Antibody-coupled T-cell Receptor Research Articles

Results from a Phase 1 Study of ACTR707 in Combination with Rituximab in Patients with Relapsed or Refractory CD20+ B Cell Lymphoma

The antibody-coupled T cell receptor (ACTR) platform is an autologous engineered T cell therapy combining the cell-killing ability of T cells and the tumor-targeting ability of coadministered antibodies. Activation of the T cell product ACTR707 is dependent on the engagement of antibody bound to target cells via the CD16 domain of the chimeric receptor (CD16V-CD28-CD3ζ). ACTR707 in combination with the anti-CD20 monoclonal antibody rituximab was evaluated in the ATTCK-20-03 study, a multisite, single-arm, open-label phase I trial in B cell non-Hodgkin lymphoma (NHL). The primary objectives of this study were to evaluate the safety of the combination of ACTR707 and rituximab and to determine a recommended phase 2 dose (RP2D). Secondary objectives included evaluation of antitumor activity and ACTR T cell persistence. The study design included an ACTR707 cell dose escalation phase and an expansion phase at the RP2D. Escalating dose levels of ACTR707 in combination with rituximab were explored in 5 dose cohorts, with 25 subjects receiving study treatment. Subjects received lymphodepleting chemotherapy (cyclophosphamide 400 mg/m2/day and fludarabine 30 mg/m2/day) for 3 days, followed by rituximab 375 mg/m2 and, 24 to 48 hours later, a single dose of ACTR707. Additional doses of rituximab were administered every 3 weeks until disease progression, unacceptable toxicity, or investigator decision. Blood samples were collected at various time points to assess levels of rituximab, cytokines, inflammatory markers, and ACTR707 T cells. The overall response rate of ACTR707 plus rituximab was 56% (14 of 25) across all dose levels. Ten subjects (40.0%) achieved a complete response, with the longest duration of 586 days (range, 85 to 586 days), and 4 subjects (16.0%) experienced a partial response, with the longest duration of 130 days (range, 44 to 130 days). Only 1 case of cytokine release syndrome (grade 2) and no events of neurotoxicity were reported. There were no dose-limiting toxicities or events leading to death. ACTR707 plus rituximab resulted in only 1 adverse event (neutropenia), leading to study discontinuation of rituximab. The ATTCK-20-03 trial serves as proof of principle regarding the ACTR approach that potentially could be used with other antibodies targeting other markers in other malignancies. Although the ACTR707 program has been discontinued, these results may support other programs in the use of similar novel approaches of antibody-coupled T cell activation.

Adaptability of Antibody-Coupled T Cell Receptor (ACTR) Engineered Autologous T Cells in Combination with Daratumumab over CAR-Based Approaches

Chimeric antigen receptor (CAR)-T cells have demonstrated profound clinical responses against CD19+ B cell malignancies, with ongoing trials in multiple myeloma (MM). Although BCMA CAR-T cells show promise in MM, development of CAR-T against other MM targets has been problematic due to target expression on T cells themselves. Specifically, efforts to develop CAR-T cell therapies targeting CD38 have been hampered by the upregulation of CD38 on activated T cells and consequent induction of CAR-T mediated autolysis that occurs during manufacturing. To overcome this challenge during CAR-T production, investigators have taken several approaches including multiple rounds of T cell expansion, addition of CD38 blocking antibody, and CD38 gene knockout in T cells prior to CAR transduction (1,2,3).The Antibody-Coupled T Cell Receptor (ACTR) technology is a universal, engineered T cell therapy that combines the Fc receptor, CD16, with endogenous T cell signaling and costimulatory domains (4). ACTR T cells function by engaging the Fc domain of therapeutic antibodies opsonized on target cells, resulting in T cell activation and tumor cell killing. In contrast to CAR-T cell constructs that express the antigen targeting single chain variable fragment (scFv), ACTR T cell activity is regulated through antibody administration and is therefore not susceptible to target-mediated autolysis during T cell production.The recent approval of the CD38-targeting antibody, daratumumab, has improved the treatment of multiple myeloma through its tumor targeting and immunomodulating capabilities. Here, we leveraged the ability of the ACTR T cell platform to decouple the targeting antibody from the engineered T cell signaling moieties, and evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. We demonstrated that in contrast to CD38-targeting CAR-T cells, ACTR T cells exhibited enhanced viability and expansion of the chimeric construct throughout the manufacturing process. We then evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. ACTR T cells mediated robust cytotoxicity, cytokine production, and proliferation in response to a range of daratumumab-opsonized target cell lines. Notably, the target specific killing and cytokine production mediated by ACTR T cells in combination with daratumumab was greater than the activity mediated by daratumumab alone. Recognizing the augmented activity of ACTR T cells in combination with daratumumab, we systematically investigated the potential for on-target off-tumor toxicity. In a peripheral blood mononuclear cell coculture assay, ACTR T cells in combination with daratumumab selectively targeted CD38-expressing multiple myeloma cells over CD38-expressing normal B cells, T cells, and natural killer cells. Furthermore, despite low levels of CD38 expression reported on erythrocytes, ACTR T cells in combination with daratumumab did not mediate hemolysis. Taken together, our findings demonstrate the adaptability of the ACTR platform to function in combination with daratumumab, and bypass the challenges of scFv-based CAR-T cell production to safely and effectively target CD38-positive multiple myeloma.

Initial Clinical Activity of FT596, a First-in-Class, Multi-Antigen Targeted, Off-the-Shelf, iPSC-Derived CD19 CAR NK Cell Therapy in Relapsed/Refractory B-Cell Lymphoma

