Avidity-Engineered CD3 Engaging DARPin ® Targeting Three Tumor Associated Antigens Induce Strong and Specific T Cell Dependent Killing of AML Cells with Potential for Improved Safety

Abstract AML is driven by leukemic stem cells (LSC) that resist conventional chemotherapies and remain unaffected in their niche, continually replenishing circulating blast cells. We postulated that an avidity-engineered CD3 engaging DARPin ® (Designed Ankyrin Repeat Protein) able to simultaneously target LSC-specific CD70 as well as CD123 and CD33 could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while preserving a therapeutic window towards healthy cells. Moreover, this simultaneous targeting of three different tumor associated antigens (TAAs) has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each single TAA. To achieve this ambitious goal we used our DARPin ® platform to build a novel class of triple targeting CD3 engaging molecules. Our DARPin ® libraries contain trillions of molecules allowing the generation of highly diverse binders against target proteins that can be easily combined into multi-specific DARPins ® to elicit desired biological effects. We leveraged this proprietary platform to screen multi-specific CD3 engaging DARPin ® molecules, including serum albumin binding DARPins ® for systemic half-life extension, and identify the optimal target affinity and molecular architecture to ensure potent avidity-driven T cell-mediated killing of AML cells while sparing healthy cells. This approach allowed the generation of CD3 engaging DARPins ® able to target simultaneously CD33, CD123, and CD70. Such DARPins ® demonstrated, in both allogenic and autologous setting, single digit pM potency against AML cell lines and primary cells expressing any combination of at least 2 of the 3 targeted TAAs, while showing low activity against single TAA-expressing cells, the latter representing cells of the healthy compartment. Higher expression of the selected TAAs on LSCs vs normal hematopoietic stem cells (HSC) can further enhance the selectivity of such an avidity driven molecule, leading to the preferential killing of LSC over HSC. Moreover, our multi-specific T cell engager (TCE) format resulted in a significant decrease in cytokine release in a whole blood test system for cytokine release syndrome (CRS) when compared to other mono-targeting TCE therapies, confirming its specificity and the potential for an improved safety profile within the normal hematopoietic system. Additionally, while showing similar anti-tumor efficacy in a mouse xenograft model using Molm-13 cell line and human PBMCs, CRS measured in serum 4 h after the initial injection of our multi-specific DARPin ® molecule was drastically reduced compared to a reference CD33 TCE, further strengthening the evidence that our multi targeting DARPins might also exhibit a good safety profile in humans. In conclusion, we were able to generate multi-specific CD3 engaging DARPin ® molecules with tailored affinities towards different TAAs showing exceptional efficacy and with the potential for superior safety over mono-specific TCE approaches, including systemic half-life extension to avoid a continuous intravenous infusion-based therapy. Disclosures Bianchi: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reschke: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reichen: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Fischer: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Grübler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Eggenschwiler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Krieg: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ioannou: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ragusa: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Looser: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Spitzli: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Herzog: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Villemagne: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kaufmann: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Matzner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Auge: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Hänggi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ali: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Franchini: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kirkin: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Schlereth: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Luethi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ochsenbein: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Riether: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Steiner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Goubier: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company.

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632 HPN601 is a protease-activated EpCAM-targeting T cell engager with an improved safety profile for the treatment of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0632 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A668-A668

Author(s):

Jack Lin ◽

Sony Rocha ◽

Kathryn Kwant ◽

Maria Dayao ◽

Tessie Ng ◽

...

Keyword(s):

