ADG153, an Anti-CD47 Monoclonal Antibody Prodrug, Has Strong In Vivo Anti-Tumor Activity, Minimal RBC-Related and Antigen Sink Liabilities, and Extended Half Life in Comparison with Benchmark Clinical Antibodies of the Same IgG Subclass

Abstract Binding of the CD47 membrane protein, overexpressed on many tumor types, to the SIRPα inhibitory receptor on myeloid cells results in the inhibition of the activation of macrophages and other phagocytes against tumors. Therapies targeting the CD47/SIRPα axis have shown success in various preclinical models and are now in clinical trials for both solid and hematologic malignancies. Although anti-CD47 therapies have demonstrated promising clinical activities, the expression of CD47 on many different normal human cell types, including red blood cells (RBCs), serves as a large antigen sink for anti-CD47 antibodies. Blocking CD47 on RBCs, such as by magrolimab (Hu5F9), has led to transient anemia, requiring step-up dosing in the clinic. To circumvent these challenges, we developed ADG153, a fully human anti-CD47 SAFEbody masked by conditionally activable peptides. In normal tissues, the SAFEbody masking moiety can function to block ADG153 from binding to CD47; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG153 antibody to bind to and inhibit CD47 function on tumor cells. For head-to-head comparisons, in vitro studies were performed to compare the activity of Hu5F9 and unmasked ADG153 parental antibody of the IgG4 isotype. Both antibodies (1) blocked human SIRPα from binding to human CD47, (2) had similar potencies for binding to human CD47 protein, CD47-positive tumor cell lines, and human RBCs, and (3) induced macrophage phagocytosis. In contrast, the masked ADG153 SAFEbody demonstrated significantly reduced activities (>450-fold) in the same in vitro assays, showing strong masking efficiencies. Unlike Hu5F9, both the ADG153 parental and SAFEbody molecules did not cause in vitro human RBC hemagglutination. Although the ADG153 SAFEbody had significantly reduced binding to CD47 in vitro as expected, it demonstrated strong anti-tumor activity in in vivo mouse tumor models. In both the disseminated and subcutaneous CD47-positive Raji tumor models, the ADG153 SAFEbody of the IgG4 isotype showed similar anti-tumor activities to Hu5F9. However, in exploratory toxicology studies in cynomolgus monkeys, the ADG153 SAFEbody showed significantly less decreases than Hu5F9 in RBCs, hemoglobin, and hematocrit. Hu5F9 at 10 mg/kg caused ~49% maximum decrease in RBCs, while ADG153 SAFEbody at 60 mg/kg showed ~23% maximum decrease in RBCs (Panel A). Pharmacokinetic (PK) studies of single intravenous dose ADG153 SAFEbody compared to Hu5F9 in monkeys demonstrated ~8-fold longer apparent half-life and ~9-fold higher Area Under the Curve at 10 mg/kg (Panel B). Collectively, ADG153 SAFEbody is a differentiated anti-CD47 antibody that has strong in vivo anti-tumor activity with reduced RBC-related and antigen sink liabilities and favorable PK properties. This preclinical profile with an enhanced therapeutic index provides a strong rationale for advancing ADG153 SAFEbody into clinical development. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Sparcbio, LLC: Ended employment in the past 24 months. Zheng: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Janssen Pharmaceuticals: Ended employment in the past 24 months. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Download Full-text

Combination of Camidanlumab Tesirine, a CD25-Targeted ADC, with Gemcitabine Elicits Synergistic Anti-Tumor Activity in Preclinical Tumor Models

Blood ◽

10.1182/blood-2020-137004 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Asma Jabeen ◽

Shiran Huang ◽

John A. Hartley ◽

Patrick H Van Berkel ◽

Francesca Zammarchi

Keyword(s):