Induced pluripotent stem cell (iPSC)-derived immune effector cells offer distinct advantages over existing patient- and donor-derived therapeutic approaches, including the use of a clonal master engineered iPSC line as a renewable source for the mass production of immune cells, which are available off-the-shelf for broad patient access. FT596 is an investigational, off-the-shelf, multi-antigen targeting, chimeric antigen receptor (CAR) natural killer (NK) cell therapy derived from a human clonal master iPSC line engineered with three anti-tumor modalities: (1) a proprietary CD19-targeting CAR; (2) a novel high-affinity, non-cleavable CD16 Fc receptor that enables tumor targeting and enhanced antibody-dependent cell cytotoxicity in combination with a therapeutic monoclonal antibody (mAb); and (3) interleukin (IL)-15/IL-15 receptor fusion promoting cytokine-autonomous persistence. Preclinical in vivo models demonstrate potent multi-antigen targeting activity of FT596 against both CD19+ and CD19- tumor cell lines when combined with the anti-CD20 agent rituximab (Goodridge et al. 2019). FT596 is currently being investigated as a monotherapy and in combination with the anti-CD20 mAbs rituximab and obinutuzumab in a multicenter, Phase I clinical trial for the treatment of relapsed/refractory B-cell lymphoma and chronic lymphocytic leukemia. The trial will test up to four FT596 dose levels ranging from 30 to 900 million cells. We describe the demonstration of early clinical benefit of FT596 in a patient who received a single administration of FT596 as a monotherapy at the first dose level in the Phase I trial. The patient is a 76-year-old female with diffuse large B-cell lymphoma, germinal center B-cell subtype, who was initially diagnosed in January 2014. The patient received eight prior treatment regimens, including autologous stem cell transplant, rituximab in combination with engineered autologous T cells expressing antibody-coupled T-cell receptor, and, most recently, was refractory to an experimental combination therapy comprised of lymphodepleting chemotherapy followed by ex vivo expanded allogeneic NK cells, IL-2, and rituximab. The patient received fludarabine and cyclophosphamide lympho-conditioning followed by a single administration of 30 million cells of FT596 as monotherapy. During the 28-day follow-up period for safety, no dose-limiting toxicities were observed. No cytokine release syndrome, neurologic toxicity, or graft-versus-host disease of any grade was observed. Grade ≥3 treatment-emergent adverse events (AEs) included decreased white blood cell count, decreased neutrophil count, anemia, urinary tract infection with neutropenic fever, and hypertension. Notably, none of these AEs were considered related to FT596 by the treating investigator physician, except for decreased neutrophil count, which was considered possibly related to FT596 and resolved. Evidence of hematologic recovery was observed by the end of the 28-day follow-up safety period. On Study Day 29, the patient's tumor response assessment showed partial response by Lugano 2014 criteria, with a greater than 70% decrease in 18F-fluorodeoxyglucose uptake and greater than 50% reduction in tumor size. The patient remains on study and, per protocol and in collaboration with the U.S. Food and Drug Administration, is being considered for retreatment with a second cycle of FT596 monotherapy. This is the first-ever demonstration of clinical activity following treatment with an off-the-shelf, iPSC-derived CAR immune cell therapy and provides direct evidence that low doses of FT596 can induce short-term responses, opening the opportunity for multi-dosing strategies to deepen response and duration. Additional clinical, pharmacokinetic, and pharmacodynamic data from this patient will be provided at the time of the meeting. The Phase I trial is ongoing and is registered on clinicaltrials.gov: NCT04245722. Disclosures Bachanova: FATE: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Karyopharma: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding. Janakiram:Takeda, Fate, Nektar: Research Funding. Payne:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cooley:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Miller:GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as lympho-conditioning therapy prior to FT596 administration.

BCMA-Targeting Therapy: Driving a New Era of Immunotherapy in Multiple Myeloma.

The treatment of multiple myeloma (MM) has entered into a new era of immunotherapy. Novel immunotherapies will significantly improve patient outcome via simultaneously targeting malignant plasma cell (PC) and reversing immunocompromised bone marrow (BM) microenvironment. B-cell maturation antigen (BCMA), selectively expressed in PCs and a key receptor for A proliferation-inducing ligand (APRIL), is highly expressed in MM cells from patients at all stages. The APRIL/BCMA signal cascades promote the survival and drug resistance of MM cells and further modulate immunosuppressive BM milieu. Impressively, anti-BCMA immunotherapeutic reagents, including chimeric antigen receptor (CAR), antibody-drug conjugate (ADC) and bispecific T cell engager (BiTE) have all shown high response rates in their first clinical trials in relapse and refractory patients with very limited treatment options. These results rapidly inspired numerous development of next-generation anti-BCMA biotherapeutics, i.e., bispecific molecule, bispecific or trispecific antibodies, a novel form of CAR T/NK cells and T Cell Antigen Coupler (TAC) receptors, antibody-coupled T cell receptor (ACTR) as well as a cancer vaccine. We here highlight seminal preclinical and clinical studies on novel BCMA-based immunotherapies as effective monotherapy and discuss their potential in combination with current anti-MM and novel checkpoint drugs in earlier disease stages to further achieve durable responses in patients.

A Phase 1 Study of ACTR087 in Combination with Rituximab, in Subjects with Relapsed or Refractory CD20-Positive B-Cell Lymphoma

Background: The Antibody-Coupled T-cell Receptor (ACTR) platform is an autologous engineered T-cell therapy that combines the cell-killing ability of T cells and the tumor-targeting ability of co-administered antibodies to exert potent antitumor immune responses. ACTR087 comprises the extracellular domain of CD16 linked to a CD3ζ-signaling domain and a 4-1BB co-stimulatory domain. Here we present the clinical experience from Study ATTCK-20-2 (NCT02776813), a multicenter, phase 1 study of ACTR087 in combination with rituximab in subjects with relapsed or refractory (R/R) CD20+ NHL. Methods: The main objectives of this first-in-human study were to evaluate the safety and antitumor activity of ACTR087+rituximab. Other objectives included evaluating ACTR T-cell persistence and other correlative biomarkers. Subjects must have had CD20+ NHL that was R/R after prior treatments, which must have included anti-CD20 antibody-containing chemotherapy. Subjects received lymphodepleting chemotherapy (cyclophosphamide and fludarabine) for 3 days, followed by rituximab and a single dose of ACTR087. Additional doses of rituximab were administered q3w until disease progression, unacceptable toxicity, or Investigator decision. The study included a dose escalation phase (increasing doses of ACTR087) and an expansion phase (ACTR087 at the preliminary recommended phase 2 dose [RP2D]); all subjects received rituximab at a fixed dose of 375 mg/m2 q3w. Results: Two dose levels (DL) of ACTR087 were evaluated during dose escalation (n=17). The MTD was exceeded at DL2, with severe cases of cytokine release syndrome (CRS) and neurotoxicity. Statistical analysis of the relationship between non-hematologic toxicity and ACTR+ T-cell doses was retrospectively performed (two-parameter Bayesian logistic regression model) to estimate an RP2D of 35×106 ACTR+ T cells. Nine subjects enrolled in an expansion cohort and received ACTR087 at this RP2D in combination with rituximab. Among all subjects treated (n=26), the majority (69%) were diagnosed with DLBCL. Subjects had received a median of 3 (range 1-9) prior lines of therapy, with 77% having received ≥3 prior lines. ACTR087 showed dose-dependent expansion with peak levels generally observed 7 to 14 days post administration. In subjects with ongoing clinical response (CR), ACTR remained detectable through the last timepoint evaluated. Across all cohorts, Grade ≥3 TEAEs reported in >3 subjects regardless of causality were limited to hematologic events. Potential T cell-mediated toxicities were observed, including 4 serious cases of CRS (Gr 4 in 2 subjects, both with fatal sepsis) and 2 serious cases of neurotoxicity (1 Gr 5, 1 Gr 4 in a subject with fatal septic shock). Elevated baseline inflammatory markers (eg, ferritin, CRP) were observed in patients who developed Gr ≥3 CRS and neurotoxicity post ACTR087. Of note, severe CRS presented without fever and events occurred >7 days post ACTR087. Clinical activity was reported with an ORR of 50% in all dose levels tested, including durable complete responses, with one subject in CR for 869+ days (Table 1). Conclusions: ACTR087+rituximab demonstrated antitumor activity, with observed safety events that are expected with other autologous T-cell products. The time to onset and clinical presentation of severe CRS and neurotoxicity events in this study informed the safety monitoring and adverse reaction management guidance across clinical studies of ACTR T-cell products. Data from this first-in-human study of ACTR087+rituximab confirm the proof of concept and will be used to guide further development for the ACTR platform. Updated clinical data, as well as expanded biomarker correlations to efficacy and safety, will be presented. Disclosures Munoz: Pharmacyclics /Janssen: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy; Fosunkite: Speakers Bureau; AstraZeneca: Speakers Bureau; Kyowa: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene/Juno: Consultancy, Research Funding; Genentech: Consultancy, Research Funding, Speakers Bureau; Kite/Gilead: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Alexion: Consultancy; Portola: Research Funding; Incyte: Research Funding; Bayer: Consultancy, Speakers Bureau; Merck: Consultancy. Jaglowski:Kite: Consultancy, Other: advisory board, Research Funding; Novartis: Consultancy, Other: advisory board, Research Funding; Unum Therapeutics Inc.: Research Funding; Juno: Consultancy, Other: advisory board. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Stiff:Gamida-Cell: Research Funding; Incyte: Research Funding; Cellectar: Research Funding; Unum: Research Funding; Gilead/Kite Pharma: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Sachs:Unum Therapeutics Inc.: Employment. Ranger:Unum Therapeutics Inc.: Employment. Harris:Unum Therapeutics Inc.: Employment. Payumo:Unum Therapeutics Inc.: Employment. Akard:Bristol-Myers Squibb: Speakers Bureau; Gilead: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Celgene: Speakers Bureau.

