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BackgroundCAR-T therapy has limited efficacy against solid tumors due to low trafficking to the tumor, limited cell expansion in patients, tumor antigen heterogeneity, and an immunosuppressive microenvironment. TTS are fusion proteins that consist of genetically engineered Superantigens (Sag) linked to Fragment antigen binding (Fab) moieties directed to tumor associated antigens. It was previously shown that TTS selectively activates a subset of T cells [1], turns ”cold tumors hot” [2] and, in preclinical models, can lead to long-term memory responses [3]. Here we present preclinical data demonstrating that the lead TTS compound, NAP (5T4 targeted Sag), enhanced the efficacy of CAR-T treatment against tumor cells in vitro, suggesting that NAP may overcome current CAR T limitations.MethodsHer2-CAR-T cells were produced in the presence of NAP or CD3&CD28, and their potency was evaluated against the FaDu cell line and by measurement of T cell activation markers (CD25, CD137, IFN-gamma, CD107a). The expression of memory markers (CCR7, CD45RA/CD45RO, CD95) and Th1 polarization (transcription factors) of the resulted CAR-T cells were analyzed by staining with specific antibodies. The combined potency of NAP with CAR-T was also tested in vitro against the FaDu cell line (which expresses both Her2 and 5T4 antigens). The chemotactic activity of T cells was assessed using a chemotactic chamber.ResultsPBMCs grown in the presence of NAP, in comparison with PBMCs grown in the presence of CD3&CD28 antibodies, resulted in the production of more potent CAR-T cells as measured both by killing assay using the FaDu cell line and by INFγ production, activation markers and T cell degranulation. Central memory (CM) percentages were increased and Th1 polarization was significantly more prominent after NAP stimulation. Following incubation with NAP, the chemotaxis towards the tumor cells was significantly enhanced. Finally, combination of CAR-T and NAP resulted in a synergistic killing effect of the tumor cell line.ConclusionsOur studies show that NAP generates more potent CAR-T cells and acts synergistically with CAR-T against tumor cell lines in vitro. The ability of NAP administration to activate T cells outside of the immunosuppressive microenvironment (in the lymphoid organs), promote T cell infiltration into the tumor and induce long-term memory responses, strongly suggests that combination of CAR-T cells with NAP may overcome the limited effect of CAR-T therapy against solid tumors. NAP is currently being evaluated in clinical studies in combination with durvalumab [NCT03983954] and docetaxel [NCT04880863].ReferencesHedlund G, Eriksson H, Sundstedt A, et al. The Tumor Targeted Superantigen ABR-217620 Selectively Engages TRBV7-9 and Exploits TCR-pMHC Affinity Mimicry in Mediating T Cell Cytotoxicity. PLoS One. 2013; 8(10): e79082.Azulay M, Lifshits S, Friedmann A, et al. Naptumomab Estafenatox induces T cell recognition, turning anti-PD-1 unresponsive “cold” tumors into “hot” responsive tumors. Cancer Research. Jul 2018, 78 (13 Supplement) abstract # 2712 AACR Annual Meeting 2018; Chicago, IL; DOI: 10.1158/1538-7445.AM2018-2712.Azulay M, Lifshits S, Shany E, et al. Selective T cell Redirection Proteins (STR) Enhance the Anti-Tumor Activity of Checkpoint Inhibitors (CPIs) and can Lead to Long-Lasting Immunity Against the Tumor. Abstract # P657. SITC 34th Annual Meeting, 2019. National Harbor, Maryland, USA.

