Abstract 1,000
Title
Leveraging B-cell Immunity to Promote PD-1 blockade Effectiveness in Glioblastoma
Presenter
David Hou, BA
Company/Institution
Northwestern University
Topic Area
Immunity Beyond T Cells
Description
Background: Contrasting other malignancies like melanoma and sarcoma, glioblastoma (GBM) remains difficult to treat with immunotherapies including checkpoint blockade. This difference is largely attributed to their differing tumor microenvironments (TME). In fact, with melanoma and sarcoma, accumulation of germinal-center-like B cells have been shown to be predictive of survival in patients treated with neoadjuvant PD1 blockade. In contrast, GBM-associated B cells previously identified by this group were shown to be scarce and immunosuppressive through interactions with tumor-associated myeloid cells (TAMCs). This study establishes how B cells are driven towards their immunosuppressive phenotypes. Methods: Using single-cell RNA sequencing (scRNA-seq) of GBM patients tumor samples treated with neoadjuvant PD-1 blockade, and CT2A murine glioma model, TGFb receptors 1 and 3 were identified as the most highly expressed inhibitory receptors on GBM-associated B cells. Furthermore, TGFb1 was identified as the most highly expressed and immunosuppressive cytokine in the TME. TAMCs and glioma cells within the TME were identified as the principal source of TGFb1. We evaluated the mechanisms of B-cell exclusion and B-cell mediated anti-tumoral effector function using genetic deletion, preclinical modeling using transgenic mice and patient-derived biospecimens. Results: Targeting the myeloid compartment using intracranial anti-Gr1 antibody in combination with PD-1 blockade exhibited the ability the rescue the functional status of the B cell compartment within the TME. B cells analyzed from the dual treatment group exhibited greater proliferation and differentiation to germinal-center-like B cells. Genetic deletion of TGFb1 in tumor cells promoted PD-1 blockade efficacy. This clinical phenotype was strictly correlated to the infiltration of B cells, as it failed to improve animal survival in B-cell deficient mice. Additionally, autologous T cells isolated from the spleen exhibited greater proliferation and more robust anti-tumor response when cocultured with isolated tumor-associated B cells from the dual treatment group. Finally, inhibiting aVb8 integrin, a key complex in releasing active TGFb, increased tumor-infiltrating proliferating B cells and conferred a long-term survival benefit in the CT2A murine model. Long-term survivors treated with aVb8+PD-1 blockade received a second injection of CT2A tumor cells in the contralateral hemisphere. B cells were depleted in a group of mice using intracranial delivery of anti-CD20 depleting Ab (rituximab). Only mice where intracranial B cells were depleted developed tumor growth. Untreated long-term survivors were able to clear tumor re-challenge. Long-term survival was not achieved in B-cell-deficient CT2A mice and B and T-cell-deficient mice (Rag KO). Our mechanistic study revealed that B cells utilize both cellular and humoral immunity to fight tumor growth. On one hand, B cells activated cytotoxic granzyme B-producing CD8 T cells with direct tumor killing activity, and IL17-producing CD4 T cells able to reprogram the tumor-associated myeloid cell compartment. On the other hand, B cells showed the ability to produce tumor-reactive Abs, with potential anti-tumoral effect. Conclusions: Our results demonstrates that the immunosuppressive TME of GBM is influenced by the vital interplay between B cells and the TME through TGFb signaling. This study highlights that dual TGFb and PD1 blockade promotes germinal-center B-cell immune response against GBM.
Abstract 1,001
Title
A Combined Strategy of CD70 CAR co-expression with Membrane-bound IL-15 and CISH Knockout Results in Enhanced NK Cytotoxicity and Persistence
Presenter
James B. Trager, PhD
Company/Institution
Nkarta Therapeutics
Topic Area
Immunity Beyond T Cells
Description
Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer(NK) cells offer an alternative to T cells for cellular immunotherapy. NK cells are well-suited for allogeneic use as they are not HLA-restricted and patients that receive NK cell treatment do not develop graft-versus-host disease (GVHD). Further, NK cells can be highly expanded, engineered, edited, and cryopreserved, thus lending themselves to 'off-the-shelf' production of a potent, targeted cell therapies. Background Here, we investigated multiple approaches to modify NK anti-tumor targeting and function via CD70 targeted chimeric antigen receptor(CAR) expression and CISH gene knockout (KO). CD70 (CD27 ligand) is highly expressed in multiple hematological malignancies and insolid tumors, such as renal cell carcinoma. CISH, cytokine-inducible SH2 containing protein, is a critical checkpoint regulator in NK cell-mediated tumor immunity. We show that a combined strategy of CAR engineering with CISH KO in NK cells resulted in superior anti-tumorpotency. Methods NK cells were generated by co-culture of purified NK cells from normal donors with genetically modified irradiated K562 feeder cells. CISHKO and CD70 KO were performed using CRISPR/Cas9 ribonucleoprotein (RNP) complexes. NK cells were transduced with a gamma-retrovirus encoding a CD70 CAR and membrane bound IL-15 (mbIL-15). CAR expression was evaluated by flow cytometry. In vitrocytotoxicity of transduced NK cells was measured using the IncuCyte S3 live cell analysis system. Results Various anti-CD70 scFv domains were incorporated into CAR constructs and evaluated in primary NK cells. CAR expression in multiple donor NK cells was typically between 50-80% and expression of these CD70 CAR constructs was also maintained over the course of several weeks. In addition, transduced NKs exhibited an increase in survival and persistence in culture. Expression of CD70 CARs enhanced NKcytotoxicity against CD70 expressing RCC cell lines, 786-O and ACHN. CISH KO further enhanced not only the cytotoxic activity of CD70CAR NK cells but also their persistence in culture and resistance to suppressive molecules that are associated with the tumor microenvironment. Conclusions A combined editing and engineering strategy to modify NK cells with a CD70 CAR, mbIL-15 and CISH gene knockout increases NKcytotoxic activity and persistence. These data support the further development of CD70 CAR NK cells with CISH KO for clinical use.