Preliminary Clinical Results from a Phase 1 Study of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ non-Hodgkin Lymphoma

Background: The Antibody-Coupled T-cell Receptor (ACTR) platform is an autologous engineered T-cell therapy that combines the cell-killing ability of T cells and the tumor-targeting ability of co-administered antibodies to exert potent antitumor immune responses. ACTR707 comprises the extracellular domain of CD16 linked to a CD3ζ signaling domain and a CD28 co-stimulatory domain. ACTR707 is in clinical development in combination with rituximab (NCT03189836) or trastuzumab (NCT03680560). Here we present clinical findings from the dose escalation phase of Study ATTCK-20-03, an ongoing, multicenter, phase 1 study of ACTR707+rituximab in subjects with relapsed or refractory (R/R) CD20+ NHL. Methods: The primary objectives of this first-in-human study are to evaluate the safety of the combination of ACTR707 and rituximab and to determine a recommended phase 2 dose (RP2D). Other objectives include evaluating antitumor activity and ACTR T-cell persistence. Subjects must have CD20+ NHL that is R/R after prior treatments, which must include anti-CD20 antibody-containing chemotherapy. Subjects receive lymphodepleting chemotherapy (cyclophosphamide and fludarabine) for 3 days, followed by rituximab and a single dose of ACTR707. Additional doses of rituximab are administered q3w until disease progression, unacceptable toxicity, or Investigator decision. The study includes a dose escalation phase (increasing doses of ACTR707 with fixed dose of rituximab at 375 mg/m2 q3w) and an expansion phase at the RP2D. Results: Six subjects received ACTR707 at Dose Level 1 (DL1; 23-38×106 ACTR+ T cells), 3 subjects at DL2 (30-50×106 ACTR+ T cells), and 5 subjects at DL3 (45-55×106 ACTR+ T cells). The majority of the subjects were diagnosed with DLBCL (93%) and had refractory disease (71%), defined as progressive disease as the best response to any prior treatment or relapse <1 year post autologous stem cell transplant. In DL1 through DL3, as of 27 May 2019, there were no dose-limiting toxicities, AEs of cytokine release syndrome (CRS), serious or severe neurologic AEs, or AEs leading to deaths on treatment. TEAEs reported in >2 subjects, regardless of causality or grade, included neutropenia, thrombocytopenia, anemia, febrile neutropenia, pyrexia, cough, constipation, diarrhea, nausea, and vomiting. SAEs considered possibly related to ACTR707 were febrile neutropenia (n=2) and cytopenia (n=1). ACTR707 expansion generally reached peak levels within 1 to 2 weeks after administration. All subjects with complete response (CR) up to 1 year had detectable ACTR at the last timepoint evaluated. Higher ACTR707 CD8:CD4 T-cell ratios were associated with clinical responses. Clinical activity was reported across DL1 through DL3, with an overall response rate of 64% including durable complete responses (CRs), with one subject in CR for 387+ days (Table 1). Conclusions: Data available from DL1 through DL3 of ACTR707+rituximab suggest that clinical responses can be achieved without severe T cell-mediated toxicities (eg, CRS and neurotoxicity) that have been reported with other autologous T-cell products. Dose escalation continues at a target dose of 80×106 ACTR+ T cells; enrollment in DL4 (n=6) was recently completed. Updated data, including identified correlates of clinical outcomes, will be presented for DL1 through DL4. Disclosures Flinn: TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; AbbVie, Seattle Genetics, TG Therapeutics, Verastem: Consultancy; TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; Acerta Pharma, Agios, Calithera Biosciences, Celgene, Constellation Pharmaceuticals, Genentech, Gilead Sciences, Incyte, Infinity Pharmaceuticals, Janssen, Karyopharm Therapeutics, Kite Pharma, Novartis, Pharmacyclics, Portola Pharmaceuticals: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding. Westin:Genentech: Other: Advisory Board, Research Funding; Janssen: Other: Advisory Board, Research Funding; Kite: Other: Advisory Board, Research Funding; Unum: Research Funding; Curis: Other: Advisory Board, Research Funding; Juno: Other: Advisory Board; MorphoSys: Other: Advisory Board; 47 Inc: Research Funding; Celgene: Other: Advisory Board, Research Funding; Novartis: Other: Advisory Board, Research Funding. Cohen:Genentech, Inc.: Consultancy, Research Funding; Takeda Pharmaceuticals North America, Inc.: Research Funding; Gilead/Kite: Consultancy; LAM Therapeutics: Research Funding; UNUM: Research Funding; Hutchison: Research Funding; Astra Zeneca: Research Funding; Lymphoma Research Foundation: Research Funding; ASH: Research Funding; Bristol-Meyers Squibb Company: Research Funding; Seattle Genetics, Inc.: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy. Akard:Celgene: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Gilead: Speakers Bureau. Jaglowski:Juno: Consultancy, Other: advisory board; Kite: Consultancy, Other: advisory board, Research Funding; Unum Therapeutics Inc.: Research Funding; Novartis: Consultancy, Other: advisory board, Research Funding. Sachs:Unum Therapeutics Inc.: Employment. Ranger:Unum Therapeutics Inc.: Employment. Harris:Unum Therapeutics Inc.: Employment. Payumo:Unum Therapeutics Inc.: Employment. Bachanova:Celgene: Research Funding; Gamida Cell: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Novartis: Research Funding.