T Cell ◽

Solid Tumors ◽

Safety Profile ◽

Half Life ◽

Active Drug ◽

Tumor Antigens ◽

Tumor Regression ◽

Life Extension ◽

Therapeutic Window ◽

Constitutively Active

BackgroundEpithelial cell adhesion molecule (EpCAM) is highly expressed in many solid tumors. However, therapeutics targeting EpCAM have had limited clinical utility or failed in clinical development likely due to the expression of EpCAM in normal tissues. For example, clinical testing of solitomab, an EpCAM-targeting T cell engager, resulted in severe dose-limiting toxicities, including elevated liver transaminases, hyperbilirubinemia, and diarrhea. Designing an EpCAM-targeting T cell engager that is only active in the tumor would expand its therapeutic window and improve its safety profile.MethodsUsing a T cell engager prodrug platform named ProTriTAC that couples therapeutic half-life extension with functional masking, we have engineered HPN601, a protease-activated EpCAM-targeting T cell engager. HPN601 is a single polypeptide with three binding domains: anti-albumin for half-life extension, anti-CD3e for T cell engagement, and anti-EpCAM for tumor cell engagement. The anti-albumin domain contains a masking moiety and a protease-cleavable linker and keeps the molecule inert outside the tumor microenvironment. Activation by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potently active drug inside the tumor. This active drug has minimal activity outside of tumor because, without an albumin binding domain, it is rapidly cleared in circulation.ResultsA humanized rodent tumor model was used to simultaneously measure anti-tumor efficacy and clinically relevant toxicity endpoints. In this model, a surrogate molecule of HPN601 was safely administered at a dose ten-fold higher than the minimal efficacious dose required for durable tumor regression. Higher doses produced toxicities including elevated ALT/AST and bilirubin, body weight loss, and evidence of tissue damage by histopathology. In contrast, a constitutively active EpCAM-targeting T cell engager could only be dosed safely up to its minimal efficacious dose. The improved safety profile of HPN601 is further supported by a toxicokinetic study in non-human primates: compared to a constitutively active EpCAM-targeting T cell engager, HPN601 had significantly attenuated cytokine production, including IFN-g, IL-2, IL-6, and IL-10.ConclusionsHPN601 is a conditionally active EpCAM-targeting T cell engager with a ten-fold improved therapeutic window compared to a constitutively active EpCAM-targeting T cell engager. An EpCAM-specific T cell engager with an improved safety profile could address unmet needs in many solid tumors and demonstrate the feasibility of using conditionally active T cell engagers to target more solid tumor antigens.Ethics ApprovalThe study was reviewed and approved by Harpoon’s Institutional Animal Care and Use Committee.

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Combinatorial Anti-Tumor Activity in Animal Models of a Novel CD123 x CD3 Bispecific Dart® Molecule (MGD024) with Cytarabine, Venetoclax or Azacitidine Supports Combination Therapy in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-153192 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1165-1165

Author(s):

Ralph F. Alderson ◽

Ling Huang ◽

Xiaoyu Zhang ◽

Haiquan Li ◽

Thomas Kaufman ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Combination Therapy ◽

