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2797 CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, drug development, Diseases, Therapies, Myeloid Malignancies
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Mariam Murtadha, PhD1,2*, Miso Park3*, Yinghui Zhu4,5*, Enrico Caserta2,6*, Ottavio Napolitano2,6*, Theophilus Tandoh2,6*, Milad Moloudizargari2,6*, Alexander Pozhitkov2,6*, Mahmoud Singer, PhD7*, Ada Alice Dona2,6*, Hawa Vahed8*, Asaul Gonzalez, Gonzalez9*, Kevin Ly10*, Ching Ouyang11*, James Sanchez6*, Lokesh Nigam2,6*, Arnab Chowdhury6*, Lucy Y. Ghoda, PhD12,13*, Ling Li, PhD5, Bin Zhang13,14*, Amrita Krishnan, MD15, Guido Marcucci, MD13,16,17, John C. Williams3* and Flavia Pichiorri2,6

1Judy and Bernard Briskin Center for Multiple Myeloma Research, City of Hope, Los Angeles, CA
2Department of Hematologic Malignancies Translational Science, City of Hope, Monrovia, CA
3Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, Duarte, CA
4TONFJI UNIVERSITY, SHANGHAI, China
5Department of Hematological Malignancies Translational Science, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
6Judy and Bernard Briskin Center for Multiple Myeloma Research, City of Hope, Duarte, CA
7UCI, Irvine, CA
8City of Hope, Duarte, CA
9Beckman Research Institute, Department of Cancer Biology and Molecular Medicine,, Duarte, CA
10Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, Duarte, CA
11Integrative Genomics Core, City of Hope Comprehensive Cancer Center, City of Hope;, Duarte, CA
12Department of Hematologic Malignancies Translational Science, City of Hope, Duarte, CA
13Department of Hematologic Malignancies Translational Science, Gehr Family Center for Leukemia Research, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA
14Department of Hematologic Malignancies Translational Science, Gehr Family Center for Leukemia Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
15Judy and Bernard Briskin Center for Multiple Myeloma Research, City of Hope, Irvine, CA
16Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
17Hematology/HCT, City of Hope National Medical Center, Duarte, CA

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation.

Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (KD 4.77xE-10 M) and CD3 (KD 4.27xE -8 M). The IC50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher.

By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05).

BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x106 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38–treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice.

In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Disclosures: Krishnan: Bristol-Myers Squibb Company: Other: Stock Options/Ownership-Public Company; Amgen Inc, Bristol-Myers Squibb Company, Takeda Pharmaceuticals USA Inc: Other: Speakers Bureau; Janssen Biotech Inc: Other: Contracted Research; Adaptive Biotechnologies Corporation, Bristol-Myers Squibb Company, GlaxoSmithKline, Regeneron Pharmaceuticals Inc, Sanofi Genzyme: Other: Consulting Agreements; Sutro Biopharma: Other: Advisory Committee. Marcucci: Ostentus Therapeutics: Current equity holder in private company, Research Funding.

*signifies non-member of ASH