Abstract OT2-07-06: Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies

Abstract Antibody-Coupled T cell Receptor (ACTR) is an autologous engineered T cell therapy developed to combine with tumor-targeting antibodies to exert potent anti-tumor immune responses and tumor cell killing. The ACTR construct is composed of the extracellular domain of CD16 fused to CD3ζ signaling and T cell co-stimulatory domains. ACTR-expressing T cells are universal in that they can be paired with a therapeutic antibody to target specific antigens on tumors. Unum has two ACTR constructs, ACTR087 and ACTR707, currently in clinical testing. ACTR087 and ACTR707 are being tested in combination with rituximab in subjects with CD20+ B cell lymphoma in two separate trials (NCT02776813 and NCT03189836, respectively). Preliminary data with ACTR087 + rituximab has demonstrated clinical proof-of-concept and a dose-response relationship in subjects with relapsed/refractory B cell lymphoma. ACTR087 is also being tested in combination with a novel BCMA-targeting antibody in subjects with multiple myeloma (NCT03266692). While T cell therapies, such as chimeric antigen receptor (CAR) T cells, have demonstrated clinical activity in hematological cancers, the therapeutic potential of this approach has yet to be established in solid tumors. Challenges associated with targeting solid tumors with CAR-T cells include tumor antigen heterogeneity and antigen expression on normal tissues. HER2 is a well-established therapeutic target that is over-expressed in a number of cancer indications. HER2 is also expressed at low levels on normal epithelial cells, creating a risk for on-target/off-tumor toxicities of HER2-targeted CAR-T cells. Here we present nonclinical studies demonstrating that ACTR T cells in combination with trastuzumab have antigen density-dependent activity on HER2-expressing tumor cell lines, while trastuzumab-based CAR-T cells do not. We observed that ACTR + trastuzumab had robust activity against HER2-amplified tumor cells and more modest activity against non-amplified tumor cells, whereas HER2-targeting CAR-T cells had comparable activity against HER2-amplified and non-amplified tumor cells. On normal human primary cells, ACTR + trastuzumab had minimal activity in comparison to HER2 CAR-T cells, suggesting that ACTR + trastuzumab may exhibit a superior clinical therapeutic index. Furthermore, the activity of ACTR T cells against HER2-amplified tumor cells was titratable with antibody concentration, allowing for control of ACTR activity by modulation of trastuzumab concentration. Together, these data demonstrate the specificity of the ACTR T cell therapeutic approach to target HER2-amplified tumors and support clinical testing in combination with trastuzumab. A phase 1, multicenter, single-arm, open-label dose escalation study, ATTCK-34-01, is proposed to evaluate ACTR T cells in combination with trastuzumab in subjects with advanced HER2-positive malignancies. The primary study objectives are to assess the safety and tolerability of the combination, and to define the recommended phase 2 dose combination for further study. Additional objectives include assessment of anti-tumor activity, ACTR T cell persistence and trastuzumab pharmacokinetics. Enrollment is expected to commence in early 2019. Citation Format: O'Callaghan KM, Shin J, Cheung AS, Cheema T, Judge C, Ranger A, Huet HA, Ettenberg SA, Sachs J, Vasconcelles M, Motz G. Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-07-06.

Abstract A003: Safety and preliminary efficacy of ACTR707, autologous T lymphocytes expressing an antibody-coupled T-cell receptor, in combination with rituximab in subjects with relapsed or refractory CD20-positive B-cell lymphoma

Abstract Background: Autologous T cells engineered to express a universal ACTR chimeric receptor kill tumors through interactions with tumor-targeting antibodies [Kudo, Cancer Res 2014]. ACTR707 was identified through rigorous preclinical screening of more than 100 different ACTR variants. It is composed of the extracellular domain of CD16, the cytoplasmic signaling domain of CD3ζ, and the costimulatory domain of CD28. Study ATTCK-20-03 (NCT03189836) is the first clinical trial of ACTR707. ACTR707 in combination with rituximab is being studied in subjects with relapsed or refractory CD20+ B-cell lymphoma previously treated with anti-CD20 monoclonal antibody (mAb) therapy. Herein we report data from the first dose level of ACTR707, where 6 subjects have been enrolled and treated.Methods: ATTCK-20-03 is a first-in-human, multicenter phase 1 dose escalation study of ACTR707 in combination with rituximab. The primary objective is to evaluate the safety of ACTR707 in combination with rituximab, and secondary objectives include evaluation of antitumor activity, assessment of T-cell expansion and persistence, cytokine levels, and rituximab pharmacokinetics. Eligible subjects must have histologically confirmed relapsed or refractory aggressive CD20+ B-cell lymphoma of DLBCL, MCL, PMBCL, Gr3b FL, or transformed FL subtype and have received prior anti-CD20 mAb therapy in combination with chemotherapy. Subjects received lymphodepleting chemotherapy (cyclophosphamide 400 mg/m2 and fludarabine 30 mg/m2) for 3 days, followed by rituximab (375 mg/m2) and a single infusion of ACTR707. Additional doses of rituximab are administered, one dose every 3 weeks in the absence of disease progression. The study is separated into two sequential phases, a dose escalation and a safety expansion phase. During dose escalation, ACTR707 will be tested at increasing doses in combination with rituximab. Results: Six subjects received ACTR707 in combination with rituximab at the first dose level: 5 diagnosed with DLBCL (83%) and 1 with Gr3b FL (17%). Median age was 61 years (range: 57-76), 83% were male, 50% were treated with ≥ 3 lines of prior therapy, and 67% had no response to or progression within 6 months of immediate prior therapy. ACTR707 was successfully manufactured for all subjects. ACTR+ T cells demonstrated expansion and were detectable at D28 post-ACTR707 infusion for all subjects tested. There were no dose-limiting toxicities observed in the 4 subjects evaluable for DLTs (2 subjects were not DLT-evaluable due to early disease progression). There were no serious adverse events of cytokine release syndrome, neurotoxicity, or autoimmune events and no deaths on study. Grade 3 or higher AEs included neutropenia (n=2), febrile neutropenia (n=2), and thrombocytopenia (n=1). Antitumor activity was assessed in all 6 subjects. Three subjects experienced disease progression and 3 subjects demonstrated investigator-reported complete responses at the first disease response assessment (D42). Responses are ongoing at the time of the data cut-off. Conclusions: In the first dose level studied in subjects with relapsed or refractory aggressive CD20+ B-cell lymphoma, ACTR707 in combination with rituximab induced complete responses in 3 out of 6 treated subjects, with no serious events of cytokine release syndrome or neurotoxicity. ACTR+ T-cells were detectable in all subjects and ACTR+ T-cells persisted in the presence of continued rituximab administration. These results support the continued dose escalation of ACTR707 in combination with rituximab. Citation Format: Veronika Bachanova, Jonathon Cohen, Luke Akard, Samantha Jaglowski, Jessica Sachs, Ann Ranger, Patricia Harris, Kathleen McGinness, Greg Motz, Ian Flinn. Safety and preliminary efficacy of ACTR707, autologous T lymphocytes expressing an antibody-coupled T-cell receptor, in combination with rituximab in subjects with relapsed or refractory CD20-positive B-cell lymphoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A003.