Half Life ◽

Stock Options ◽

Myeloid Leukemia ◽

Cytokine Release ◽

Publicly Traded ◽

Acute Myeloid ◽

Privately Held

Abstract Introduction: Notwithstanding recent progress, acute myeloid leukemia (AML) remains an incurable disease, particularly in patients (pts) with relapsing/refractory disorder or ineligible for intensive induction therapy (unfit pts). Redirected T-cell mediated killing via CD3-engaging bispecific molecules may offer an alternate therapeutic opportunity for aggressive disease or unfit pts. Flotetuzumab, a continuous infusion CD123 x CD3 DART molecule, has shown preliminary single-agent activity in pts refractory to induction therapy (Uy et al., Blood. 2021, 137:751). CD123, the IL-3 receptor alpha chain, is expressed by both leukemic blasts and leukemic stem cells and is a suitable therapeutic target in AML (Testa et al., Cancers (Basel). 2019, 11:1358). MGD024 is an Fc-bearing CD123 x CD3 DART molecule designed for prolonged circulating half-life and intermittent delivery. MGD024 was also designed with a CD3-binding arm with reduced affinity to diminish the propensity for cytokine release compared to flotetuzumab. The potentially improved tolerability and dosing convenience of MGD024 may provide a framework for introducing T-cell immunotherapy in early-stage AML or unfit pts. To explore whether MGD024 could complement AML standard of care (SOC), we investigated combination therapy in mouse models. Materials and Methods: The DART molecules, flotetuzumab and MGD024, shared identical CD123 (humanized 7G3) and CD3 (humanized XR32) Fv arms, save for a mutation in the anti-CD3 arm of MGD024 that decreases its affinity for the CD3-epsilon chain. While flotetuzumab has no Fc domain, MGD024 includes an ala-ala-mutated human IgG1 Fc that extends its circulating half-life via the neonatal Fc receptor-mediated salvage pathway together with impairing binding to Fc-gamma receptors and complement. An IgG1-ala-ala Fc-bearing version of flotetuzumab (RES234M1.1) was also engineered to allow delivery at identical time intervals as MGD024 and avoid continuous infusion in experimental animals. MHC class I-null, NOD/SCID/IL2R-gamma-null mice were reconstituted with human PBMC (8x10 6 cells/mouse, retro orbital). Two human AML cell lines expressing low or high levels of CD123 (KG1a < MOLM-13) were implanted SC at 2.5 x 10 6 (KG1a) or 5 x 10 6 (MOLM-13) cells/mouse. Treatments (IV, IP or PO by gavage, as indicated) were initiated when tumor volumes reached ~150 mm 3, with volumes recorded weekly or twice weekly thereafter. Results and Conclusions: Consistent with its decreased affinity for CD3, MGD024 demonstrated reduced in vitro potency in killing CD123-positive target cells compared to flotetuzumab or RES234M1.1, but proportionally greater reduction in cytokine release. MGD024, however, achieved maximal cytolytic activity as flotetuzumab or RES234M1.1, albeit at increased concentrations. Similarly, MGD024 showed reduced potency in vivo against CD123-positive tumors compared to RES234M1.1; nevertheless, tumor growth reduction of the same magnitude as that observed with RES234M1.1 was attained at higher doses of MGD024 (0.5-1 mg/kg IV 2QW MGD024 vs. 0.05-0.1 mg/kg IV 2QW RES234M1.1, depending on the model). Reduced cytokine release was also observed with MGD024 compared to RES234M1.1 in vivo. To explore MGD024 suitability for combination therapy, sub-active doses of cytarabine (CYT, 10 mg/kg IV 2QW or 7.5-10 mg/kg IP QD), venetoclax (VEN, range 10-80 mg/kg PO QD), or azacitidine (AZA, 2 mg/kg PO QD) were co-administered with suboptimal regimens of MGD024 (range 0.005-0.1 mg/kg IV 2QW, depending on the model). Complete or near complete tumor elimination was observed with the combination of suboptimal MGD024 and CYT or VEN. In contrast, AZA, at the dose tested, did not contribute to the antitumor effect of MGD024. CYT, VEN or AZA did not inhibit a fully active dose of MGD024, confirming no detrimental impact of the SOC agents at the doses employed on the effector cell population engaged by the DART molecule. All treatments were well tolerated, as indicated by body weigh profiles across treatment groups. These data support clinical exploration of the combination of MGD024 with SOC in patients with AML. An investigational new drug (IND) application of MGD024 in pts with selected relapsed or refractory hematologic malignancies is planned. Disclosures Alderson: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Huang: MacroGenics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Zhang: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Li: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kaufman: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Diedrich: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Moore: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Bonvini: MacroGenics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

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Safety profile of chimeric antigen receptor T-cell immunotherapies (CAR-T) in clinical practice

European Journal of Clinical Pharmacology ◽

10.1007/s00228-021-03106-z ◽

2021 ◽

Author(s):

Giulia Bonaldo ◽

Nicola Montanaro ◽

AlbertoVaccheri ◽

Domenico Motola

Keyword(s):