Abstract 1,002
Not Available
Abstract 1,003
Title
AlloCAR TTM Cells Built for Enhanced Expansion, Persistence, and the Ability to Overcome the Solid Tumor Microenvironment
Presenter
Barbra J. Sasu, PhD
Company/Institution
Allogene Therapeutics
Topic Area
CAR T and other Cell Therapies in Solid Tumors
Description
CAR T cell therapy has attained unprecedented success in the treatment of hematological malignancies. However, clinical benefit in solid tumor indications has been limited, potentially due to a lack of targetable tumor-associated antigens (TAAs) and suppressive solid tumor microenvironments (TMEs) that inhibit T cell effector function and persistence. An essential initial step in development of solid tumor CAR T cells is identification of TAAs with high expression on tumor tissue and acceptable expression on normal tissues. Two targets meeting these criteria, CD70 for RCC and DLL3 for SCLC, where identified and extensive evaluation performed to identify lead allogeneic AlloCAR T candidates. Large panels of unique CAR T cells were screened both in vitro and in vivo in RCC and SCLC cell line and xenograft models. Candidates with robust activity were identified and advanced into safety studies, including tissue-cross reactivity off-target binding analysis and exploratory animal toxicity studies. No unexpected results were observed in the safety studies and findings included elimination of CD70-positive lymphocytes for CD70 CAR T and reversible infiltration into pituitary for DLL3 CAR T, thus supporting advancement of both CAR T programs into clinical testing. To further address the challenges of solid tumors, novel cytokine- stimulated allogenic TurboCARTM T cells co-expressing a CAR and a Turbo domain (i.e. an inducible or constitutively active homodimeric cytokine receptor chimera) that transmits CAR T cell-intrinsic cytokine signals were designed. The addition of cytokine signaling resulted in enhanced potency, expansion, and persistence in preclinical studies with both BCMA and EGFRvIII CAR T cells. Negative signaling through the PD1 receptor is an additional immune barrier CAR T cells must overcome, thus TurboCARs were augmented with a PD1 ectodomain to serve as a dominant-negative receptor. The PD1 ectodomain was modified for high-affinity binding to PD1 ligands, allowing for preferential ligand sequestration and more effective inhibition of endogenous PD1 signaling. Anti-EGFRvIII PD1 TurboCAR T cells directed towards PDL1-expressing GBM solid tumor target cells showed increased functionality compared to CAR T cells combined with PD1 blockade or to the parental TurboCAR T cells alone. Similar results were also observed using a DN TGFBR2 construct. In conclusion, promising solid tumor candidate CAR T cells were developed for the antigens CD70 and DLL3 for the treatment of RCC and SCLC. In addition, PD1 TurboCARs were developed to conferred CAR T cells with resistance to PD1-mediated inhibition, while simultaneously transmitting a cytokine signal in a CAR T cell-intrinsic fashion to enhance potency, persistence, and prevent exhaustion. The identification of suitable targets and development of novel technologies to overcome suppressive TMEs are two major steps toward making the success of CAR T in solid tumors a reality.
