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
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.
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