Solid Tumor ◽

Synergistic Activity ◽

Publicly Traded ◽

Tumor Models ◽

Antibody Drug Conjugate ◽

Drug Conjugate ◽

Anti Tumor Activity ◽

Antibody Drug

Camidanlumab tesirine (ADCT-301) is an antibody-drug conjugate (ADC) comprised of HuMax®-TAC, a monoclonal antibody directed against human CD25, conjugated to the pyrrolobenzodiazepine dimer payload tesirine[1]. Currently, camidanlumab tesirine is being evaluated in a pivotal Phase 2 clinical trial in patients with relapsed or refractory Hodgkin lymphoma (HL) (NCT04052997) and in a Phase 1b clinical trial in patients with advanced solid tumors (NCT03621982). In pre-clinical studies, camidanlumab tesirine demonstrated strong and durable single agent activity in CD25-expressing lymphoma xenograft models[1] and in vitro it synergised with selected targeted agents[2]. Moreover, CD25-ADC, a mouse CD25 cross-reactive surrogate of camidanlumab tesirine, induced potent anti-tumor immunity against established syngeneic solid tumor models by depleting CD25-positive tumor-infiltrating T regulatory cells (Tregs) and it showed synergistic activity when combined with PD-1 blockade[3]. Here, we investigated the in vitro and in vivo anti-tumor activity of camidanlumab tesirine combined with gemcitabine, a common standard-of-care chemotherapeutic agent used both in a hematological and solid tumor clinical setting. In vitro, the combination of camidanlumab tesirine and gemcitabine was evaluated in three human-derived cancer cell lines (two HL and one anaplastic large cell lymphoma, ALCL) and resulted in synergistic activity as determined by the Chou-Talalay method. In vivo, camidanlumab tesirine was tested either alone (0.05 or 0.1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4) in the CD25-expressing ALCL Karpas299 xenograft model. At both ADC dose levels, combination with gemcitabine resulted in synergistic anti-tumor activity (coefficient of drug interaction (CDI) 0.51 and 0.17, respectively), better response rates and increased survival compared to monotherapy with camidanlumab tesirine. In order to extend the investigation to solid tumor models, CD25-ADC was tested in the CT26 syngeneic model, a colorectal cancer model with CD25-expressing tumor-infiltrating Tregs. CD25-ADC was administered either alone (0.1, 0.5 or 1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4). At the 0.1 mg/kg dose of CD25-ADC, combination with gemcitabine resulted in synergistic anti-tumor activity (CDI 0.68). Moreover, at 0.5 and 1 mg/kg, the combination of CD25-ADC and gemcitabine resulted in more durable anti-tumor activity and better response rates compared to both monotherapy treatments. In conclusion, the combination of camidanlumab tesirine and gemcitabine was synergistic both in vitro and in vivo in lymphoma preclinical models. Synergistic anti-tumor activity was also demonstrated in a colorectal cancer model using CD25-ADC, a mouse-cross-reactive version of camidanlumab tesirine, in combination with gemcitabine. Altogether, these novel pre-clinical data warrant translation of the combination between camidanlumab tesirine and gemcitabine into the clinic. 1.Flynn, M.J., et al., ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Mol Cancer Ther, 2016. 15(11): p. 2709-2721. 2.Spriano, F., et al., The anti-CD25 antibody-drug conjugate camidanlumab tesirine (ADCT-301) presents a strong preclinical activity both as single agent and in combination in lymphoma cell lines. Hematological Oncology, 2019. 37(S2): p. 323-324. 3.Zammarchi, F., et al., A CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity. Journal for ImmunoTherapy of Cancer, 2020. In press. Disclosures Jabeen: ADC Therapeutics: Current Employment. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

The Anti-Tumor Activity of Igm-8444, an Agonistic Death Receptor 5 (DR5) IgM Antibody, Is Sensitized in Combination with Chemotherapy and Bcl-2 Inhibitors in NHL and AML

Blood ◽

10.1182/blood-2020-141449 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 43-44

Author(s):

Beatrice T. Wang ◽

Thomas J. Matthew ◽

Ling Wang ◽

Tasnim Kothambawala ◽

Susan E. Calhoun ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Single Agent ◽