T Cell ◽

Chimeric Antigen Receptor ◽

Clinical Setting ◽

Safety Profile ◽

Antigen Receptor ◽

The United States ◽

Reporting Odds Ratio ◽

Cytokine Release ◽

Car T ◽

Age Range

Abstract Purpose Two chimeric antigen receptor T-cell (CAR-T) therapies have been approved in the United States (USA) in 2017 and Europe (EU) in 2018: axicabtagene ciloleucel and tisagenlecleucel. They contain the patient’s own T cells, which are extracted, genetically modified, and reinfused. Alongside the good efficacy results, the assessment of safety profile of these new therapies represents a great challenge. Our aim was to analyze the reports of the adverse drug reactions (ADR) after CAR-T administration as occurred in the real clinical setting. Methods We performed a retrospective observational study, collecting all the reports in EU (EudraVigilance, EV) and US (FAERS) databases of ADRs regarding axicabtagene ciloleucel and tisagenlecleucel. Both descriptive and statistical analyses were performed, the latter by using Reporting Odds Ratio (ROR). Results A total number of 1426 reports of suspected ADRs were retrieved in EudraVigilance and FAERS. Patients’ reported age reflected the age range for which the drugs are approved (18–64 years for axicabtagene ciloleucel and patients aged under 25 years for tisagenlecleucel). The most reported event was cytokine release syndrome (CRS), 185 events for tisagenlecleucel and 462 for axicabtagene ciloleucel in FAERS and 137 and 498, respectively, in EudraVigilance. A disproportionality was found comparing axicabtagene ciloleucel with tisagenlecleucel for the above-mentioned event: EV ROR 2.47, 95% CI 2.22–2.74, FAERS 1.89, 1.70–2.10. Conclusion CRS represents the major problem with the administration of CAR-T therapies. Our analysis has not revealed new ADRs; however, it supports the safety profile of CAR-T with new data from real clinical setting.

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Efficacy of rFVIIIFc for First-Time Immune Tolerance Induction (ITI) Therapy: Final Results from the Global, Prospective VerITI-8 Study

Blood ◽

10.1182/blood-2021-155156 ◽

2021 ◽

Vol 138 (Supplement 2) ◽

pp. LBA-5-LBA-5

Author(s):

Lynn Malec ◽

An Van Damme ◽

Anthony Chan ◽

Mariya Spasova ◽

Nisha Jain ◽

...

Keyword(s):