TPS3160 Background: Immunotherapy with the anti-PD-(L)1 checkpoint inhibitors (CPIs) has been largely ineffective in so-called non-immunogenic “cold tumors”. Facilitating T cell infiltration is necessary to invoke an immune response which may be augmented or complemented by the activity of CPIs like durvalumab. Selective T cell Redirection Proteins (STRs) are fusion proteins that consist of genetically engineered Superantigen (Sag) linked to Fragment antigen binding (Fab) moieties directed to tumor-associated antigens. Nap is a first in class STR compound, recognizing the tumor-associated oncofetal antigen 5T4, whereas the SAg moiety selectively engages the T cell receptor β variable (TRBV) 7-9. Nap has been shown to induce specific T cells expansion, activation and infiltration into the tumor in pre-clinical and clinical studies. Pre-clinical data demonstrated that the combination of STR with CPI may lead to long term durable responses not possible in most patients receiving single agent CPI therapy and suggests that combining CPIs with STR may be a promising therapeutic strategy for patients with solid tumors. Methods: Patients will be treated with the combination of Nap and durvalumab using a flat dose of durvalumab (1120 mg) and the 3+3 design for Nap dose escalation (2, 5, 10, 15 and 20 mcg/kg). The MTD of Nap for the combination treatment will be established based on DLTs occurring during the first treatment cycle. The dose escalation part will be followed by MTD expansion cohort in which 10–15 patients will be treated with MTD of Nap and 1120 mg durvalumab (Clinical trial registry number NCT03983954). Major eligibility criteria include patients with pretreated advanced or metastatic, 5T4 expressing solid tumors, including patients previously progressed on CPI therapy. As of January 2020, enrollment to dose levels 2, 5 and 10 mcg/kg has been completed without DLT, enrollment to dose level 15 mcg/kg will start on February 2020. Clinical trial information: NCT03983954 .

Tumor recognition is a key factor in checkpoint inhibitors (CPI) efficacy and acquired resistance. Lack or loss of tumor antigens expression or inefficient presentation prevents tumor cell recognition by T cells, inhibiting CPI anti-cancer effect. Naptumomab estafenatox (Nap) is a tumor-targeted superantigen (TTS) protein that increases tumor recognition by both coating tumor cells with bacterial-derived superantigens (SAg) as well as selectively expanding T cells lineages that can recognize it. Nap consists of a genetically engineered SAg, staphylococcal enterotoxin A (SEA/E-120), linked to a fragment antigen binding (Fab) moiety directed to the 5T4 oncofetal tumor-associated antigen expressed on many tumors. Here we present new pre-clinical results of synergistic anti-tumor effect of Nap (or its murine equivalent TTS) with anti-PD1, under conditions which mimic poor tumor recognition. In vitro studies with Nap investigated the effect of co-culturing SAg activated T cells with HCC827 NSCLC or MDA-MB 231 Triple-Negative Breast Cancer cells. Co-cultures of T cells with either cancer cell line resulted in an increase in tumor staining for PDL1, which was further elevated in the presence of Nap, probably due to increase in IFNγ secretion. The viability of HCC827 or MDA-MB-231 cells co-cultured with the SAg activated T cells was examined in the presence or absence of the PD1 inhibitor, Nap or the combination. Anti-PD1 alone had no effect on T cell mediated cytotoxicity, whereas Nap induced a significant cytotoxic effect, with the combination of anti-PD1 plus Nap producing the most significant reduction in tumor cell viability. Similar to the in vitro studies, the in vivo mouse studies using low immunogenic tumor models showed limited or no effect of anti-PD1 monotherapy. Although TTS alone increased T cells activation and infiltration into the tumor, the combination with anti-PD1 was significantly more effective in increasing serum cytokines, increasing the CD8:CD4 ratio in the tumor, reducing tumor burden and prolonging median survival. In summary, our studies show that combination of anti-PD1 with tumor-targeted superantigens overcomes the limited effect of anti-PD1 monotherapy in low immunogenic tumor models in vitro and in vivo. This novel combination may offer clinical benefit for CPI unresponsive patients, particularly in cases where tumor recognition is lost or restricted. Clinical phase 1-2 trials are planned with the combination of naptumomab estafenatox and anti-PD1/PDL1 blockade. Citation Format: Meir Azulay, Sveta Lifshits, Adam Fridman, Gunnar Hedlund, Marie Törngren, Michal Shahar. Naptumomab Estafenatox induces T cell recognition, turning anti-PD-1 unresponsive "cold" tumors into "hot" responsive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2712.

