Complete Durable Remission Research Articles

Abstract 601: Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity

Abstract Unlike the tumor targets associated with initial CAR T cell successes, the vast majority of tumor associated antigens are not unique, but rather expressed at elevated levels compared to normal tissue. For example, while CD123 is overexpressed on many AML tumor cells, concerns regarding on-target, off-tumor toxicities - often associated low-level CD123 expression on normal endothelial cells - has hindered clinical success. Therefore, future generations of CAR T therapies will require novel strategies that have the ability to discriminate between normal and elevated antigen levels - potentially through mechanisms that involve 1) the simultaneous or sequential targeting of multiple tumor associated antigens and 2) agents that contain affinity-tuned targeting domains. Due to the large inventory of validated antibodies against a variety of therapeutic targets, scFv naturally emerged as the obvious and justifiable targeting domain for chimeric antigen receptors. However, many of the characteristics that have made antibodies versatile and effective recombinant therapeutics (e.g., high affinity, bivalency, antibody-dependent cytotoxicity, complement-dependent cytotoxicity, FcRn recycling, and low renal filtration rates) are not advantageous for membrane associated chimeric receptors. Furthermore, scFv are not native protein structures and their development, particularly as it pertains to solubility and aggregation, can be challenging. Therefore, we sought to develop a simple, highly selective targeting domain that could be engineered into complex, potentially multispecific therapeutics. We describe the design and development of non-scFv-derived binding domains. Using phage display and targeted mutagenesis, we identify a series of binding domains that target CD123 with high specificity - as characterized by functional and tissue binding studies. These domains exhibit affinities that range over 2 logs and demonstrate in vitro potencies that correlate with their affinities. CARs comprised of higher affinity binding domains mediate potent T-cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We also describe a strategy of engineering less immunogenic binding domains through the identification and removal of putative T cell epitopes and demonstrate that the resultant variants retain biological activity. Finally, we demonstrate further potential of our binding domains by generating functional, bi-specific CARs comprised of a CD123-specific binding domain and a CD19-specific scFv. The ability to incorporate our target-specific binding domains with a range of affinities into complex chimeric receptors, affords a viable alternative to scFv as targeting domains in CAR T cell therapeutics. Citation Format: David W. LaFleur, Haiying Qin, Justin P. Edwards, Liubov Zaritskaya, Ankit Gupta, C. Jenny Mu, Laura K. Richman, Terry J. Fry, David M. Hilbert. Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 601.

MiR-371a-3p, miR-373-3p and miR-367-3p as Serum Biomarkers in Metastatic Testicular Germ Cell Cancers Before, During and After Chemotherapy.

Background: LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and β-HCG (human chorionic gonadotropin) are used in diagnosis and follow-up of testicular germ cell cancer (TGCC) patients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p and miR-367-3p and clinical features in metastatic TGCC. Methods: relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TGCC patients. A prospectively included and a retrospectively selected cohort were studied (total patient number = 109). Blood samples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) levels were determined using the ampTSmiR test. Results: at start of chemotherapy, miR-371a-3p, miR-373-3p and miR-367-3p levels were positively correlated to LDH. The median level of these miRs was higher in patients who developed a relapse after complete biochemical remission (n = 34) than in those who had complete durable remission (n = 60). Higher levels of miR-367-3p were found in patients with refractory disease (n = 15) compared to those who had complete response. miR levels decreased during the first week of chemotherapy in patients with complete response and stayed below threshold after one year of treatment. Conclusion: high miR levels at start of chemotherapy are associated with worse clinical outcome and can assist in early diagnosing of relapses.

THOR-707: Using synthetic biology to reprogram the therapeutic activity of interleukin-2 (IL-2).