Abstract 1,004
Title
Beyond T cells and Bispecific Antibodies for Solid Tumors: Odyssey of Bispecific Antibody (BiAb) Armed Activated T cells (BATs)
Presenter
Lawrence G. Lum, MD, DSc
Company/Institution
University of Virginia
Topic Area
Novel Combinations
Description
Conventional cancer therapy for most locally advanced or metastatic solid tumor is associated with poor responses and survival rates. Retargeting anti-CD3 activated T cells (ATC) with anti-CD3 x anti-HER2 (HER2Bi) or anti-CD3 x anti-EGFR (EGFRBi), bispecific antibody (BiAb) makes every ex vivo activated T cell (ATC) into a non-MHC restricted HER2 or EGFR-specific cytotoxic T lymphocyte. Engaging CD3 on T cells and tumor associated antigens (TAA) induces cytokine secretion, proliferation, and cytotoxicity. Infusions of ATC armed with HER2Bi (HER2 BATs) in phase I study in 23 heavily pretreated metastatic breast cancer (MBC) showed that the OS for all, HER2 3+, and HER2 - were 36, 57, and 27 mos, respectively, with no DLTs (Lum CCR, 2016). In phase I study of hormone refractory metastatic prostate cancer (PC), there was 1 partial responder and 3 of 7 patients had significant decreases in their PSA levels (Vaishampayan, 2015 Prostate Cancer). In a phase II trial in 32 HER2- MBC the median OS was for the all patients, HER2-HR+, and TNBC 13.1 , 15.2 , and 12.3 mos for the entire group, HER2-HR+, and TNBC patients, respectively(Lum JITC,2021). The phase II trial involving 9 patients who received pembrolizumab and HER2 BATs (Vaishampayan 2020,GU ASCO). Five patients demonstrated a PSA decline of >=25%; 4 of 9 patients were progression free at 6 months; 2 patients progressed after 12 weeks of therapy; 1 patient died due to unrelated causes; and 1 patient died of progressive disease. In 9 unresectable or metastatic pancreatic cancer who received ATC armed with anti-CD3 x anti-EGFR BiAb, the median TTP is 7 months, and OS is 31 months. One patient had stable disease for 6.5 and a second is disease free 66+ months, and 2 patients developed complete responses after restart of chemotherapy (Lum, 2020,OncoImmunol). In the phase I clinical trial in 5 hypermethylated and 4 unmethylated primary glioblastoma patients, 8 infusions EGFR BATs in combination with Temozolomide/radiation therapy (TMZ)/RT) showed that EGFR BATs infusions were safe, no DLTs, and the MTD was not reached. The TTP and OS were 20 months and 31 months, respectively (unpublished). Targeting with HER2 or EGFR BATs infusions in breast, prostate, pancreatic, and brain cancer: 1) is safe without DLTs or cytokine release syndrome associated with BiAb infusions alone or CAR-T cell infusions; 2) there was no prolonged use of high dose steroids; 3) provides evidence for induction of endogenous specific anti-tumor immunity; 4) remarkable post-chemotherapy responses. These data suggest that HER2 and EGFR BATs infusions can be performed in the inexpensively in the outpatient clinic, provide an antitumor effect, may sensitize tumors to subsequent chemotherapy, and can be combined with checkpoint inhibitors to enhance anti-tumor effects. There were no dose limiting toxicities (DLTs), need for intubation, prolonged use of steroids, need for pressors, or cell-based toxicities in over 150 patients. This approach for arming ex vivo expanded T cells without the need for extraordinary cost of vectors may provide a potent tumor specific cytotoxicity to improve clinical outcomes while avoiding CRS.
Abstract 1,005
Title
INO-5401 and INO-9012 delivered intramuscularly (IM) with electroporation (EP) in combination with cemiplimab (REGN2810) in newly diagnosed glioblastoma
Presenter
David A. Reardon, MD
Company/Institution
Dana Farber Cancer Institute
Topic Area
Novel Combinations
Description
Background: Novel T cell-enabling therapies, in combination with checkpoint inhibition, may improve OS in GBM. INO-5401 (synthetic DNA plasmids encoding for hTERT, WT-1 and PSMA), plus INO-9012 (synthetic DNA plasmid encoding IL-12), with the PD-1 checkpoint inhibitor cemiplimab, was given to patients with newly-diagnosed GBM to evaluate tolerability, efficacy and immunogenicity. This abstract reports on efficacy; a companion abstract reports on immunogenicity. Methods: This is a phase I/II, single arm, two cohort study (A: MGMT unmethylated, B: MGMT methylated). Primary endpoint is safety; efficacy and immunogenicity are secondary. Nine mg INO-5401 plus 1 mg INO-9012 (every 3 weeks x 4 doses, then Q9W) was given IM with EP by CELLECTRA 2000 with cemiplimab (350 mg IV Q3W). RT was given as 40 Gy over 3 weeks. TMZ was given with radiation (all patients), followed by maintenance (Cohort B only). Results: Fifty-two subjects were enrolled: 32 in Cohort A; 20 in Cohort B. 35% women and median age 60 years (range 19-78 years). The adverse event profile was consistent with known single-agent (INO-5401, INO-9012, EP and cemiplimab) reported events; most events were <=Grade 2, and there were no related Grade >=4 events. OS at 18 months was 50% and 70% (Cohort A, Cohort B), and OS at 24 months was 22% and 55% (Cohort A, Cohort B). Median OS in Cohort A was 17.9 months (95% CI 14.5-19.8); Median OS in Cohort B has not been reached yet but will be presented (Figure). Assessment of gene transcripts from tumors at diagnosis, peripheral immune responses and paired biopsy from diagnosis and suspected progression demonstrated evidence of treatment effect and are presented elsewhere at this meeting. Conclusions: INO-5401 + INO-9012, a novel DNA plasmid vaccine/IL-12 immunotherapeutic approach has shown an acceptable safety profile and generates robust systemic immune responses to vaccine encoded tumor antigens when administered with cemiplimab and RT/TMZ among newly diagnosed GBM patients. Clinical trial information: NCT03491683. This study was approved by The New York University Institutional Review Board (IRB); approval number i17-00764.