced IFN gamma. Interestingly, in response to CD19+HY- ALL, both Matahari and Marilyn expressed CD107a and produced IFN gamma indicating that CD4 T cells can acquire CD8-like lytic activity when stimulated through a CAR receptor. When CD19 CAR transduced Marilyns and Mataharis were stimulated in the presence of HY and CD19, CD8 Mataharis had an attenuated effect against CD19, suggesting that the presence of antigen activated TCR adversely affects the potency of the CAR receptor. Efficacy of the HY and polyclonal CAR T cells were next tested in-vivo in male and female B6 mice. Mice were given 1E6 E2aPBx ALL leukemia cells on day 1, and received 500 rads sub-lethal total body irradiation on day 4 as a lymphodepleting regimen. On day 5, mice were given a low (1E5) or high (5E6) dose of CAR T cells. There was a statistically significant (p=0.0177) improvement in the survival of female versus male mice after treatment with the CD4+ HY specific anti-CD19 CAR T cells, and female mice that received HY anti-CD19 CAR T cells survived longer than untreated control females (p=0.01). Remarkably, the survival of male mice that received HY anti-CD19 CAR T cells was statistically worse than untreated control males (p=0.008). This suggests that the presence of TCR antigen negatively impacts the function of CAR T cells. Furthermore, in a separate experiment using an equally mixed population of Marilyn (CD4+) and Matahari (CD8+) HY specific T cells, males has a statistically significantly (p=0.0116) worse survival compared to females after receiving 5E5 HY specific T cells. In conclusion, simultaneous stimulation through both CAR and TCR results in attenuated cytokine production and degranulation by CD8 T cells. In vivo, in the presence of the endogenous TCR antigen, both CD4 and CD8 CAR T cells are less potent at eradicating leukemia. These have implications for the development of universal donors for CAR T cell therapy. Disclosures No relevant conflicts of interest to declare.

Breast cancer is a multi-faceted disease giving rise to five distinct subtypes. Emerging data show that each of these subtypes has distinct interactions with the surrounding stroma and immune cell populations present in the microenvironment. We hypothesized that macrophages have distinct interactions with breast cancer in a subtype-dependent manner. Using a coculture system, we evaluated changes in THP-1 differentiation, morphology and gene expression during the monocyte-to-macrophage transition in response to cell lines representing the basal-like (HCC1937, MDA-MB-468, SUM149), luminal (MCF-7, T47D, ZR-75-1), epidermal growth factor receptor/HER2-enriched (BT474, SKBR3) and claudin-low (Hs578T, MDA-MB-231, SUM159) breast cancer subtypes. Macrophage polarization phenotypes were validated by immunofluorescence staining for M1 (CD163) and M2 (CD36) macrophage markers in normal tissue sections adjacent to representative breast tumors. We further investigated differences in genomic and cytokine expression profiles, proliferation, and chemosensitivity (to Doxorubicin) of the breast cancer cell lines in response to THP-1 coculturing. Greatest levels of THP-1 differentiation was induced in basal-like cocultures (mean differentiation 79%, compared to differentiation Citation Format: Delisha A. Stewart, Yinmeng Yang, Xuezheng Sun, Liza Makowski, Kristen Brantley, Jean Gowen Cook, Melissa A. Troester. Characterizing breast cancer subtype-specific responses to macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2013-502