Mouse Tumor ◽

Targeted Agents ◽

Publicly Traded ◽

Tumor Models ◽

In Vivo Efficacy

Background: Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that multimerizes when bound to its ligand, TNF-related apoptosis inducing ligand (TRAIL), to activate the extrinsic apoptotic pathway. DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have been unsuccessful due to lack of efficacy or hepatotoxicity. We have developed IGM-8444, a pentameric IgM with 10 binding sites specific for DR5, that multimerizes DR5 to selectively and potently induce tumor cell apoptosis while maintaining tolerability. We have previously presented the in vitro and in vivo efficacy of IGM-8444 in solid tumor models, demonstrating low picomolar potency across multiple tumor cell lines, strong tumor regressions in cell line and patient derived xenograft mouse tumor models, and dose-dependent increases in apoptotic biomarkers. Here, we evaluate the activity of IGM-8444 in hematologic malignancies in combination with chemotherapy or targeted agents including Bcl-2 inhibitors targeting the intrinsic apoptotic pathway. Methods: Human hematologic cancer cell lines and primary human hepatocytes were evaluated in vitro for dose-dependent IGM-8444-induced cytotoxicity. Cell lines were further evaluated using IGM-8444 in combination with chemotherapy or targeted agents including Bcl-2 inhibitor ABT-199. In vivo efficacy was evaluated using IGM-8444 in combination with ABT-199 in cell line-derived xenograft mouse tumor models. Results: In a previous cancer cell line screen profiling single agent IGM-8444 cytotoxicity across 190 solid and hematologic cell lines, 25 (13%) were classified as highly responsive and 75 (39%) as moderately responsive to IGM-8444 induced cell death. Here the in vitro activity of IGM-8444 was evaluated across a subset of 32 NHL and AML cell lines. 5/21 (24%) of NHL cell lines and 5/11 (45%) of AML cell lines tested were classified as highly responsive or moderately responsive to IGM-8444-induced cytotoxicity. The DOHH-2 and JEKO1 NHL cell lines were amongst the most sensitive, with growth-normalized EC50 values as low as 0.03 ng/mL (0.03 pM) for JEKO1. Combinations with chemotherapy including cytarabine and doxorubicin or targeted agents such as Bcl-2 inhibitor ABT-199 resulted in synergistic in vitro cytotoxicity in multiple cell lines, as determined by Bliss synergy scores. IGM-8444 demonstrated minimal to no in vitro cytotoxicity to primary human hepatocytes at doses several log-fold higher than efficacious doses, and this favorable in vitro safety profile was maintained in combination with chemotherapeutic agents and ABT-199. Combination of IGM-8444 with ABT-199 also resulted in synergistic in vivo efficacy. In a DOHH-2 NHL model, IGM-8444 and ABT-199 showed modest tumor growth inhibition as single agents. However the combined treatment regimen led to tumor regressions during the first 2 weeks of treatment, with 3 of 10 animals showing a partial response and 2 of 10 animals achieving a complete response. The combined treatment also extended median overall survival compared to the control group, which was a significant improvement compared to either agent alone. Collectively, these results provide a strong rationale for simultaneously targeting the extrinsic and intrinsic apoptotic pathways to achieve enhanced efficacy. Conclusions: These data support the clinical development of IGM-8444 in hematological malignancies as a single agent, in combination with standard of care chemotherapy, and in combination with targeted agents that impact the intrinsic signaling pathway such as Bcl-2 inhibitor ABT-199. Initiation of a Phase I clinical study evaluating the safety of IGM-8444 is anticipated in 2020. Disclosures Wang: IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Matthew:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Wang:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Kothambawala:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Calhoun:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Humke:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Sinclair:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Keyt:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

Bispecific anti-PD-1/PD-L1 antibody CTX-8371 to promote cellular clustering and PD-1 shedding, antitumor activity and tolerability.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15056 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15056-e15056

Author(s):

Diana I. Albu ◽

Yan Qin ◽

Xianzhe Wang ◽

Vivian Li ◽

Taeg Kim ◽

...

Keyword(s):

T Cell ◽

Transgenic Mice ◽

T Cell Activation ◽

Cell Activation ◽

Mouse Tumor ◽

Tumor Models ◽

Cynomolgus Monkeys ◽

Range Finding

e15056 Background: Checkpoint blockade therapies targeting PD-1 and PD-L1 have shown great success for the treatment of various malignancies. However, a substantial fraction of patients with PD-L1-positive tumors remain unresponsive to these therapies. Novel therapy with significantly greater activity than the leading PD-1/PD-L1 inhibitors is expected to bring additional clinical benefit to patients. Here we describe the preclinical evaluation of CTX-8371, which combines anti-PD-1 and anti-PD-L1 monoclonal antibodies in one bispecific tetravalent molecule. Methods: The immune-enhancing activity of CTX-8371 was tested in vitro in T cell activation assays and tumor cell killing assay. CTX-8371 anti-tumor efficacy in vivo was assessed using mouse tumor cells expressing human PD-L1 implanted in transgenic mice humanized at the PD-1 and PD-L1 loci. CTX-8371 anti-tumor activity was also tested in xenograft tumor models. The mechanism of action of CTX-8371 was investigated in vitro using Jurkat cells expressing PD-1 or PD-L1, human PBMCs, and in vivo in tumor-bearing, chimeric PD-1/PD-L1 transgenic mice. CTX-8371 PK was determined in mice using an MSD ELISA-based assay and in cynomolgus monkeys using a qualified ELISA method. Dose range finding and toxicokinetic studies were performed in cynomolgus monkeys. Results: CTX-8371 potently enhanced T cell activation and function in vitro and showed curative efficacy as monotherapy in multiple solid tumor models, isografts or xenografts. Furthermore, CTX-8371 demonstrated superior anti-tumor efficacy compared to Keytruda or atezolizumab in checkpoint inhibitors-sensitive and resistant syngeneic mouse tumor models. Mechanistically, in addition to blocking PD-1 interaction with PD-L1, CTX-8371 bispecific antibody facilitated cell to cell bridging between cells expressing PD-1 and cells expressing PD-L1. Furthermore, we show that simultaneous binding of CTX-8371 to both PD-1 and PD-L1 resulted in long term PD-1 shedding. This suggests that CTX-8371 may prevent or overcome T cell exhaustion within the tumor microenvironment, thus providing additional advantage over existing therapies. Lastly, excellent tolerability was observed in non-human primates given 2 weekly drug infusions at up to 50 mg/kg dose. Conclusions: CTX-8371 displays multiple mechanisms of action over monoclonal PD1/PD-L1 blockade. These unique pharmacological properties of CTX-8371 could explain the enhanced T cell responses to tumor antigens and superior efficacy over current monoclonal antibody therapies. With favorable PK/PD and toxicology profiles in mice and cynomolgus monkeys, CTX-8371 warrants further advancement to clinical testing.