2603 Background: Recombinant interleukin-2 (rIL-2; aldesleukin) is an approved immunotherapy in melanoma and renal cell carcinoma based on complete durable remissions. The anti-neoplastic properties of IL-2 are mediated by interactions with the beta-gamma chain (IL-2Rβγ) on naïve CD8+ T cells, which lead to their expansion and differentiation into T effector and T memory cells directed against the tumor. However, the widespread use of IL-2 in oncology is limited by interaction with the high affinity IL-2 receptor alpha chain (IL-2R⍺) on regulatory CD4+ T cells (Tregs), which leads to immunosuppression, and on innate lymphoid cells in the vascular endothelium, which leads to eosinophilic recruitment and activation, and the sometimes fatal complication of vascular leak syndrome (VLS). A rIL-2 biased toward IL-2βγ affinity with no IL-2R⍺interaction could fill unmet needs in oncology. Methods: Using a synthetic biology platform, we have engineered THOR-707, a rIL-2 that contains a novel amino acid encoded in the IL-2 gene via a new DNA base pair (X-Y). The novel amino acid serves as a hook for site specific pegylation that extends half-life, blocks IL-2R⍺ engagement and binds to the IL-2Rβγ. Results: In non-human primates, THOR-707 can be dosed to maximize the level of cytotoxic CD8+ T lymphocytes without elevation of VLS-inducing eosinophils. In murine tumor models, THOR-707 induced the expansion of peripheral and intratumoral CD8+ T cells without expansion of suppressive Tregs. Single-agent dose-dependent anti-tumor efficacy was observed in two syngeneic mouse models. In combination with a PD-1 inhibitor, survival of tumor-bearing mice was longer than either agent as monotherapy. Efficacy in tumor models was durable, suggesting activation of CD8+ memory T cell populations. Conclusions: THOR-707 is a reprogrammed, site-directed, singly-pegylated rIL-2 that changes the pharmacologic profile of IL-2, potentially providing a favorable risk-benefit profile. First-in-human studies are expected to begin this year evaluating THOR-707 as monotherapy and in combination with a PD-1 inhibitor. Based on preclinical evidence to-date, THOR-707 may potentially address existing and emerging unmet needs across multiple solid tumors.

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Chimeric antigen receptor (CAR) Tcell therapies have demonstrated impressive initial response rates in hematologic malignancies.However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent Tcell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative Tcell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.

Gastrointestinal Stromal Tumor Mimicking as Ovarian Tumor in Gynaecologic Oncology.

To report the clinical presentation and outcomes of a series of patients who presented with abdominal/pelvic mass or pelvic pain and were diagnosed with a gastrointestinal stromal tumor (GIST). Retrospective data were collected of all patients who presented with an abdominal/pelvic mass or pelvic pain between January 2010 and July 2015 and who were ultimately diagnosed with a GIST. The patients' medical records were reviewed. A literature review was also conducted. The event free survival and overall survival was calculated for all patients using Kaplan Meier curve (SPSS19-SPSS Inc. USA). A total ten patients were identified with GIST during the study period. Eight of ten patients had a tumor in the small intestine, one in sigmoid colon and one in base of small bowel mesentry. The mean tumor size was 13.9cm (range, 3.9 to 24cm). A complete resection was achieved in all 10 patients. No patient had distance metastasis. There were no intraoperative complications. One patient developed postoperative intestinal fistula and was managed conservatively. All patients were treated with imatinib after surgery. The mean follow-up time was 18months (range, 2 to 47months). The seven of the 10 patients (70%) with no evidence of disease, two (20%) lost follow up and one patient developed recurrence during follow up period and was started on sunitinib and patient died during follow up period because of disease. Gastrointestinal stromal tumors should be considered in the differential diagnosis of patients presenting with an abdominal/pelvic mass or pelvic pain in Gynaecologic oncology department. In such unusual circumstances the complete resection and appropriate adjuvant treatment results in complete durable remission.

MALT lymphoma of the lacrimal gland in the context of systemic lupus erythematosus: complete remission after treatment with rituximab

Mucosa-associated lymphoid tissue (MALT) lymphoma of the lacrimal glands has been reported in only two patients with systemic lupus erythematosus (SLE) in the literature. We describe a 41-year-old female with SLE who had multiple relapses and remissions of her disease during the last 20 years and developed a right eyelid swelling. Magnetic resonance imaging showed a lesion in the right lacrimal gland with increased enhancement on T1- and T2-weighted images after intravenous contrast administration, and the biopsy of lacrimal gland was consistent with the diagnosis of marginal zone B-cell lymphoma of MALT type. The patient received treatment with four once-weekly doses of rituximab 375 mg/m(2) every 6 months for 2 years resulting in complete remission. Lacrimal gland MALT lymphoma is mainly treated with local radiotherapy, or chemotherapy in cases with systemic lymphoma. This is the first case of rituximab treatment in a patient with SLE who developed lacrimal gland MALT lymphoma, resulting in complete durable remission.