A Phase 1 Study of Two Investigational Agents, ACTR087, an Autologous T Cell Product Expressing an Antibody-Coupled T Cell Receptor, in Combination with SEA-BCMA, a Novel Non-Fucosylated Monoclonal Antibody, in Subjects with Relapsed or Refractory Multiple Myeloma

Background: B cell maturation antigen (BCMA) has recently emerged as a promising candidate antigen for therapeutic targeting in multiple myeloma (MM), with several targeted agents in clinical studies including antibody-drug conjugates and bispecific T cell engagers as well as CAR T cells. The Antibody-Coupled T cell Receptor (ACTR) platform is a universal, engineered autologous T cell therapy developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR construct is composed of the ectodomain of CD16 fused to intracellular co-stimulatory and CD3ζ signaling domains (Kudo et al., Cancer Res. 2014), which allow ACTR T cells to exert antibody-dependent cell-mediated cytotoxicity, a function otherwise physiologically limited to CD16-expressing natural killer cells and macrophages.ACTR087 expresses a 4-1BB-containing receptor and has been evaluated in combination with rituximab in subjects with relapsed or refractory (R/R) CD20+ B cell lymphoma as previously reported (Akard et al., Blood 2017). SEA-BCMA is a novel, humanized non-fucosylated anti-BCMA IgG1 antibody that has been demonstrated pre-clinically to bind to ACTR087 T cells to mediate ACTR T cell activation, cytotoxicity, cytokine release, and proliferation in the presence of BCMA-expressing MM cell lines. These functional activities were demonstrated to be BCMA-specific and SEA-BCMA dose-dependent (Cheema et al., AACR 2017). Here we present preliminary findings from the first 2 single-subject cohorts of the ATTCK-17-01 study (NCT03266692), an ongoing Phase 1 study of ACTR087 in combination with the first-in-human administration of SEA-BCMA.Methods: ATTCK-17-01 is a multicenter, Phase 1, dose-escalation study of ACTR087 in combination with SEA-BCMA. The primary objectives are to characterize the safety and to determine the recommended Phase 2 dose of ACTR087 in combination with SEA-BCMA in subjects with R/R MM. The secondary objectives include evaluation of anti-myeloma activity, ACTR T cell persistence, cytokines, and SEA-BCMA pharmacokinetics (PK); exploratory objectives include the anti-myeloma activity of SEA-BCMA alone. Subjects must have measurable disease and must have received at least 3 prior lines of therapy including treatment with a proteasome inhibitor and an immunomodulatory agent, and hematopoietic stem cell transplant (HSCT) for HSCT-eligible subjects. BCMA expression on MM cells was not a condition of eligibility. Dose escalation of the 2 investigational agents, ACTR087 and SEA-BCMA, is determined according to adaptive design principles.After study enrollment and leukapheresis, subjects receive SEA-BCMA by IV infusion once every 3 weeks until disease progression or treatment discontinuation. After the third dose of SEA-BCMA and lymphodepleting chemotherapy (cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2, each daily for 3 days), subjects receive a single dose of ACTR087.Results: Two subjects enrolled and received ACTR087 at the first dose level in combination with the first 2 dose levels of SEA-BCMA. First-in-human dosing of SEA-BCMA was well tolerated, with no reported SEA-BCMA-related adverse events (AEs) or dose-limiting toxicities (DLTs).Following ACTR087 infusion, ACTR+ T cells were detectable in the peripheral blood and demonstrated expansion post-infusion. No DLTs were observed with the combination of ACTR087 and SEA-BCMA in the first 2 cohorts. Grade 3 or higher treatment-emergent AEs experienced by at least 1 subject, regardless of causality assessment, include cytopenias, increased ALT, and bone pain.Conclusions: ACTR087 in combination with SEA-BCMA was well tolerated in the first 2 subjects treated, with no DLTs or AEs leading to treatment discontinuation. These results support the continued dose escalation of ACTR087 in combination with SEA-BCMA. Enrollment in Cohort 3 is ongoing. Updated data, including SEA-BCMA PK, biomarkers, and preliminary Cohort 3 data, will be presented. DisclosuresHolmes:Unum: Research Funding; Seattle Genetics: Research Funding, Speakers Bureau; Novartis: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Rigel: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy. Hari:Kite Pharma: Consultancy, Honoraria; Janssen: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Sachs:Unum Therapeutics Inc.: Employment. Exter:Unum Therapeutics Inc.: Employment. Ranger:Unum Therapeutics Inc.: Employment. Cheema:Unum Therapeutics Inc.: Employment. Sienczylo:Unum Therapeutics Inc.: Employment. O'Meara:Seattle Genetics: Employment, Equity Ownership. Sussman:Seattle Genetics: Employment. Akard:Gilead: Speakers Bureau; Celgene: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Preliminary Clinical Results of a Phase 1 Study Evaluating the Safety and Anti-Tumor Activity of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ B-Cell Lymphoma

Preliminary Clinical Results of a Phase 1 Study Evaluating the Safety and Anti-Tumor Activity of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ B-Cell Lymphoma

Abstract A163: Effective targeting of HER2-amplified cancers with trastuzumab in combination with T cells expressing a novel Antibody-Coupled T-cell Receptor (ACTR)

Abstract HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancers, and is associated with poor prognosis. The standard of care for HER2-positive early breast cancer includes the HER2-targeting antibody, trastuzumab, administered with chemotherapy. In patients with advanced HER2-positive disease, the standard of care as first-line therapy includes dual blockade of HER2 with trastuzumab and pertuzumab, an antibody that binds to an alternative epitope of HER2. Despite the clinical success of targeting HER2, many patients are intrinsically resistant or develop resistance during treatment regardless of continued expression of HER2, highlighting the need for novel therapeutic paradigms. Engineered cell therapies, including chimeric antigen receptor (CAR) T cells, have demonstrated promising clinical activity in advanced cancers. Despite success, CAR-T cells have shown substantial toxicity, and in initial clinical trials of HER2-amplified cancer CAR-T cells demonstrated acute toxicities, presumably due to "on target/off tumor" activity. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy developed to mediate antitumor activity when administered with tumor-targeting antibodies. The ACTR construct is composed of the extracellular domain of the high-affinity CD16 (FCGR3A) V158 variant fused with costimulatory and CD3ζ intracellular signaling domains. ACTR is engineered to engage the Fc domain of human IgG1 antibodies when opsonized to target cells, which delivers an activation signal to the engineered T cells. Two distinct ACTR variants, ACTR087 and ACTR707, are currently being evaluated clinically in combination with rituximab in patients with B cell lymphoma (NCT02776813, NCT03189836). The ACTR707 variant was selected from a panel of constructs based on its strong performance in preclinical tests to assess activity not only in hematologic cancers, but also in solid tumors. When combined with trastuzumab, ACTR707 mediated potent cytotoxicity against HER2-amplified tumor cell lines in vitro. Cytokine response and proliferation of ACTR707 was also observed in the presence of trastuzumab and target cells. ACTR707 activity was specific to trastuzumab-treated HER2-positive cells as no cytotoxicity was observed in the absence of trastuzumab, or with a nontargeting antibody. In HER2-amplified xenograft models, such as the N87 gastric cancer model, ACTR707 and trastuzumab combination treatment mediated tumor regressions, demonstrating significantly enhanced activity compared to trastuzumab alone. Finally, the activity of ACTR707 on a panel of normal cells representing normal tissue expression of HER2 was tested in the presence of trastuzumab. ACTR707 in combination with trastuzumab demonstrated marginal cytotoxicity on these cells. In contrast, trastuzumab-based CAR-T cells mediated potent cytotoxicity against these cells. Taken together, our data demonstrate that ACTR707 in combination with trastuzumab has robust antitumor activity in vitro and in vivo. Furthermore, this combination has differential cytotoxicity on HER2-amplified tumor cells compared to HER2-expressing nonmalignant cells, suggesting that a favorable therapeutic index may be established in the clinical treatment of HER2-amplified cancers. Citation Format: Katie M. O'Callaghan, John Shin, Eugene Choi, Greg Motz, Casey B. Judge, Heather A. Huet, Birgit C. Schultes, Seth A. Ettenberg. Effective targeting of HER2-amplified cancers with trastuzumab in combination with T cells expressing a novel Antibody-Coupled T-cell Receptor (ACTR) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A163.