Half Life ◽

Stock Options ◽

Research Funding ◽

Hemophilia A ◽

High Titer ◽

High Dose ◽

Severe Hemophilia ◽

First Time ◽

Inhibitor Titer ◽

Privately Held

Abstract Introduction: Inhibitor development is a major complication of factor VIII (FVIII) replacement therapy, affecting approximately 30% of people with severe hemophilia A (Peyvandi et al Lancet 2016). Inhibitor eradication is the standard of care to restore responsiveness to FVIII; however, ITI regimens often require frequent high-dose factor injections over a long period (DiMichele et al Haemophilia 2007; Carcao et al Haemophilia 2021). Median (interquartile range [IQR]) time (months) to negative titer in the International ITI Study with high-dose FVIII was 4.6 (2.8-13.8) (n=31); negative titer to normal recovery was 6.9 (3.5-12.0) (n=23); and normal recovery to tolerance was 10.6 (6.3-20.5) (n=22) (Hay and DiMichele Blood 2012). Recombinant factor VIII Fc fusion protein (rFVIIIFc) is an extended half-life (EHL) FVIII that showed potential benefits for ITI in retrospective clinical data and case reports (Malec et al Haemophilia 2016; Groomes et al Pediatr Blood Cancer 2016; Carcao et al Haemophilia 2021). VerITI-8 (NCT03093480) is the first prospective study of rFVIIIFc in first-time ITI and follows on from the reITIrate (NCT03103542) study of rFVIIIFc for rescue ITI (Königs et al Res Pract Thromb Haemost, ISTH 2021). Aim: Describe outcomes in the verITI-8 study of first-time ITI with rFVIIIFc over 48 weeks in subjects with severe hemophilia A and high-titer inhibitors. Methods: VerITI-8 is a prospective, single-arm, open-label, multicenter study exploring efficacy of rFVIIIFc for first-time ITI in people with severe hemophilia A with high-titer inhibitors. Initial screening was followed by an ITI period in which all subjects received rFVIIIFc 200 IU/kg/day until tolerization or 48 weeks had elapsed (Figure). This was followed by tapered dose reduction to standard prophylaxis and follow-up. Key inclusion criteria included males with severe hemophilia A, high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL), and prior treatment with any plasma-derived or recombinant standard half-life or EHL FVIII. Key exclusion criteria included coagulation disorder(s) other than hemophilia A and previous ITI. The primary endpoint was time to tolerization (successful ITI) with rFVIIIFc defined by inhibitor titer <0.6 BU/mL, incremental recovery (IR) ≥66% of expected IR (IR ≥1.32 IU/dL per IU/kg) (both at 2 consecutive visits), and t ½ ≥7 hours (h) within 48 weeks. Secondary endpoints included number of subjects achieving ITI success, annualized bleed rates (ABR), and adverse events (AEs). Results: Sixteen subjects were enrolled and received ≥1 rFVIIIFc dose. Median (range) age at baseline was 2.1 (0.8-16.0) years, and historical peak inhibitor titer was 22.4 (6.2-256.0) BU/mL (Table). Twelve (75%), 11 (69%), and 10 (63%) subjects, respectively, achieved a negative inhibitor titer, an IR >66%, and a t½ ≥7 h (ie, tolerance) within 48 weeks. Median (IQR) times in weeks to achieve these markers of success were 7.4 (2.2-17.8), 6.8 (5.4-22.4), and 11.7 (9.8-26.2) (ie, 2.7 [2.3-6.0] months to tolerance), respectively. One subject achieved partial success (negative inhibitor titer and IR ≥66%), and 5 subjects failed ITI, of which 2 had high inhibitors throughout, 2 experienced an increase in inhibitor levels, and 1 recorded a negative inhibitor titer at 282 days. Most bleeds occurred in the ITI period when median (IQR) ABRs (n=13) were 3.8 (0-10.1) overall, 0 (0-2.6) for spontaneous, 1 (0-4) for traumatic, and 0 (0-3.1) for joint. During tapering, median (IQR) ABRs (n=10) were overall, 0 (0-2.4); spontaneous, 0 (0-0); traumatic, 0 (0-1.3); and joint, 0 (0-0). All 16 subjects experienced ≥1 treatment-emergent AE (TEAE), the most frequent of which was pyrexia in 7 subjects (44%). One subject reported ≥1 related TEAE (injection site pain). Nine subjects (56%) experienced ≥1 treatment-emergent serious AE (TESAE). TESAEs occurring in ≥2 subjects included vascular device infection, contusion, and hemarthrosis. No treatment-related TESAEs, discontinuations due to AEs, or deaths were reported. Conclusions: rFVIIIFc is the first EHL FVIII with prospective data for first-time ITI in patients with severe hemophilia A with historical high-titer inhibitors. Evaluated within a 48-week timeframe, rFVIIIFc offered rapid time to tolerization (median 11.7 weeks; 2.7 months) with durable responses in almost two-thirds of subjects and was well tolerated. Optimizing ITI to eradicate inhibitors remains a priority. Figure 1 Figure 1. Disclosures Malec: CSL Behring: Consultancy; Genentech: Consultancy; HEMA Biologics: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy, Research Funding; Takeda: Consultancy; Bioverativ: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy; Bayer: Consultancy. Van Damme: Pfizer: Consultancy; Shire: Consultancy; Bayer: Consultancy. Chan: Bioverativ: Consultancy. Jain: Sanofi: Ended employment in the past 24 months; Takeda: Current Employment, Current holder of stock options in a privately-held company. Sensinger: Sanofi: Current Employment, Current holder of stock options in a privately-held company. Dumont: Sanofi: Current Employment, Current holder of stock options in a privately-held company. Lethagen: Sobi: Current Employment, Current holder of stock options in a privately-held company. Carcao: Bayer, Bioverativ/Sanofi, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Roche, and Shire/Takeda: Research Funding; Bayer, Bioverativ/Sanofi, CSL Behring, Grifols, LFB, Novo Nordisk, Pfizer, Roche, and Shire/Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Ablynx, Grifols, Kedrion, Novo Nordisk, Roche, Shire, and Sobi: Other: Personal Fees. OffLabel Disclosure: adheres to routine clinical practice

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Preclinical evaluation of AMG 160, a next-generation bispecific T cell engager (BiTE) targeting the prostate-specific membrane antigen PSMA for metastatic castration-resistant prostate cancer (mCRPC).