P101 - Azathioprine-associated lymphopenia in IBD – are our patients in danger? A prospective multi-centre study

s of the 4th Congress of ECCO the European Crohn’s and Colitis Organisation S51 P101 Azathioprine-associated lymphopenia in IBD are our patients in danger? A prospective multi-centre study A. Al-Rifai1, N. Prasad1 *, E. Shuttleworth2, A. Robinson2, S. Campbell1. 1Manchester Royal Infirmary, Manchester, United Kingdom, 2Salford Royal Hospital, Manchester, United Kingdom Introduction: Azathioprine (AZA) is frequently used to manage IBD patients and opportunistic infections are well documented in long-term use. Amongst non-HIV patients on immunosuppressants for other conditions, there are reports of lymphopenia (LP) related infections. LP is a recognised effect of treatment with AZA but LP related complications in IBD patients have not been widely reported. This raises the question of whether antibiotic prophylaxis is indicated for IBD patients on immunomodulators. However, there is no consensus on how to manage LP in this group. Aims: To assess the incidence of LP and any related complications in a cohort of IBD patients started on AZA. Methods: This is a prospective observational study of all patients started on AZA between 1/8/2007 and 30/9/2008 in two tertiary gastroenterology units in the UK. Data was collected on demographics, diagnosis, disease anatomy, LP episodes, relapses and concurrent therapy. LP was defined as 3 and the need for corticosteroids or anti-TNF therapy. Results: The median duration of follow-up was 51 months (10 142). Eighteen patients (12%) had a clinical recurrence within the first year following surgery before a colonoscopy was performed; 90 patients (Group C) had a systematic colonoscopy within the year following surgery (median time: 7 months, 2 13) and in 82 of them (91%), treatment was tailored according to the severity of endoscopic recurrence; 42 patients (Group NC) did not have endoscopic evaluation (8 patients declined and in 34 patients, colonoscopy was not performed in time). At time of surgery, there was no difference between Group C and Group NC except that surgery was more often curative in Group C (96%) than in Group NC (83%; p = 0.02). Time to clinical recurrence was shorter in Group NC than in Group C, with probabilities of clinical recurrence of 31% and 52% at 3 and 5 years in Group NC, and of 21% and 26% respectively in Group C (p = 0.01; logrank test). In multivariate analysis (Cox model), the following factors were associated with a higher risk of clinical recurrence: Group NC, presence of extraintestinal manifestations, and non penetrating disease behaviour. Conclusion: In our experience, the majority of patients accepts to have a colonoscopy within the year following surgery for decision of a tailored treatment. This strategy reduced clinical recurrence rate by 50% compared to absence of colonoscopy, but 12% of patients had an early recurrence before colonoscopy. P103 Mesenchymal stem cells administered via novel selective mesenteric artery cannulation for the treatment of severe refractory Crohn’s disease L. Dinesen1 *, A. Wang1, F. Vianello2, P. Tait3, E. Leen3, J. Davis2, F. Dazzi2, S. Ghosh1. 1Dept of Gastroenterology, Hammersmith Hospital, Imperial College London, United Kingdom, 2Dept of Haematology, Hammersmith Hospital, Imperial College London, United Kingdom, 3Dept of Imaging, Hammersmith Hospital, Imperial College London, United Kingdom Background: Mesenchymal stem cells (MSC) are a population of stromal origin that, in adult life, resides primarily in the bone marrow. MSC have the ability to self-renew and differentiate into tissues of mesodermal origin and produce a potent immunosuppressive effect. Recent results from multicentre clinical trials have described the successful use of MSC in severe graft-versus-host disease (GvHD), in which 60% of patients obtained complete durable remission with no toxicity. We tested their therapeutic potential in a patient with refractory Crohn’s disease. We administered MSC for the first time via selective mesenteric artery cannulation to ensure that the cells reached their target. Method: A 35 year old gentleman with severe refractory fistulizing Crohn’s disease failing all conventional therapies, biological therapies and surgical defunctioning ileostomy received mesenchymal stem cells from a haplo-identical donor, after informed consent. MSC were injected after by gest on July 1, 2016 http://eccoxfordjournals.org/ D ow nladed from