Abstract B105: ACTR707: a novel T-cell therapy for the treatment of relapsed or refractory CD20+ B cell lymphoma in combination with rituximab

Abstract The Antibody-Coupled T-cell Receptor (ACTR) platform is an autologous engineered T-cell therapy developed to combine with tumor-targeting antibodies to exert potent antitumor immune responses and tumor cell killing. The ACTR construct is composed of the extracellular domain of CD16 (FCGR3A; high-affinity V158 variant) linked to CD3ζ signaling and costimulatory domains. ACTR-expressing T cells are universal, and can be flexibly paired with desired therapeutic antibodies to target tumor antigens. Unum’s most advanced clinical candidate, ACTR087, is currently in clinical testing in combination with rituximab for the treatment of CD20+ B cell lymphoma (NCT02776813). To expand upon the ACTR platform, over 90 different domain-modified variant ACTRs were generated and evaluated through rigorous in vitro and in vivo testing in combination with a broad range of tumor-targeting antibodies for use in hematologic and solid tumor indications. ACTR T-cell cytotoxicity, cytokine release and proliferation assays, combined with performance in an in vitro repeated stimulation “stress test,” led to the identification of several lead ACTR candidates. The final ACTR candidate was identified on the ability to mediate tumor regressions of aggressive CD20+ lymphomas and HER2+ solid tumors in vivo when combined with rituximab or trastuzumab, respectively. These efforts identified ACTR707, an ACTR construct containing a CD28 costimulatory domain with additional molecular elements. Here we present nonclinical data supporting an ongoing first-in-human phase 1 clinical trial of ACTR707 in combination with rituximab. When combined with rituximab, ACTR707 T cells had potent activation, proliferation, cytokine production, and tumor-directed cytotoxicity in the presence of CD20+ lymphoma cell lines. ACTR707 T-cell in vitro activity was dependent on rituximab and was dose-titratable. ACTR707 T cells had potent antitumor efficacy in vivo in an aggressive Raji lymphoma xenograft model in NSG mice; ACTR707 T cell antitumor activity was ACTR T cell and rituximab dose-dependent, with higher T-cell numbers and antibody concentrations mediating improved responses. Further, ACTR707 in combination with rituximab outperformed a CD19 targeted CAR-T against aggressive Raji tumors in vivo. Taken together, these nonclinical data demonstrate the specificity and versatility of the ACTR707 T-cell therapeutic approach to target diverse cancer antigens and directly supports clinical testing in combination with rituximab. The phase I study, ATTCK-20-03 (NCT03189836), in subjects with relapsed or refractory CD20+ B cell lymphoma is a multicenter, single-arm, open-label dose escalation study evaluating the safety and antilymphoma efficacy of ACTR707 T cells in combination with rituximab. Subjects who meet eligibility requirements with histologically confirmed relapsed or refractory CD20+ B cell lymphoma of one of the following histologic subtypes are eligible: DLBCL, MCL, PMBCL, Gr3b-FL, TH-FL. The study’s primary objective is to assess the safety of the combination and define dose recommendations for further study. Additional study objectives include the assessment of antilymphoma activity, ACTR707 persistence, ACTR707 product characterization, inflammatory laboratory assessments, and rituximab pharmacokinetics. Enrollment is ongoing. Citation Format: Greg Motz, Kathleen Whiteman, John Shin, Tapasya Pai, Casey Judge, Anthony Barnitz, James Hemphill, James Kim, Ann Ranger, Heather Huet, Kathleen McGinness, Birgit Schultes, Geoffrey Hodge, Michael Vasconcelles, Seth Ettenberg. ACTR707: a novel T-cell therapy for the treatment of relapsed or refractory CD20+ B cell lymphoma in combination with rituximab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B105.

Abstract 4605: Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody

Abstract B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody-Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co-stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti-tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93-103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti-BCMA antibody, SEA-BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA-BCMA - opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA-BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi-faceted activity of ACTR T cells in combination with the anti-BCMA antibody, SEA-BCMA, for clinical consideration in multiple myeloma patients. Citation Format: Tooba Cheema, Taylor Hickman, Katie O'Callaghan, Lori Westendorf, Luke Manlove, Shyra Gardai, Allison Nelson, Ryan Boomer, Kathleen McGinness, Birgit Schultes, Seth Ettenberg, Django Sussman, Heather Huet. Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2017-4605

Abstract 3762: Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell

Abstract Glypican-3 (GPC3) is a GPI-anchored member of the heparan sulfate proteoglycan family. GPC3 is an oncofetal antigen expressed transiently during fetal development with re-expression during malignant transformation. GPC3 is an ideal tumor target as expression has been found in numerous epithelial malignancies, with highest expression in hepatocellular carcinoma (HCC) and non-small cell lung carcinoma (NSCLC), and normal tissue expression is highly restricted (Baumhoer D., Am J Clin Pathol, 2008.). Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy, but the broad applicability of this approach has been limited in part by safety concerns due the constitutive expression of a biologically active targeting receptor. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy designed to engage the Fc domain of therapeutic antibodies opsonized to tumor cells to mediate anti-tumor activity. ACTR activity is therefore both regulatable and flexible, providing enhanced therapeutic control and improved safety of the T cell therapy. Using both HCC and NSCLC tumor cell lines, we tested a panel of wild-type and afucosylated antibodies with similar binding affinities that bound to regions spanning the GPC3 protein across unique epitopes. We found that for GPC3 targeting antibodies, the greatest activity in a Jurkat-NFAT reporter assay was observed for the afucosylated antibody that bound GPC3 most proximal to the membrane. Further, the antibody that bound membrane proximal also had the most potent activity in primary ACTR T cell cytotoxicity and cytokine release assays. The physical distance between T cells and tumor targets has been previously determined to impact T cell activation for both peptide-MHC and CAR-T interactions. Similarly, our results demonstrate a potential relationship between spatial distance of tumor targets and T cells in determining the activity ACTR transduced T cells when targeting GPC3. Our data demonstrate that ACTR T cell activity is antibody-specific and dose-titratable, highlighting both efficacy and improved safety of the ACTR T cell platform when targeting GPC3+ solid tumor malignancies. Citation Format: Greg Motz, John Shin, Kathleen Whiteman, Birgit Schultes, Tapasya Pai, Lori Westendorf, Seth Ettenberg, Travis Biechele, Django Sussman, Heather Huet. Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2017-3762