Download Full-text

Combination of Loncastuximab Tesirine and Polatuzumab Vedotin Shows Increased Anti-Tumor Activity in Pre-Clinical Models of Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2021-146800 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2273-2273

Author(s):

Nikoleta Sachini ◽

Asma Jabeen ◽

Patrick H van Berkel ◽

Francesca Zammarchi

Keyword(s):

Monoclonal Antibody ◽

Single Dose ◽

Hodgkin Lymphoma ◽

Annexin V ◽

Publicly Traded ◽

Non Hodgkin Lymphoma ◽

Clinical Models ◽

Anti Tumor Activity

Abstract Loncastuximab tesirine-lpyl (formerly ADCT-402) is an antibody-drug conjugate (ADC) comprising a humanised anti-CD19 monoclonal antibody conjugated to the pyrrolobenzodiazepine (PBD) dimer-based payload tesirine. Once bound to CD19 on the cell membrane, loncastuximab tesirine is rapidly internalised and the released PBD dimer warhead causes interstrand DNA crosslinks which ultimately trigger cell death. Pre-clinically, loncastuximab tesirine has shown potent and specific anti-tumor activity in lymphoma models both as single agent and in combination with other approved drugs, like venetoclax, idelalisib and bendamustine (Zammarchi, Corbett et al. 2018, Tarantelli, Spriano et al. 2019). Loncastuximab tesirine has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL) and it is currently being tested in multiple clinical trials, either as monotherapy or in combination with other anti-lymphoma drugs. Polatuzumab vedotin is an ADC composed of a humanized anti-CD79b monoclonal antibody conjugated to monomethyl auristatin E (vcMMAE) and it is approved by the FDA for treatment of r/r DLBCL when used in combination with bendamustine and rituximab. Here, we investigated the in vitro and in vivo anti-tumor activity of loncastuximab tesirine combined with polatuzumab vedotin in pre-clinical models of non-Hodgkin lymphoma (NHL). In vitro, the combination of loncastuximab tesirine and polatuzumab vedotin was tested in three human-derived, CD19 and CD79b-positive NHL cell lines (WSU-DLCL2, TMD8 and Ramos) and it resulted in synergistic (TMD8 and Ramos) and additive (WSU-DLCL2) activity, as assessed by the Chou-Talalay method. Quantification of cell viability (propidium iodide [PI]-negative and Annexin V-negative) and early/late apoptosis (Annexin V-positive and PI-negative/ Annexin V-positive and-PI positive) on TMD8 and Ramos cells treated with loncastuximab tesirine, polatuzumab vedotin or the combination of the two agents showed a significant reduction of viable cells accompanied by an increase in apoptotic cells in the combination setting compared to the single agents. In vivo, loncastuximab tesirine was tested either alone (0.25 or 0.5 mg/kg, single dose) or in combination with polatuzumab vedotin (1 mg/kg, single dose) in the WSU-DLCL2 xenograft model. At the highest dose of loncastuximab tesirine, combination with polatuzumab vedotin resulted in improved anti-tumor activity and superior response rate compared to the 2 agents in monotherapy. All treatment regimens were well tolerated by the mice, as assessed by body weight measurements and frequent observation for signs of treatment-related side effects. In conclusion, the combination of loncastuximab tesirine and polatuzumab vedotin resulted in improved anti-tumor activity both in vitro and in vivo in lymphoma preclinical models and it was well tolerated. Altogether, these novel pre-clinical data warrant translation of the combination of loncastuximab tesirine and polatuzumab vedotin into the clinic for the treatment of NHL. Disclosures Sachini: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Jabeen: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. van Berkel: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Zammarchi: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Download Full-text

Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2021-151147 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1142-1142

Author(s):

Jennifer Totman ◽

Dorothy Brach ◽

Vinny Motwani ◽

Selene Howe ◽

Emily Deutschman ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

B Cell Lymphoma ◽

Publicly Traded ◽

The Past ◽

Molecule Inhibitor

Abstract Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types. Methods: Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays. Results: Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to >10 μM. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated > 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting > 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. In vitro synergistic antiproliferative activity was also observed when EZM0414 was combined with certain SOC agents for MM and DLBCL. Conclusions: Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL. Disclosures Totman: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Brach: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Motwani: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Howe: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Deutschman: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Lampe: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Riera: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Eckley: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Alford: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Duncan: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Farrow: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Dransfield: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Raimondi: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Thomeius: Foghorn Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cosmopoulos: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Kutok: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

Induction of Fetal Hemoglobin By FTX6058, a Novel Small Molecule Development Candidate

Blood ◽

10.1182/blood-2020-142056 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-1

Author(s):

Peter Rahl ◽

Ivan Efremov ◽

Billy Stuart ◽

Keqiang Xie ◽

Mark Roth ◽

...