Proteomics To Identify Novel Immune-Targeted CML Stem Cell Antigens.

Eradication of residual disease remains a critical problem in patients with chronic myeloid leukemia (CML). Donor lymphocyte infusion (DLI) for the treatment of relapsed CML after transplantation is a robust example of the graft-versus-leukemia (GvL) effect, producing complete durable remission in 75% of patients. Since CML originates as a clonal stem cell disorder, we hypothesize that the curative effect of DLI results from immunologic targeting of the self-renewing CML progenitor cell. We have focused on B cell responses as a critical element in effective tumor immunity after observing significant B cell lymphocytosis in 7 responders of DLI at 6 and 9 months following DLI compared to pre-treatment levels (p=0.03; p=0.04, respectively), which was not seen in 5 DLI nonresponders. Moreover, some DLI responders showed marrow plasmacytosis at the time of cytogenetic response. To identify targets of this visible B cell response, we used post-DLI plasma as a source of immunoglobulin to probe two complementary immunoproteomic platforms and identify antigens eliciting increased reactivity compared to pre-DLI plasma. In the first approach, plasma from 7 individual responders was screened for binding to proteins expressed from a CML cDNA phage library (SEREX). In the second approach, plasma from 3 of the 7 responders was screened for binding to proteins expressed from approximately 5000 open reading frames spotted on a protein microarray (ProtoArray, Invitrogen). Screening by SEREX yielded 24 and by ProtoArray 58 candidate antigens. Analysis of datasets generated from normal and malignant myeloid cells on HG-Focus and HG-U133A Affymetrix microarrays confirmed that most candidate targets were expressed in CML progenitor cells. Of the antigens represented on the microarrays, 83% (10 of 12) and 64% (18 of 28) of the SEREX- and ProtoArray-identified antigens, respectively, were expressed in CML CD34+ cells. EXOSC5 was expressed on CML CD34+ cells and was identified by both platforms. Four of 16 ProtoArray-identified antigens (RAB38, TBCE, DUSP12, RPS6KC1) were expressed at higher levels in CML compared to normal CD34+ and mature myeloid lineage cells (p<0.001; p<0.002, respectively). SEREX screening identified exclusively intracellular antigens, whereas ProtoArray analysis yielded four surface-expressed proteins (TGFBR2, TNFRSF19, ACVR2B, and RET). Our studies have thus uncovered a broad panel of novel antigens expressed in CML progenitor cells that are targeted by the polyclonal B cell response associated with GvL. Compared to SEREX, the ProtoArray is a less disease-specific platform that can enable rapid identification of many antigens with compelling attributes for vaccine development in CML, including cell-surface expression and differential expression between normal and tumor stem cells. We are incorporating the entire panel of CML-associated stem cell antigens onto a custom protein array. A CML antigen array provides a powerful new tool for monitoring antigen-specific immunity in the context of immunotherapies designed to eliminate persistent residual disease in CML.

1,1-Bis(3'-indolyl)-1-(p-biphenyl)methane inhibits basal-like breast cancer growth in athymic nude mice