Abstract A77: Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells

Abstract HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancer diagnoses, often signifying poor prognosis. The current standard of care for HER2 amplified cancer is the HER2 targeting antibody trastuzumab with chemotherapy. In the setting of HER2 amplified breast cancer, the combination of HER2 targeting antibodies trastuzumab and pertuzumab with the chemotherapeutic docetaxel is the frontline therapy. Despite the success of targeting the HER2 pathway, there are still many patients who are refractory or relapse following HER2 targeting regimens. Outside of the setting of HER2 overexpression or gene amplification, targeting HER2-positive cancers has thus far been ineffective. Engineered autologous T-cells, including chimeric antigen receptors (CARs) and high affinity T-cell receptors (TCRs), have gained attention recently due to their potent efficacy, with overall response rates reaching 80% and examples of long lasting remissions, particularly in advanced lymphoma and leukemia. However, initial clinical attempts to target HER2-amplified breast cancer with CAR-T cell therapy met with either acute toxicities or lack of efficacy. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR chimeric construct is composed of the high-affinity CD16 (FCGR3A) V158 variant with the signaling domains from CD3z and 4-1BB. ACTR is designed to engage the Fc domain of human IgG1 antibodies when opsonized to the cognate target cells and deliver an activation signal to the engineered T cells. ACTR T cells combined with either HER2 targeting trastuzumab or pertuzumab exhibited potent cytotoxic activity, cytokine response and proliferation on a HER2 amplified tumor cell lines. ACTR activity was specific to antibody treated cells, and had little activity on HER2 low or negative tumor lines. Furthermore, the effectiveness of ACTR T cells in a non-amplified HER2 (HER2 low) setting was tested in the presence of a combination of trastuzumab and pertuzumab. This multi-antibody combination increased the cytotoxicity by ACTR T cells, whereas trastuzumab or pertuzumab as single antibody combinations with ACTR had little effect, suggesting a potential therapeutic approach for this disease setting with high unmet medical need. Importantly, the ACTR platform decouples the targeting moiety from the engineered T cell allowing for an antibody dose response which may spare cytotoxicity on normal cells. The specific response to HER2 positive tumor cells in the presence of trastuzumab, pertuzumab or the combination of HER2 directed antibodies demonstrates the potential therapeutic activity of ACTR T cells and supports consideration of clinical testing of ACTR T cells in HER2-positive cancers. Furthermore, combining multiple targeting antibodies to achieve greater potency and efficacy is unique to the ACTR technology and demonstrates the potential for such activity across many other tumor targets and tumor targeting antibodies. Citation Format: Casey B. Judge, Rachel DeBarge, Eugene Choi, Katie O'Callaghan, Lindsay Edwards, Birgit Schultes, Seth Ettenberg, Heather A. Huet. Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A77.

Targeting CD20+ Relapsed Refractory B Cell Lymphoma with ACTR087, Antibody-Coupled T-Cell Receptor (ACTR) Engineered Autologous T Cells, in Combination with Rituximab

The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal engineered T cell therapy developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR construct is composed of the extracellular domain of CD16 (FCGR3A; high-affinity V158 variant) linked to CD3ζ signaling and 41BB co-stimulatory domains. ACTR-expressing T cells recognize antibodies bound to antigen on the surface of target cells, and this receptor engagement results in T cell activation, proliferation, and cytotoxic attack of target cells. Previous preclinical research has demonstrated that ACTR T cells elicit tumor cell cytotoxicity in combination with rituximab, trastuzumab, and hu14.18K322A against CD20, HER2, and GD2 expressing tumor cell lines, respectively (Kudo et al., Cancer Res 2014; 74:93-103). The safety of ACTR T cells in combination with rituximab for subjects with B-cell malignancies is currently being explored in a pilot phase 1 clinical study (ATTCK-20) sponsored by National University Hospital, Singapore. Electroporation of mRNA is being used to deliver the ACTR transgene to study subjects’ T-cells, creating a transiently active form of the therapy.In this study, we present non-clinical data supporting the first-in-human clinical trial of ACTR087, the ACTR transgene (CD16V-41BB-CD3ζ) delivered by γ-retrovirus to human T cells, in combination with rituximab in relapsed refractory B cell lymphoma (NCT02776813). In vitro data demonstrate that in the absence of rituximab or in the presence of non-targeting antibodies, no increase in cytotoxicity is observed, demonstrating that ACTR signaling is target- and antibody-specific. ACTR T cells proliferate in response to rituximab-bound lymphoma cells, but this proliferation is antibody-dependent and self-limiting in the absence of antibody. In vivo efficacy and pharmacokinetic studies further support the dependence on adequate rituximab exposure for ACTR T cell response, suggesting that ACTR T cell activity can be modulated by antibody dose and schedule, which is a potential advantage over other chimeric antigen receptor (CAR) T cell therapies.Additional in vitro studies were conducted to explore the effects of potential interventional antibody therapies used to mitigate adverse events reported for CAR-T therapies in similar patient populations. These include tocilizumab, used to treat cytokine release syndrome (CRS), and intravenous immunoglobulin (IVIG), used to treat hypogammaglobulinemia complicated by recurrent sinopulmonary infections. At therapeutically relevant doses, rituximab was able to compete with substantial excess IVIG to induce a CD20-specific ACTR T cell response. Importantly, IVIG did not induce greater cytokine release from ACTR T cells. Likewise, tocilizumab did not mediate ACTR T cell activation or cytotoxicity of IL-6R+ target cells, consistent with reports that tocilizumab is ADCC-deficient. These data illustrate an important principal of Fc receptor biology: not all IgG1 antibodies mediate ADCC despite binding to CD16, and support the considered use in the presence of ACTR T cells under certain circumstances. Taken together, these non-clinical data demonstrate the specificity and versatility of the ACTR T cell therapeutic approach to target diverse cancer antigens.The Phase 1 study (ATTCK20-2, ClinicalTrial.gov No. NCT02776813) of ACTR087 plus rituximab in subjects with relapsed or refractory CD20-positive B cell lymphoma is a multi-center, single-arm, open-label study evaluating the safety and efficacy of an autologous T-cells, culture expanded and transduced ex vivo with a γ-retrovirus containing an ACTR expression construct (CD16V-41BB-CD3ζ). Subjects who meet eligibility requirements with histologically-confirmed relapsed or refractory CD20+ B-cell lymphoma of one of the following types are eligible for the study: DLBCL, MCL, PMBCL, Gr3b-FL, TH-FL. Following a conditioning regimen with fludarabine and cyclophosphamide, and prior to ACTR087 infusion, subjects will receive rituximab at the standard IV clinical dose, 375 mg/m2. Subjects may receive up to 7 additional cycles of rituximab 375 mg/m2 at 3-week intervals and continuing as long as they show evidence of response. DisclosuresHuet:Unum Therapeutics: Employment. Judge:Unum Therapeutics: Employment. Barnitz:Unum Therapeutics: Employment. Boomer:Unum Therapeutics: Employment. McGinness:Unum Therapeutics: Employment. Shin:Unum Therapeutics: Employment. Cheema:Unum Therapeutics: Employment. Whiteman:Unum Therapeutics: Employment. Schultes:Unum Therapeutics: Employment. Ranger:Unum Therapeutics: Employment. Cao:Unum Therapeutics: Employment. Hodge:Unum Therapeutics: Employment. Vasconcelles:Unum Therapeutics: Employment. Ettenberg:Unum Therapeutics: Employment.