Keyword(s):

Small Molecule ◽

Plasma Concentrations ◽

Fetal Hemoglobin ◽

Target Engagement ◽

Publicly Traded ◽

In Vivo Models ◽

Α Subunit ◽

The Past

Red blood cell disorders like Sickle Cell Disease (SCD) and β-thalassemias are caused by mutations within the gene for the hemoglobin β (HBβ) subunit. A fetal ortholog of HBβ, hemoglobin γ (HBγ) can prevent or reduce disease-related pathophysiology in these disorders by forming nonpathogenic complexes with the required hemoglobin α-subunit. Globin expression is developmentally regulated, with a reduction in production of the fetal ortholog (γ)occurring shortly after birth and a concomitant increase in the levels of the adult ortholog (β). It has been postulated that maintaining expression of the anti-sickling γ ortholog may be of therapeutic benefit in children and adults with SCD. Indeed, individuals with the SCD mutation who also have genetic variants that maintain HBγ expression at clinically meaningful levels do not present with SCD-related symptoms. Parallel target identification efforts using CRISPR and the Fulcrum proprietary, annotated chemical probe screening set in HUDEP2 cells identified a protein complex as a key regulator of HbF expression. Structure-guided medicinal chemistry optimization led to the design of FTX-6058, a novel, potent and selective small molecule with desirable DMPK properties suitable for clinical testing. FTX-6058 treatment of differentiated primary CD34+ cells from multiple healthy donors demonstrated target engagement and potent upregulation of HBG1/2 mRNA and HbF protein. Across multiple healthy and SCD donors, FTX-6058 treatment resulted in a clinically desirable globin profile (e.g., up to 30% absolute HbF) accompanied by pancellular HbF expression, resembling the phenotype of SCD mutation carriers with hereditary persistence of fetal hemoglobin. FTX-6058 demonstrated a superior pharmacological profile relative to hydroxyurea and other small molecule compounds whose putative mechanism of action is to induce HbF. FTX-6058 treatment resulted in robust target engagement and subsequent elevation of the endogenous mouse Hbb-bh1 mRNA in wildtype CD-1 mice and, importantly, also elevation of the human HBG1 mRNA and HbF protein in the Townes SCD mouse model. Preclinical studies using a variety of in vitro and in vivo models have demonstrated the potential of FTX-6058 as a novel HbF-inducing small molecule that could be beneficial to patients with SCD and β-thalassemias. FTX-6058 was shown to be potent and selective in vitro, was well tolerated and elicited a desirable exposure-response relationship in multiple preclinical rodent models with once-a-day oral dosing and at plasma concentrations predicted to be achievable in patients. IND enabling studies for FTX-6058 have been completed. Disclosures Rahl: Fulcrum Therapeutics: Ended employment in the past 24 months. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stuart:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Xie:Fulcrum Therapeutics: Current Employment. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Barnes:Fulcrum Therapeutics: Ended employment in the past 24 months. Appiah:Fulcrum Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Peters:Fulcrum Therapeutics: Current Employment. Li:Fulcrum Therapeutics: Ended employment in the past 24 months. Kazmirski:Fulcrum Therapeutics: Ended employment in the past 24 months. Bruno:Fulcrum Therapeutics: Current Employment. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Cadavid:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

Methionine gamma-lyase-encapsulated into red blood cells (ERY-MET) antitumor activity in gastric carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.4_suppl.78 ◽

2017 ◽

Vol 35 (4_suppl) ◽

pp. 78-78

Author(s):

Vanessa Bourgeaux ◽

Karine Sénéchal ◽

Karine Aguera ◽

Fabien Gay ◽

Françoise Horand

Keyword(s):