Antibody-targeted superantigens have a potential to become useful drugs for tumor therapy. However, clinical practice has identified several issues that need to be addressed to optimize such molecules. On the basis of the experience from superantigen products in clinical trials, a novel tumor-targeted superantigen, naptumomab estafenatox (5T4FabV18-SEA/E-120 or ABR-217620) has been designed. Critical properties, such as tumor reactivity, therapeutic window, and seroreactivity were all improved. The engineered 5T4Fab moiety recognizes the 5T4 antigen expressed on a large number of solid tumor forms with an affinity in the order of 1 nM. The fusion protein induces T-cell mediated killing of tumor cells at concentrations around 10 pM. Compared with a construct with a wild-type superantigen, it is more potent in mediating killing of tumor cells but a 10,000-fold less active in mediating killing of MHC class II positive cells. The target epitopes for naturally occurring antibodies toward bacterial superantigens are reduced. Only large excesses of human anti-SEA antibodies neutralize the antitumor effects of the antibody-targeted superantigen. Naptumomab estafenatox induces dramatic reduction of established human tumors in Severe Combined Immunodeficient mice grafted with human lymphocytes. Thus, naptumomab estafenatox is a novel optimized tumor-targeted superantigen currently investigated in clinical trials.

PurposeTwo phase I studies were conducted of ABR-217620 alone or in combination with docetaxel. This is a recombinant fusion protein consisting of a mutated variant of the superantigen staphylococcal enterotoxin E (SEA/E-120) linked to fragment antigen binding moiety of a monoclonal antibody recognizing the tumor-associated antigen 5T4.Patients and MethodsPatients with non–small-cell lung cancer (NSCLC), pancreatic cancer (PC), and renal cell cancer (RCC) received 5 daily boluses of ABR-217620 (3-month cycles) in escalating doses to determine the maximum-tolerated dose (MTD; ABR-217620 dose escalation monotherapy [MONO] study). Doses were selected based on individual patient anti–SEA/E-120 titers pretreatment. Patients with NSCLC received 4 daily, escalating doses of ABR-217620 followed by docetaxel in 21-day cycles (ABR-217620 dose escalation combination with docetaxel [COMBO] study).ResultsThirty-nine patients were enrolled in the MONO study and 13 were enrolled in the COMBO study. The monotherapy MTD was 26 μg/kg (NSCLC and PC) and 15 μg/kg (RCC). Dose-limiting toxicities (DLTs) in the MONO study were fever, hypotension, acute liver toxicity, and vascular leak syndrome. In the COMBO study, the MTD was 22 μg/kg (neutropenic sepsis). Adverse events included grade 1 to 2 fever, hypotension, nausea, and chills. Treatment caused a systemic increase of inflammatory cytokines and selective expansion of SEA/E-120 reactive T-cells. Tumor biopsies demonstrated T-cell infiltration after therapy. Fourteen patients (36%) had stable disease (SD) on day 56 of the MONO study. Two patients (15%) in the COMBO study had partial responses, one in a patient with progressive disease on prior docetaxel, and five patients (38%) had SD on day 56.ConclusionABR-217620 was well tolerated with evidence of immunological activity and antitumor activity.