Introduction1,1-Bis (3'-indolyl)-1-(p-biphenyl) methane (CDIM9) has been identified as a new peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits both receptor dependent and independent antitumor activities. CDIM9 has not previously been studied with respect to its effects against basal-like breast cancer. Our goal in the present study was to investigate the anti-basal-like breast tumor activity of CDIM9 in vitro and in vivo.MethodsThe effects of CDIM9 on cell protein and DNA syntheses were determined in basal-like breast cancer MDA-MB231 and BT549 cells in vitro. Maximum tolerated dose and dose-limited toxicity were determined in BalB/c mice, and antitumor growth activities were assessed in MDA-MB231 basal-like breast tumor xenografts in athymic nude mice.ResultsCDIM9 exhibited selective cell cytotoxicity and anti-proliferation effects on basal-like breast cancer lines. In MDA-MB231 cell, CDIM9 induced caveolin-1 and p27 expression, which was significantly downregulated by co-treatment with the PPAR-γ antagonist GW9662. Nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3 were upregulated by CDIM9 through a PPAR-γ independent pathway. CDIM9 (40 mg/kg daily, intraperitoneally, for 35 days) inhibited the growth of subcutaneous MDA-MB231 tumor xenografts by 87%, and produced a corresponding decrease in proliferation index. Nearly half of the treated mice (46%) had complete durable remissions, confirmed by histology. The growth of an established tumor was inhibited by CDIM9 treatment (64 mg/kg daily, intraperitoneally, for 10 days), with a mean tumor growth inhibition of 67% as compared with controls. CDIM9 induced increases in tumor caveolin-1 and p27 in vivo, which may contribute to its antitumor activity in basal-like breast cancer.ConclusionCDIM9 showed potent antiproliferative effects on basal-like breast cancer cell in tissue culture and dramatic growth inhibition in animal models at safe doses. These findings justify further development of this drug for treatment of basal-like breast cancer.

Developing a Rational Tumor Vaccine Therapy for Renal Cell Carcinoma: Immune Yin and Yang

In patients with progressive malignancy, the natural balance between proinflammatory (Yang) and inhibitory (regulatory or Yin) immune pathways is disrupted and favors cancer-specific immune suppression. Therapy with interleukin 2 (IL-2) can mobilize immune effector cells that recognize and destroy cancer. High-dose IL-2 is the only therapy that has consistently induced complete durable remissions in patients with metastatic renal cell carcinoma (RCC) but only in a few of them. The lack of benefit in most metastatic RCC patients is likely due to the ineffective manipulation of other immune circuits critical in regulating tumor cytotoxic pathways. The limited clinical activity of IL-2, RCC vaccines, and other immune therapies to date leads us to postulate that effective clinical treatment strategies will need to simultaneously enhance proinflammatory pathways and disrupt regulatory pathways. We present preliminary studies in RCC patients to highlight the complexity of the regulatory pathways and our approach to shifting the balance of proinflammatory and regulatory immune pathways using dendritic cell-tumor lysate vaccine followed by cytokine therapy.

Progress in the systemic treatment of cancer.Concepts, trials, drugs, and biologics

The effective treatment of systemic cancer began in the 1950s on two fronts, i.e., childhood leukemia and choriocarcinoma. These two diseases were successfully treated as a direct result of the use of antifolate methotrexate. The demonstration of complete durable remissions in these diseases quickly led to development of other anticancer drugs, tested using the prospective clinical trials. In the 1960s as the number of active drugs increased, combination chemotherapy was introduced. Other systemic cancers, such as Hodgkin's, large cell lymphoma, and testicular cancer, became curable in the 1970s. For the common low-growth fraction solid tumors, the curability of systemic disease remained elusive until the introduction of adjuvant therapy to treat micrometastases. The past decade of the 1980s has seen improvement in the outcomes for breast cancer, osteosarcoma, and possible colon cancer utilizing adjunctive chemotherapy. The 1980s also saw the introduction of biologic therapies that have further improved the outcomes of several leukemias and produced consistent responses in patients with renal cell and melanoma. The 1990s will undoubtedly see more improvements as the effects of current drugs will be enhanced not only by improved integration of systemic and local therapies but also by utilizing cytokines and biologic response modifiers in concert with cytotoxics. Moreover, as we understand more about the process of cancer induction, promotion, and progression, more specific anti-cancer approaches will be developed to control cancer even before clinical cancer is diagnosed. Underlying and facilitating the improvement in cancer therapy have been not only the experimental results of many laboratory scientists but also the outcomes from many controlled clinical trials, the laboratory of clinical scientists.