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.7_suppl.301 ◽

2019 ◽

Vol 37 (7_suppl) ◽

pp. 301-301 ◽

Cited By ~ 5

Author(s):

Julie Bailis ◽

Petra Deegen ◽

Oliver Thomas ◽

Pamela Bogner ◽

Joachim Wahl ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Half Life ◽

Cell Activation ◽

Phase 1 ◽

Xenograft Model ◽

Cytokine Release ◽

Single Chain

301 Background: mCRPC is a disease of high unmet medical need, especially for patients who fail novel hormonal therapies and chemotherapy. BiTE molecules provide an off the shelf therapy that activates a patient’s own immune system and redirects T cells to kill tumor cells. The BiTE mechanism of action is distinct from other immunotherapies and may unlock immune response in mCRPC. PSMA is a compelling BiTE target that is highly expressed on PCa compared to normal tissue and has increased expression in mCRPC. Methods: AMG 160 is a fully human, half-life extended (HLE) BiTE that targets PSMA on tumor cells and CD3 on T cells. AMG 160 comprises two tandem single chain variable fragments fused to an Fc domain. Results: AMG 160 binds human and non-human primate (NHP) PSMA and CD3, leading to T cell activation and proliferation and cytokine production. AMG 160 redirects T cells to kill PSMA-positive cancer cell lines in vitro, including those with low PSMA levels or androgen-independent signaling. Weekly dosing of AMG 160 induces significant antitumor activity in established PCa xenograft model. The pharmacokinetics (PK) and pharmacodynamics of AMG 160 were tested in NHP. AMG 160 treatment led to BiTE target engagement in vivo, including transient T cell activation and cytokine release in blood, and mixed cellular infiltrates in multiple organs known to express PSMA. AMG 160 treatment was well tolerated. Cytokine release associated with the first dose could be attenuated using a step dose regimen. The half-life of AMG 160 in NHP was about one week. Based on allometric scaling, the PK profile of AMG 160 may be projected to enable dosing every other week in humans. Conclusions: AMG 160 is a potent HLE BiTE with specificity for PSMA-positive tumor cells. A Phase 1 study is planned to evaluate the safety and efficacy of AMG 160 in patients with mCRPC.

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In Vitro Generation of Human Pluripotent Stem Cell-Derived T Cells for Immunotherapy

Blood ◽

10.1182/blood.v130.suppl_1.691.691 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 691-691

Author(s):

Amélie Montel-Hagen ◽

Christopher S. Seet ◽

Suwen Li ◽

Brent Chick ◽

Patrick Chang ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Pluripotent Stem Cells ◽

Research Funding ◽

Cytokine Release ◽

Highly Efficient ◽

Car T ◽

Engineered T Cells

Abstract Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19. PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.

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Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity

Blood ◽

10.1182/blood-2021-148687 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1693-1693

Author(s):

Somayeh Pouyanfard ◽

Manuel Fierro ◽

Dan S Kaufman

Keyword(s):

Ovarian Cancer ◽

Stem Cells ◽

Tumor Cells ◽

Pluripotent Stem Cells ◽

Stock Options ◽

Ovarian Cancer Cells ◽

Anti Tumor Activity ◽

Privately Held

Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing. Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

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Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer Immunotherapy

Blood ◽

10.1182/blood-2021-149095 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2771-2771

Author(s):

Mark A Wallet ◽

Toshinobu Nishimura ◽

Christina Del Casale ◽

Andriana Lebid ◽

Brenda Salantes ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Stock Options ◽