Purpose: To prospectively determine the efficacy of naptumomab estafenatox (Nap) + IFNα versus IFN in metastatic renal cell carcinoma (RCC). Experimental Design: In a randomized, open-label, multicenter, phase II/III study, 513 patients with RCC received Nap (15 μg/kg i. v. in three cycles of four once-daily injections) + IFN (9 MU s.c. three times weekly), or the same regimen of IFN monotherapy. The primary endpoint was overall survival (OS). Results: This phase II/III study did not meet its primary endpoint. Median OS/PFS for Nap + IFN patients was 17.1/5.8 months versus 17.5/5.8 months for the patients receiving IFN alone (P = 0.56; HR, 1.08/P = 0.41; HR, 0.92). Post hoc exploratory subgroup and trend analysis revealed that the baseline plasma concentrations of anti-SEA/E-120 (anti-Nap antibodies) for drug exposure and IL6 for immune status could be used as predictive biomarkers. A subgroup of patients (SG; n = 130) having concentrations below median of anti-SEA/E-120 and IL6 benefitted greatly from the addition of Nap. In SG, median OS/PFS for the patients treated with Nap + IFN was 63.3/13.7 months versus 31.1/5.8 months for the patients receiving IFN alone (P = 0.02; HR, 0.59/P = 0.02; HR, 0.62). Addition of Nap to IFN showed predicted and transient immune related AEs and the treatment had an acceptable safety profile. Conclusions: The study did not meet its primary endpoint. Nap + IFN has an acceptable safety profile, and results from post hoc subgroup analyses showed that the treatment might improve OS/PFS in a baseline biomarker-defined RCC patient subgroup. The results warrant further studies with Nap in this subgroup. Clin Cancer Res; 22(13); 3172–81. ©2016 AACR.

Traditionally, the major objective in phase I trials is to iden- tify a working-dose for subsequent studies, whereas the major endpoint in phase II and III trials is treatment efficacy. The dose sought is typ- ically referred to as the maximum tolerated dose (MTD). Several sta- tistical methodologies have been proposed to select the MTD in cancer phase I trials. In this manuscript, we focus on a Bayesian adaptive design, known as escalation with overdose control (EWOC). Several aspects of this design are discussed, including large sample properties of the sequence of doses selected in the trial, choice of prior distri- butions, and use of covariates. The methodology is exemplified with real-life examples of cancer phase I trials. In particular, we show in the recently completed ABR-217620 (naptumomab estafenatox) trial that omitting an important predictor of toxicity when dose assignments to cancer patients are determined results in a high percent of patients experiencing severe side effects and a significant proportion treated at sub-optimal doses.

Improvement of cancer therapy by introducing new concepts is still urgent even though there have been major advancements lately. Immunotherapy is well on the way to becoming an established tool in the cancer treatment armory. It seems that a combination of (1) activation of immune effector cells and selective targeting of them to tumors and (2) the inhibition of immune suppression often induced by the tumor itself are necessary to achieve the therapeutic goal. The immunotoxin naptumomab estafenatox was developed in an effort to activate and target the patient’s own T cells to their tumor, by fusing a superantigen (SAg) variant that activates T lymphocytes to the Fab moiety of a tumor-reactive monoclonal antibody. Naptumomab estafenatox targets the 5T4 tumor antigen, a 72-kDa oncofetal trophoblast protein expressed on many carcinomas, including renal cell carcinoma. The therapeutic effect is associated with activation of SAg-binding T cells. The SAg-binding T lymphocytes expand, differentiate to effector cells, and infiltrate the tumor. The therapeutic efficacy is most likely related to the dual mechanism of tumor cell killing: (1) direct lysis by cytotoxic T lymphocytes of tumor cells expressing the antigen recognized by the antibody moiety of the fusion protein and (2) secretion of cytokines eliminating antigen-negative tumor cell variants. Naptumomab estafenatox has been clinically tested in a range of solid tumors with focus on renal cell carcinoma. This review looks at the clinical experience with the new immunotoxin and its potential.