Actr Platform As an Adaptable, Universal T Cell Therapy That Can Target Multiple Tumor Antigens to Overcome Antigen Escape

Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy. Nevertheless, treatment failure can occur and is often associated with loss of the targeted antigen-an outcome affecting nearly 65% of patients that have failed CD19-targeted CAR T cell therapy (1). Targeting more than one tumor antigen expressed on the surface of tumor cells to mitigate antigen loss is a strategy that has been tested preclinically with CAR-T therapy. This approach is challenging due to the complexities of validating numerous CAR constructs or generating functional CARs which contain several tandem scFvs. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR construct, derived from human CD16 and coupled to T-cell signaling domains, is designed to engage the Fc domain of therapeutic antibodies, resulting in a novel platform for T-cell targeted cancer therapy. In contrast to CAR T-cell constructs that are restricted to a single antigen, the ACTR platform is highly adaptable, which can be targeted against a diverse set of tumor antigens-thus circumventing the need to generate and characterize multiple CAR-T therapeutics.We first determined whether ACTR expressing T cells could be combined with a diverse array of tumor-targeting antibodies directed against B cell malignancies. Using cell lines derived from B cell lymphomas or multiple myeloma, we found that ACTR-expressing T cells could be activated with antibodies against multiple B-cell targets including CD19, CD20, CD22, CD38, and CS1. One approach to address CD19 antigen loss in patients relapsing following CD19 CAR-T therapy is to treat with a CD22 CAR-T therapy (2). Given that ACTR can be paired with single antibodies as described above, a flexible sequential administration of targeted antibodies could be utilized. However, we also sought to determine whether combinations of antibodies against CD19 and CD22, when used concurrently, could improve ACTR-activity. We found that dual antigen targeting with anti-CD19 and anti-CD22 antibodies could enhance both ACTR-mediated cytotoxicity and cytokine production in an antibody dose-dependent manner. Taken together, our results provide preclinical evidence for ACTR as a universal, chimeric receptor that could engage multiple tumor antigens with potential to improve patient outcomes by eliminating antigen negative relapse.

A First-in-Human Study of Autologous T Lymphocytes with Antibody-Dependent Cell Cytotoxicity (ADCC) in Patients with B-Cell Non-Hodgkin Lymphoma (NHL)

Recent trials in patients with B-cell malignancies have demonstrated the clinical potential of chimeric antigen receptor (CAR)-directed T cells. We developed a chimeric receptor (antibody-coupled T-cell receptor, ACTR) consisting of high affinity CD16 V158, CD8α transmembrane domain, 4-1BB and CD3ζ (Kudo et al. Cancer Res 2014). Expression of ACTR conferred ADCC capacity to T cells. In vitro and in immunodeficient mice, ACTR T cells specifically killed B-NHL and chronic lymphocytic leukemia cells tagged with the anti-CD20 antibody rituximab. Trastuzumab induced ACTR-mediated T cell killing of breast and gastric cancer cells, and anti-GD2 of neuroblastoma and osteosarcoma cells. Thus, infusion of ACTR T cells might significantly augment the anti-tumor potential of immunotherapeutic antibodies, and expand options for cell therapy of cancer.To assess the safety of this strategy and explore its efficacy, we began a pilot study in patients with relapsed/refractory CD20+ B-NHL (ATTCK20; ClinicalTrials.gov No. NCT02315118). In this first-in-human study, ACTR expression is achieved by mRNA electroporation and, hence, is transient. In Phase 1a, 3 subjects receive one dose of autologous ACTR T cells (0.5 x 106/kg) 2 days after rituximab (500 mg/m2); a second dose of ACTR T cells at 0.5 x 107/kg 28 days later is allowed if there is no toxicity and evidence of response. Interleukin-2 (IL-2) is given subcutaneously at 1 million IU/m2/d x 3 during the first week after infusion. Phase 1b plans up to 4 infusions of ACTR T cells (0.5 x 107/kg) at 10 day intervals, each preceded by rituximab at 375 mg/m2; IL-2 (as in Phase1a) is given after the first 2 infusions only.Six subjects have been enrolled to date (3 in Phase 1a, 3 in Phase 1b) and received a total of 14 ACTR T cell infusions. All had advanced CD20+ B-NHL (2 diffuse large B cell lymphoma, 2 primary mediastinal B cell lymphoma, 1 mantle cell lymphoma, and 1 mediastinal grey zone lymphoma) and had failed multiple lines of therapy. As a lymphodepleting regimen prior to the first ACTR T cell infusion, 5 of the 6 patients received fludarabine (25 mg/m2/d x 5) plus cyclophosphamide (60 mg/kg/d x 1, n=3; 900 mg/m2/d x 1, n=2); the remaining patient received bendamustine (90 mg/m2/d x 2).After leukapheresis, T cells were cultured under cGMP conditions with anti-CD3 and -CD28 antibodies, and IL-2. After 10 days, cells were electroporated with ACTR mRNA using the MaxCyte GT system. The next day, median ACTR expression in T cells, as shown by flow cytometry, was 95.8% (range, 82.8%-99.3%; n=14). With Rituximab, this produced a median cytotoxicity of 77.5% (67.6%-97.6%; n=14) against CD20+ Daudi cells at a 4:1 effector : target ratio in 4 hours. As expected, ACTR expression in vitro remained high for the first 3-4 days, declining thereafter.We met or exceeded the estimated minimum target levels of CD3+ ACTR+ cells in 13 of the 14 infused cell products. CD3+ ACTR+ cells (mostly CD8+) were detectable in peripheral blood 1 day after infusion and reached their peak on day 3-4 when they represented 0.43-11.20 (median, 1.27) cells per µL. They became undetectable after day 6, reflecting the kinetics of ACTR expression in vitro.As of July 2016, 5 of the 6 patients are evaluable, with a median follow-up of 60 weeks (range, 6-83) after the last ACTR-T cell infusion; the remaining patient has received 3 of the 4 scheduled cycles. The main toxicities were grade 3-4 cytopenias: these were observed in all patients and likely due to the lymphodepleting regimen. There was no non-hematologic toxicity, nor evidence of cytokine release syndrome. In Phase 1a, 2 patients had progressive disease (PD) and 1 had stable disease (SD) on CT scan following the first and second infusions, with PET-CT showing reduced FDG avidity and central areas of photopenia suggestive of necrosis. Of the 2 Phase 1b patients evaluable for response, 1 had PD after the third infusion and was taken off study; the other had SD 1 month after 4 ACTR-T infusions; the 2nd month assessment was interpreted as PD.The initial results of this ongoing trial using autologous T cells with transient ACTR expression are encouraging and warrant additional studies with more potent formulations of this technology. To this end, a clinical trial for patients with relapsed/refractory CD20+ B-NHL in which ACTR is permanently expressed in autologous T cells via viral vector transduction is planned (ClinicalTrials.gov No. NCT02776813). DisclosuresCampana:Unum Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Juno Therapeutics: Patents & Royalties; Nkarta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.