Γδ T Cells ◽

Differentiation Process ◽

Cell Therapies ◽

Car T Cells ◽

Car T ◽

Privately Held

Abstract Introduction Allogenic CAR-T cell therapies for cancer provide a new option to reduce barriers faced by autologous cell therapies, but several challenges remain. One challenge is the risk of graft versus host disease (GvHD) caused by the infused T cells. A potential solution is the use of a subset of gamma delta (γδ) CAR-T cells whose T cell receptors (TCRs) recognize invariant antigens rather than hypervariable MHC molecules. Here we describe an off-the-shelf, induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ CAR-iT) for treatment of cancer and a process for deriving such cells. Methods T cell-derived iPSCs (TiPSC) are generated by reprogramming γδ T cells to yield pluripotent stem cells. For proof-of-concept studies, TiPSC were engineered using CRISPR gene editing to deliver a CD19 CAR transgene. TiPSC are then subjected to a two-stage differentiation process. First, TiPSC are differentiated into CD34-expressing hematopoietic progenitor cells (HPCs). HPCs are then exposed to a feeder-free differentiation process that results in uniform γδ CAR-iT cells. The purity and identity of γδ CAR-iT cells were assessed by flow cytometry and the ability of γδ CAR-iT cells to respond to homeostatic growth factors was determined by intracellular staining of phosphorylated signaling proteins and mRNA transcriptome analysis. Cytokine production by CAR-iT cells was measured by immunoassays following stimulation of the CAR. Tumor cell killing by γδ CAR-iT cells was performed using IncuCyte cytotoxicity assays. In vivo control of tumors by γδ CAR-iT in immunodeficient mice was determined using a NALM-6 B cell lymphoblastic xenograft model. Results A research-grade γδ TiPSC line was used to develop an iT differentiation process. This γδ TiPSC line was engineered to express a CD19 CAR molecule and then subjected to the differentiation process after which >95% of cells were CD3 + γδ TCR + CAR + iT cells. These γδ CAR-iT cells responded to IL-2 and IL-15. STAT5 phosphorylation levels were similar but STAT3 phosphorylation levels were greater in response to IL-15 compared to IL-2 at equimolar concentrations of cytokine. IL-2 and IL-15 elicited qualitatively similar transcriptional responses, but the magnitude of cytokine-induced gene expression was generally greater in IL-15-treated cells. Upon activation, γδ CAR-iT cells released markedly less IFN-γ and other inflammatory cytokines than conventional blood-derived ab CAR-T cells. In an IncuCyte serial killing assay, γδ CAR-iT cells exhibited sustained killing of NALM-6 tumor cells for at least one week in the presence of IL-15. In vivo, γδ CAR-iT cells caused a significant reduction in NALM-6 tumor burden with a single dose of γδ CAR-iT resulting in >95% tumor growth inhibition. To establish an efficient method for derivation of clinical grade γδ TiPSC lines, we investigated methods to isolate, expand, and reprogram human γδ T cells. When γδ T cells were expanded by exposure to the chemical zoledronic acid (zoledronate) and IL-2, we found a large disparity between donors; some donors exhibit robust expansion while others are seemingly resistant to zoledronate. In order to enhance γδ T cell expansion we screened dozens of activation conditions and eventually established a universal activation protocol that can elicit robust expansion of γδ T cells from all donors tested. When expanded γδ T cells were subjected to reprogramming conditions, dozens to hundreds of individual TiPSC colonies were obtained from each donor. The identity of the rearranged γδ TCR locus was confirmed using molecular assays. New γδ TiPSC lines were engineered with a CD19 CAR molecule and killing activity was confirmed in an in vitro serial killing assay. Conclusions γδ CAR-iT cells provide a new opportunity to treat cancers with an off-the-shelf universal T cell platform without the risk for GvHD. γδ CAR-iT cells are readily manufacturable, and we have derived an end-to-end process that enables new TiPSC line reprogramming, genetic modification of TiPSC lines, and feeder-free differentiation. γδ CAR-iT cells exhibit potent antigen-specific tumor killing and they release less inflammatory cytokine than conventional CAR-T cells, potentially reducing the risk for cytokine-mediated toxicities. We believe that this off-the-shelf platform will enable safer and more accessible allogenic cell therapies for hematologic and solid cancers. Disclosures Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Nishimura: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Del Casale: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lebid: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Salantes: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Santostefano: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bucher: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Thompson: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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A Phase II Trial of Anakinra for the Prevention of CAR-T Cell Mediated Neurotoxicity

Blood ◽

10.1182/blood-2021-146927 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2814-2814

Author(s):

Matthew J. Frigault ◽

Kathleen M.E. Gallagher ◽

Marc Wehrli ◽

Betsy Valles ◽

Keagan Casey ◽

...

Keyword(s):