Traditionally, the major objective in phase I trials is to identify a working-dose for subsequent studies, whereas the major endpoint in phase II and III trials is treatment efficacy. The dose sought is typically referred to as the maximum tolerated dose (MTD). Several statistical methodologies have been proposed to select the MTD in cancer phase I trials. In this manuscript, we focus on a Bayesian adaptive design, known as escalation with overdose control (EWOC). Several aspects of this design are discussed, including large sample properties of the sequence of doses selected in the trial, choice of prior distributions, and use of covariates. The methodology is exemplified with real-life examples of cancer phase I trials. In particular, we show in the recently completed ABR-217620 (naptumomab estafenatox) trial that omitting an important predictor of toxicity when dose assignments to cancer patients are determined results in a high percent of patients experiencing severe side effects and a significant proportion treated at sub-optimal doses., Published in at http://dx.doi.org/10.1214/10-STS333 the Statistical Science (http://www.imstat.org/sts/) by the Institute of Mathematical Statistics (http://www.imstat.org)

Importance of the field: New agents that specifically engage the immune system are being tested in a variety of malignancies. This review provides an overview of naptumomab, an immunotoxin, with encouraging clinical activity in Phase I trials.Areas covered in this review: This review examines the preclinical and the published clinical data with regards to naptumomab.What the reader will gain: This review provides the reader with an understanding of the mechanism of action, immunology, pharmacokinetics and clinical activity of this agent.Take home message: Naptumomab has a unique mechanism of action and appears to be an active agent in the treatment of refractory solid tumors such as renal cell carcinoma.

3073 Background: Naptumomab estafenatox/ANYARA (Nap) is a fusion protein of an antibody (5T4) and a superantigen (SEA/E-120). After phase I studies (Borghaei. J Clin Oncol. 2009, 27:4116) a prospective, randomized phase II/III trial of Nap + IFN-α (A) vs IFN-α (I) was conducted. Methods: Patients (pts) with RCC were randomized in an open label study to receive A or I. The primary endpoint was OS. Secondary endpoints were PFS, response rate and safety. Baseline (bl) plasma IL-6 was predictive of pazopanib (Tran. Lancet Oncol. 2012, 13:827) and MVA-5T4 vaccine (Harrop. Cancer Immunol Immunother. 2012, 61:2283) benefit in RCC pts. IL-6 and anti-SEA/E-120 antibodies (a-S) were analyzed. A subgroup SG1 had bl levels below median for IL-6 (

3083 Background: ABR-217620 (naptumomab estafenatox) is a recombinant fusion protein that consists of the 5T4Fab moiety genetically fused to the engineered superantigen variant SEA/E-120. This fusion protein is a new generation tumor-targeted superantigen based on the previously described ABR-214936 (anatumomab mafenatox). ABR-217620 was designed to reduce antigenicity and toxicity. The 5T4 antigen is expressed on more than 95 % of tumors from patients with non-small cell lung (NSCLC), renal cell (RCC) and pancreatic cancer (PC). In clinical PET studies 124I-labeled ABR-217620 has been shown to localize to 5T4 positive tumors. Methods: The compound was administered as a 5 min bolus infusion for 5 consecutive days. Patients with disease control at day 28 were offered a second cycle of therapy. Dose escalation has been performed using a Bayesian model starting at 0.5 μg/kg/day. The primary endpoint is determination of MTD. Secondary endpoints include characterization of side effects, immunological response, efficacy and pharmacokinetics. Results: 31 patients to date have been treated (19 NSCLC, 8 RCC, 4 PC). 3 patients have had dose limiting toxicities (fever, hypotension and nausea, grade 3) at doses between 23 and 28 μg/kg/day. The side effects were resolved quickly. Based on the experience from ABR-214936, these side effects were expected, but the MTD is ∼ 200 times higher. ABR-217620 leads to a dose dependent systemic increase of cytokines including IL-2 and IFN-γ after infusion. It also leads to an expansion of the superantigen reactive T cell population. 16/29 evaluable patients investigated had SD while 13 patients had PD. In contrast to ABR-214936, the first cycle of ABR-217620 treatment can be given without factoring in the titer of preformed anti-superantigen antibodies. Conclusions: ABR-217620 treatment had predicted and manageable side effects with fever, hypotension and nausea being dose limiting toxicities. Treatment with ABR-217620 resulted in a restricted systemic activation of the immune system. A large fraction of the patients have had stable disease. [Table: see text]