Gastric Carcinoma ◽

Tumor Growth ◽

Red Blood Cells ◽

Cell Lines ◽

Blood Cells ◽

Half Life ◽

Anti Tumor Activity ◽

Methionine Gamma Lyase

78 Background: Methionine (Met) requirement is a cancer specific–metabolic defect that seems a promising target, especially in gastric cancers. Methionine gamma–lyase (MGL), a pyridoxal–5′–phosphate (PLP)–dependent enzyme, is an emerging approach consisting in tumors Met starvation via systemic Met depletion. ERY-MET is a new therapeutic product overcoming the short in vivo half-life of free MGL by its encapsulation into Red Blood Cells (RBCs). Indeed, ERY-MET works as a bioreactor degrading Met that passively diffuses inside the RBC. In addition, entrapped MGL activity can be controlled by supplying Vitamin B6 (PN), the precursor of MGL’s cofactor (PLP), converted inside RBCs. ERY-MET anti-tumor activity was evaluated in vivo in NMRI nudemice bearing subcutaneous gastric carcinoma. Methods: First, in vitro sensitivity of NCI-N87 and AGS human gastric cell lines to free MGL was assessed by IC50 determination using CCK–8 assay. MGL encapsulated into mouse RBCs by hypotonic dialysis was injected once in CD1 mice to determine PK-PD parameters with or without PN supplementation. The anti-tumor activity of weekly ERY-MET injections (x5) at 116 IU/kg ± 25% was assessed with or without PN supplementation in female NMRI nudemice (n = 10/group) xenografted with NCI-N87 cells. Met depletion was determined 6 days after each cumulative injection while tumor growth was followed twice a week by caliper measurement. Results: In vitro studies showed that NCI-N87 as well as AGS cell lines displayed a sensitivity to free MGL with IC50 of 0.35 ± 0.01 and 0.12 ± 0.02 IU/mL, respectively. ERY-MET with daily PN supplementation significantly increased active MGL half-life in vivo (from < 24h to 8–9 days). ERY-MET induced 80% inhibition of tumor growth at day 45 (p < 0.0001). Response rate obtained was 76% of treated mice (15/20). Besides, PN supplementation induced a slow-down of tumor growth during the supplementation period and improved ERY-MET efficacy. Conclusions: Theses results suggest that ERY-MET can induce tumor growth inhibition in mice bearing human gastric adenocarcinoma and that its effect can be regulated by PN supplementation. As such, ERY-MET seems a promising anti-tumor drug to treat gastric cancers.

Download Full-text

Novel CAR T Modules (Tmod) to Target HLA-Class I Loss in Lymphoma

Blood ◽

10.1182/blood-2020-134041 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Agnes E. Hamburger ◽

Breanna DiAndreth ◽

Mark E. Daris ◽

Melanie L. Munguia ◽

Kiran Deshmukh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Publicly Traded ◽

Private Company ◽

Car T Cells ◽

The Past ◽

Car T Cell ◽

Car T

Background: Chimeric Antigen Receptor (CAR) T-cell therapy is a proven, powerful clinical modality. However, it is still limited by the fundamental obstacle of cancer therapy: discriminating cancer from normal cells. Current FDA-approved CAR T-cell therapies eliminate normal B cells, leaving patients with B cell aplasia, hypogammaglobulinemia, and susceptible to infection. HLA-Class I loss of heterozygosity (LOH) occurs at an average frequency of ~13% among cancers and specifically ~13% in DLBCL (Broad Institute TCGA database). These losses are irreversible and distinguish the cancer from normal cells. To exploit LOH at the HLA locus, we target the remaining allelic product in tumors with LOH. We evaluated a novel AND NOT Boolean logic gate CAR T module (Tmod) T-cell system to target HLA-A*02 (A2) LOH in lymphoma using both in vitro and in vivo models. Methods: To model tumor cells that have lost A2 via LOH, we used CD19+ Raji lymphoma tumor cells. To model the corresponding "normal" cells, we established CD19+ Raji cells stably expressing A2 (CD19+/A2+ Raji). We then engineered human primary T cells to express a modular signal-integration circuit designed to be activated only by CD19+ lymphoma that do not express A2 (CD19+/A2- Raji). Each primary Tmod CAR T cell expresses both a CD19 activator (A) module using a CD19-targeting 3rd generation CAR, and a separate A2-targeting blocker (B) module using a novel A2-targeting inhibitory receptor. Human primary Tmod CAR T cells were engineered to co-express the A/B modules. First, T cells were stimulated via CD3/CD28 activation, followed by A/B module lentivirus transduction, and enriched for the B module. In vitro Tmod CAR T cells were evaluated for selective killing of CD19+/A2- Raji compared with CD19+/A2+ Raji. For in vivo proof of concept, both CD19+/A2- Raji and CD19+/A2+ Raji cell lines were injected and established into flanks of immunocompromised NGS mice and challenged with adoptive transfer of engineered human primary Tmod CAR T cells. Results: Engineered primary Tmod CAR T cells selectively killed CD19+/A2- Raji and spared CD19+/A2+ Raji (Figure 1). Tmod CAR T cells reversibly cycled from a state of non-killing, "block", to cytotoxicity and back, depending on the CD19+/A2- Raji vs. CD19+/A2+ Raji cells to which they were exposed. Importantly, primary Tmod CAR T cells selectively eliminated only the CD19+/A2- Raji cells in mixed cultures. In vivo, Tmod CAR T cells selectively eradicated CD19+/A2- Raji. More importantly, Tmod CAR T cells did not eradicate CD19+/A2+ Raji in vivo. Conclusions: CD19-targeting Tmod CAR T cells demonstrated robust and selective killing, distinguishing Raji lymphoma lines, one with A2 (CD19+/A2+) and one without (CD19+/A2-), both in vitro and in vivo. A critical requirement for Tmod CAR T-cell therapy is to determine reversibility and lack of anergy in the kill-"block"-kill and "block"-kill-"block" scenarios. This result demonstrates that Tmod CAR T cells do not terminally differentiate into one state (blockade or activation), but rather can switch back and forth as they integrate signals from "normal" and tumor cells. Furthermore, because Tmod CAR T cells can selectively target malignant B cells, it may increase the clinical therapeutic window for CAR T. Tmod CAR T cells may provide a powerful system to address hematologic malignancies and solid tumors with HLA-Class I LOH. Disclosures Hamburger: A2 Biotherapeutics: Current Employment, Current equity holder in private company. DiAndreth:A2 Biotherapeutics: Current Employment. Daris:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Munguia:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Deshmukh:A2 Biotherapeutics: Current Employment. Mock:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Asuelime:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Lim:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Kreke:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Gilead: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Tokatlian:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Maloney:A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Bioline Rx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; Gilead Science: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties, Research Funding. Go:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Amgen: Current equity holder in publicly-traded company; Allogene: Divested equity in a private or publicly-traded company in the past 24 months; Gilead: Current equity holder in publicly-traded company; Illumina: Divested equity in a private or publicly-traded company in the past 24 months. Kamb:A2 Biotherapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Download Full-text