T Cell ◽

Stock Options ◽

Research Funding ◽

Leadership Role ◽

Publicly Traded ◽

Car T Cell ◽

Car T ◽

Post Infusion ◽

Grade 3 ◽

Privately Held

Abstract Introduction: Chimeric antigen receptor (CAR)-T cell therapy is limited in most cases to inpatient use due to risk of severe treatment-related toxicities. The two primary toxicities observed with CAR-T therapy, cytokine release syndrome (CRS) and neurotoxicity, are associated with increased circulating inflammatory cytokines such as IL-6 and IL-1. Targeting IL-6 with tocilizumab is effective for treating CRS but not neurotoxicity. Anakinra is an FDA-approved recombinant IL-1 receptor antagonist that competitively inhibits IL-1 receptor signaling and therefore blocks downstream production of inflammatory cytokines including IL-6. Leveraging support from Kite Pharma, we opened an investigator-initiated clinical trial (NCT04150913) with the hypothesis that anakinra could be administered prophylactically to prevent severe CRS and neurologic events (NE) in patients receiving axicabtagene ciloleucel (axi-cel). Here we report preliminary outcomes of this study. Study Design and Methods: This is a phase II single center, open-label study for patients ≥18 years old with relapsed or refractory large cell lymphoma. Patients must have progressed after ≥2 lines of systemic therapy but could not have CNS disease or have been previously treated with CAR-T therapy. Following leukapheresis and manufacturing, patients received 3 days of lymphodepleting chemotherapy (LDC, cyclophosphamide 500mg/m 2 and fludarabine 30 mg/m 2) and 200 mg of subcutaneously administered anakinra starting 4 hours prior to axi-cel infusion and daily thereafter for a total of 7 days. CRS and NE were graded based on the Lee 2013 criteria and the CTCAE 4.03 criteria, respectively, to enable direct comparison to the pivotal Zuma-1 cohorts. The primary endpoint is the rate and severity of NE within the first 30 days of infusion; secondary endpoints include the incidence and severity of CRS and disease response. CAR-T cell expansion, serum cytokines, and circulating biomarkers of toxicity were measured at baseline, day 3, 7, 14, 21, and 28 post CAR-T cell infusion. Results: Interim analysis of the first 6 patients demonstrated a median age of 68 (range 59-72). Patients included a diverse group of histologies including double-hit lymphoma (n=2), transformed indolent NHL (n=3), and DLBCL NOS (n=1). Two patients were considered primary refractory at time of enrollment. Pre-LDC baseline characteristics included a median SPD of 2819 mm 2 (range 1063-5802), median LDH of 415 (range 147-497) which were comparable to the pivotal ZUMA-1 cohorts. Baseline ferritin, CRP, SAA and IL-15 were similar to the pivotal ZUMA-1 cohorts. While low-grade CRS was observed in 5/6 patients, no patients experienced severe CRS and median onset occurred on day +8 (range 1-8). Four patients did not experience any NE, while two patients experienced grade 3 NE on days +6 till +9 (somnolence) and +12 (global aphasia only, for one day) respectively. With a median follow-up of 4 months, the day +28 overall response rate was 100% (4 CRs, 2 PRs), with 4/6 patients having an ongoing complete response at last disease assessment. One patient was re-infused at progression and remains in a CR 3 months from re-infusion. Responses were seen despite varying CAR-T peak level with most patients demonstrating expansion in the lower quartile of the historic ZUMA-1 cohort. Median post-infusion peak of CRP, ferritin, IL-2, GM-CSF, IFNγ, IL-10, IL-6 and SAA were lower than that observed in the pivotal ZUMA-1 cohorts. All patients remain alive at time of data analysis. Conclusions: With a limited number of patients analyzed thus far, anakinra appears to provide benefit to the toxicity profile of axi-cel, presenting reduced and/or delayed CRS and NE and a decrease in post-infusion inflammatory analytes, when compared to ZUMA-1 pivotal cohorts. No severe CRS was observed in this initial analysis and 2/6 patients experienced grade 3 NE (somnolence and global aphasia) after day 6. Despite CAR-T expansion in the lower quartile of that of ZUMA-1, we observed a 100% ORR with 4 patients remaining in CR at a median follow-up of 4 months. Additional subjects will be assessed to investigate the role of prophylactic anakinra in the management of CRS and NE, which has potential for making axi-cel treatment an outpatient therapy. Disclosures Frigault: BMS: Consultancy; Editas: Consultancy; Iovance: Consultancy; Arcellx: Consultancy; Takeda: Consultancy; Kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Wehrli: CSL Behring: Patents & Royalties; Nestle: Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Chou: Kite Pharma: Current Employment. Shen: Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties; Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties. Filosto: Kite, a Gilead Company: Current Employment; Gilead Sciences: Other: stock or other ownership ; Tusk Therapeutics: Patents & Royalties: or other intellecular property. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

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