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Blood ◽

10.1182/blood-2021-150593 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2779-2779

Author(s):

Marina Gergues ◽

Irene Raitman ◽

Joseph Gleason ◽

Valentina Rousseva ◽

Shuyang He ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Cell Lines ◽

B Cell Lymphoma ◽

Publicly Traded ◽

B Cell Malignancies ◽

Cd34 Cells ◽

Anti Tumor Activity

Abstract Background: Natural killer (NK) cells exhibit anti-tumor activity in a non-antigen-specific manner without causing graft-versus-host disease. T cell and cord blood NK cells expressing chimeric antigen receptor (CAR) targeting CD19 have demonstrated remarkable clinical efficacies against B cell lymphomas (Maude et al, N Engl J Med 2018; Neelapu et al, N Engl J Med 2017; Liu et al, N Engl J Med 2020). Celularity has developed a platform for the expansion and differentiation of human placental CD34 + stem cells towards NK cells. The introduction of CD19 CAR enables generation of CAR19-CYNK cells that can be used as an off-the-shelf, cryopreserved, allogeneic cell therapy for CD19 + B cell malignancies. Reported here are the in vitro and in vivo results evaluating anti-tumor activity of CAR19-CYNK against CD19 + B cell malignancies. Methods: CAR19-CYNK cells were generated by retroviral transduction of human placental CD34 + cells with an anti-CD19 CAR (CD19scFv-CD28CD3ζ, Sorrento Therapeutics), followed by culture expansion in the presence of cytokines. CD19 CAR expression and phenotype of CAR19-CYNK cells were characterized by flow cytometry using the following surface markers: CD56, CD3, CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46. The in vitro anti-tumor activity of CAR19-CYNK against the B cell lymphoma cell lines, Daudi and Nalm-6, was assessed at various effector to target (E:T) ratios using a flow cytometry-based cytotoxicity assay and multiplex Luminex analysis for cytokine profiling. Non-transduced (NT) NK cells were used as control. In vivo efficacy of CAR19-CYNK was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CAR19-CYNK persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after the mice were preconditioned with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CAR19-CYNK cells (1x10 7) were IV injected on Day 7. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group). Results: Placental CD34 + cells were genetically modified using a retroviral vector and achieved an average of 29.2% ± 12.4% (range 17.5% to 50.1%; n=5 donor lots) CD19 CAR expression on CAR19-CYNK cells at the end of 35-day culture. The average fold expansion of CAR19-CYNK was 6186 ± 2847 with the range of 2692 to 10626 (n=5 donor lots). Post-thaw evaluation of CAR19-CYNK (n=5 donor lots) revealed 93.8 ± 3.9% of CD56 +CD3 - NK cells, and transduction of CD19 CAR on CYNK did not significantly alter NK cell phenotype based on various activation and lineage markers (CD226, CD16, CD11a, CD94, NKG2D, NKp30, NKp44, NKp46). CAR19-CYNK displayed enhanced in vitro cytotoxicity against lymphoma cell lines, Daudi and Nalm-6, compared to that of NT NK cells. At the E:T ratio of 10:1, CAR19-CYNK (n=5 donor lots) elicited significant increased cytotoxicity against Nalm-6 compared to that of NT NK cells, with 75.9 ± 14.8% vs. 0.00 ± 0.00% at 24h (p<0.005). Under the same condition, CAR19-CYNK (n=4 donor lots) showed higher cytotoxicity against Daudi compared to that of NT NK cells with 23.6 ± 18.9% vs. 4.9 ± 4.0%. When cocultured with tumor cell lines, CAR19-CYNK showed increased secretion of the proinflammatory cytokines GM-CSF (p<0.05 for both Nalm-6 and Daudi), IFN-g (p<0.05 for Nalm-6), and TNF-a compared to that of NT NK cells at an E:T ratio of 1:1 for 24h. To evaluate the in vivo efficacy of CAR19-CYNK, a disseminated Daudi xenograft B-NDG-hIL15 model was used. CAR19-CYNK treated mice demonstrated a significant survival benefit with a median survival of 39 days versus a median survival of 28 days for the vehicle treated group (p<0.05). Conclusions: In summary, we have successfully established a process for generating CAR19-CYNK cells from human placental CD34 + cells. CAR19-CYNK demonstrated enhanced in vitro cytotoxicity against CD19 + B cell malignancies and in vivo survival benefit in a disseminated lymphoma xenograft B-NDG-hIL15 model. Further development of CAR19-CYNK for CD19 + B cell malignancies is warranted. Disclosures Gergues: Celularity Inc: Current Employment, Current equity holder in publicly-traded company. Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Van Der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Pai: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ji: Sorrento Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

Download Full-text

Mechanisms of the Anti-Tumor Activity of STAT3 Degraders in Lymphoma

Blood ◽

10.1182/blood-2020-137778 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-42

Author(s):

Haojing Rong ◽

Kirti Sharma ◽

Fred Csibi ◽

Bin Yang ◽

Scott Rusin ◽

...

Keyword(s):

Cell Cycle ◽

T Cell ◽

Tumor Regression ◽

Deep Understanding ◽

Cytokine Signaling ◽

Publicly Traded ◽

The Past ◽

The Relationship

STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family that is activated through a variety of different cytokine and growth factor receptors via JAKs, as well as through oncogenic fusion proteins and gain-of-function (GoF) mutations in STAT3 itself. STAT3 hyperactivation and GoF mutations are found in numerous cancers, including clinically aggressive hematologic malignancies with high unmet medical need, such as peripheral T cell lymphomas (PTCLs) (Andersson et al., 2020). We have previously shown that a potent and selective STAT3 heterobifunctional degrader, KTX-201, strongly represses cell growth in models of STAT3-dependent heme malignancies (Csibi et al., 2019). Herein, we report on the cellular mechanisms underlying the anti-tumor effect of STAT3 degradation in PTCL and provide a model for the relationship between pharmacokinetics/ pharmacodynamics (PK/PD) and activity of KTX-201 in vivo. The relationship between STAT3 degradation by KTX-201, anti-tumor mechanism of action and in vivo activity were investigated in anaplastic large T cell lymphoma (ALCL) models, a subset of PTCLs. In vitro, a decrease of STAT3 by 90% for 48hr was required for ALCL cells to commit to death. To identify anti-tumor mechanism(s) of KTX-201 at the systems level, we performed a time-resolved analysis of the proteomic changes of SU-DHL-1 cells undergoing growth inhibition mediated by KTX-201 at GI95. We measured the abundance of 10,000 proteins and confirmed selective degradation of STAT3 by KTX-201 after 8h of treatment. Significant changes in several marker proteins known to be involved in STAT3-mediated proximal signaling in ALCL including SOCS3, Myc and Granzyme B were observed after 16h. Functional annotation analysis of proteins identified pathways that were significantly enriched in at least one time point. Using unsupervised hierarchical clustering of annotations, we found that proteins that increased in abundance over 48h of exposure to KTX-201 were associated with markers of apoptosis and those that decreased in abundance by 24h and 48h were associated with cytokine signaling and cell cycle, respectively. Based on these data, this study identifies inhibition of cytokine signaling, G1 cell cycle arrest and induction of apoptosis as key anti-tumor mechanisms associated with KTX-201 consistent with observed cell phenotypes. STAT3 degradation in tumor was characterized in mice bearing SU-DHL-1 tumors following single dose IV administration. The STAT3 PD response in tumor was correlated with exposures in tumor. At the dose of 25 mg/kg weekly where complete tumor regression was achieved, KTX-201 achieves >90% STAT3 degradation at 24h post dosing in SUDHL1 xenografts. STAT3 degradation was maintained at 90% at 4 days post dosing. The results from the PK/PD study suggests that STAT3 degradation in tumor of >90% is necessary for anti-tumor efficacy in vivo of KTX-201, but only for a limited duration, such as 4 days out of a weekly dosing cycle. Collectively, our data demonstrate that significant STAT3 degradation for a limited time during dosing interval with KTX-201 in ALCL promotes early changes in key signaling nodes involved with proliferation and cytokine stimulation, followed by profound changes in apoptotic proteins. By integrating mechanistic biology with a deep understanding of PK/PD and efficacy, this study provides a foundation for the clinical development of STAT3 degraders using intermittent dosing regimen for treatment of PTCL and other STAT3-dependent heme malignancies. Disclosures Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Csibi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months. Yang:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chutake:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ji:Kymera Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Li:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Desavi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Download Full-text